The Aptamer Handbook: Functional Oligonucleotides and Their Applications

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The Aptamer Handbook Edited by Sven Klussmann

Related Titles Hartmann, R. K., Bindereif, A., Schn, A., Westhof, E. (eds.)

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The Aptamer Handbook Functional Oligonucleotides and Their Applications Edited by Sven Klussmann

The Editor Dr. Sven Klussmann Noxxon Pharma AG Max-Dohrn-Str. 8–10 10589 Berlin Germany

All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate. Library of Congress Card No.: Applied for British Library Cataloguing-in-Publication Data: A catalogue record for this book is available from the British Library. Bibliographic information published by Die Deutsche Bibliothek Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the Internet at < http://dnb.ddb.de >. c 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Printed in the Federal Republic of Germany. Printed on acid-free paper. Typesetting Hagedorn Kommunikation, Viernheim Printing Betz Druck GmbH, Darmstadt Bookbinding Litges & Dopf Buchbinderei GmbH, Heppenheim Cover Christian Mihm, Berlin ISBN-13: ISBN-10:

978-3-527-31059-3 3-527-31059-2

Contents

Contents Part 1

History and Theoretical Background

1

In Vitro Selection of Functional Oligonucleotides and the Origins of Biochemical Activity 3 James M. Carothers and Jack W. Szostak Introduction 3 A Brief History of In Vitro Selection 4

1.1 1.2 1.3 1.4 1.5 1.6

Lessons from the Aptamers, Ribozymes, Deoxyribozymes Generated by In Vitro Selection 6 Synthetic Approaches to Understanding the Natural Origins of Function 14 Recent Technological Developments and Future Directions 17 Conclusion 22 Acknowledgments 23 References 23

2

Mathematical Models on RNA Evolution, Simulations In Silico, and Concepts for In Vitro Selection 29 Peter Schuster

2.1

From Early Experiments and Theories to the Concept of Neutral Networks 29 Evolution in the Test Tube 29 Kinetic Theory of the Evolution of Molecules 30 Sequence Space and Shape Space 31 RNA Structures, Thermodynamics and Kinetic Folding 33 Secondary Structures of Minimum Free Energies 33 Inverse Folding 34 Suboptimal Conformations and Kinetic Folding 36 Cofolding and DNA Parameters 38 Neutral Networks and In Silico Evolution of Molecules 39 Neutral Networks in Sequence Space 39 RNA Evolution In Silico 42 Lessons from Evolution In Silico 45

2.1.1 2.1.2 2.1.3 2.2 2.2.1 2.2.2 2.2.3 2.2.4 2.3 2.3.1 2.3.2 2.3.3

The Aptamer Handbook. Edited by S. Klussmann Copyright c 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-31059-2

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2.4 2.5

Designed and Natural RNA Switches 47 Outlook on Future Problems in RNA Design 49 Acknowledgments 50 References 50

3

Fitness Landscapes, Error Thresholds, and Cofactors in Aptamer Evolution 54 dam Kun, Marie-Christine Maurel, Mauro Santos, and Ers Szathmry Introduction 54 Functionality Landscapes Inferred from Examples 57 Fitness Landscape 57 Damage Selection Experiments with Ribozymes 58 Construction of the Fitness Landscape 62 Compatible Structure 63 Mispairs 63 Critical Sites 63 Predicted Structure 64 Case Study: The Fitness Landscape of the Neurospora VS Ribozyme 64 Compatible Structure of the VS Ribozyme 64 Allowed Mispairs in the VS Ribozyme 66 Critical Sites in the VS Ribozyme 66 Predicted Structure for the VS Ribozyme 67 Properties of the Estimated Fitness Landscape for the VS Ribozyme 68

3.1 3.2 3.2.1 3.2.2 3.2.3 3.2.3.1 3.2.3.2 3.2.3.3 3.2.3.4 3.2.4 3.2.4.1 3.2.4.2 3.2.4.3 3.2.4.4 3.2.4.5 3.3 3.4 3.4.1 3.4.2 3.5 3.6

Error Thresholds Inferred from Functional Landscapes: The “Realistic” Error Threshold of the Neurospora VS Ribozyme 68 Looking for Catalytic Partners: Cofactors and Aptamers 71 Co-ribozymes (cofactor-assisted ribozymes) 74 Aptazymes 79 The Use of Coenzymes: From the RNA World to the Protein World via Translation and the Genetic Code 79 Outlook 84 Acknowledgments 85 References 85

Part 2

In Vitro Selection of Target-binding Oligonucleotides

4

Aptamers to Small Molecules 95 Heiko Fickert, Iris G. Fransson, and Ulrich Hahn Introduction 95

4.1 4.2 4.3 4.4 4.5 4.6

Aptamers Aptamers Aptamers Aptamers Aptamers

to to to to to

Nucleotides/Nucleosides/Nucleobases 95 Cofactors 97 Amino Acids 99 Carbohydrates 102 Natural Products 105

Contents

4.7 4.8 4.9

Aptamers to Organic or Fluorescent Dyes 109 The Chimeric Approach for Aptamer Selection 112 Conclusion 112 Acknowledgments 113 References 113

5

Aptamers to Antibiotics 116 Christina Lorenz and Rene Schroeder Introduction 116 RNA-binding Antibiotics 119 Aptamers to Tetracyclines 119 Aptamers to Streptomycin 122 Aptamers to Aminoglycosides 124 Aptamers to Chloramphenicol 125

5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.7.1 5.8

6

6.1 6.2 6.3 6.3.1 6.3.2 6.3.3 6.3.4 6.3.5 6.4 6.4.1 6.4.2 6.4.3 6.4.4 6.4.5 6.5 6.5.1 6.5.2 6.5.3 6.5.4 6.5.5 6.6 6.6.1 6.6.2

Aptamers to the Peptide Antibiotic Viomycin 126 The Peptide Antibiotic Viomycin as a Primordial Lead Molecule 127 What Have We Learned From the Antibiotic-binding Aptamers? 128 Acknowledgments 128 References 128 Aptamers to Proteins 131 Shahid M. Nimjee, Christopher P. Rusconi, and Bruce A. Sullenger Introduction 131 Properties of Aptamers as Protein Inhibitors 134 Cytokines/Growth Factors 140 Vascular Endothelial Growth Factor (VEGF) 140 Human Interferon g 142 Angiopoietin-2 142 Basic Fibroblastic Growth Factor 142 Platelet-derived Growth Factor 143 Nucleic Acid Binding Proteins 144 HIV-1 Tat 144 HIV-1 Rev 145 HIV Reverse Transcriptase 145 Transcription Factor E2F 145 Nuclear Factor Kappa B 146 Serine Proteases 147 Hepatitis C Virus–NS3 (HSV–NS3) 147 Human Neutrophil Elastase 148 Thrombin 149 Factor VIIa 151 Factor IXa 151 Antibodies/Immunoglobulins 152 Anti-insulin Receptor Antibody MA20 152 Monoclonal Antibody (MAb) to Acetylcholine Receptor 153

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Contents

6.6.3 6.6.4 6.7 6.7.1 6.7.2 6.7.3 6.7.4 6.8 6.9 6.10 6.11 6.12 6.12.1 6.12.2 6.12.3 6.12.4 6.13

Immunoglobulin E 153 Cytotoxic T Cell Antigen 4 154 Cell Surface Receptor/Cell Adhesion Molecules 155 P-Selectin 155 L-Selectin 155 Prostate-specific Membrane Antigen 156 Trypanosoma cruzi 156 Complement Proteins – Human Complement C5 157 Extracellular Membrane Protein – Tenascin-C 157 Lipoproteins – Human Non-pancreatic Secretory Phospholipase A2 157 Prion Proteins – Prion Protein PrPSc 158 Peptides 158 Ghrelin 158 Neuropeptide Calcitonin Gene-related Peptide 1 159 Gonadotropin-releasing Hormone 159 Neuropeptide Nociceptin/Orphanin FQ 160 Conclusion 160 References 161

7

Aptamers to Nucleic Acid Structures 167 Jean-Jacques Toulm, Fabien Darfeuille, Carmelo Di Primo, and Eric Dausse Introduction 167 Targeting Double-stranded Nucleic Acids 168 Loop–Loop Interactions 170 RNA–RNA Kissing Complexes 170 DNA–RNA Kissing Complexes 173 Double RNA–RNA Kissing Loops 176 Apical Loop–Internal Loop Interactions 178 Chemically Modified Aptamers Recognizing RNA Targets 180 Biological Properties of Aptamers Targeted to Nucleic Acids 184 Conclusion 185 Acknowledgments 187 References 187

7.1 7.2 7.3 7.3.1 7.3.2 7.3.3 7.3.4 7.4 7.5 7.6

8

8.1 8.2 8.3 8.4 8.5 8.6

Riboswitches: Natural Metabolite-binding RNAs Controlling Gene Expression 191 Adam Roth, Rdiger Welz, and Ronald R. Breaker Introduction 191 Genetic Control by Riboswitches 192 Aptamer Domains of Riboswitches 194 Natural Aptamers Specific for Guanine and Adenine 196 High-resolution Aptamer Structures 200 The Glycine Riboswitch 201 References 205

Contents

Part 3

In Vitro Selection of Short, Catalytically Active Oligonucleotides

9

Catalytically Active RNA Molecules: Tools in Organic Chemistry 211 Barbara-Sylvia Weigand, Andreas Zerressen, Jrg C. Schlatterer, Mark Helm, and Andres Jschke Introduction 211 Catalytic Biopolymers 212 De Novo Creation of Ribozymes 213 The Catalytic Spectrum of Ribozymes 215 Summary and Outlook 224 References 224

9.1 9.2 9.3 9.4 9.5

10

10.1 10.1.1 10.1.2 10.1.3 10.2 10.2.1 10.2.2 10.2.3 10.2.4 10.2.5 10.3 10.3.1 10.3.2 10.3.3 10.3.4 10.3.5 10.3.6 10.3.7 10.4

Deoxyribozymes: Catalytically Active DNA Molecules 228 Kenny Schlosser, Simon A. McManus, and Yingfu Li Initial Demonstration of DNA’s Catalytic Ability 228 DNAzymes that Cleave RNA 229 Deoxyribozymes that Join DNA 231 Catalytic DNA for Porphyrin Metallation 232 A Tale of Two Deoxyribozymes that Cleave RNA 233 In Vitro Selection and Secondary Structures of 10-23 and 8-17 234 10-23 as Gene Therapeutics 235 Other Uses of 10-23 239 Utilities of 8-17 240 Recurrence of 8-17 from Several In Vitro Selection Experiments 241 Other Deoxyribozymes 245 Other RNA-cleaving Deoxyribozymes 245 RNA-ligating Deoxyribozymes 246 DNA-cleaving DNA Enzymes 248 DNA-modifying DNA Enzymes 249

DNA Enzymes that Catalyze the Formation of Phosphorothioester Bond 252 Deoxyribozymes for Thymine Dimer Repair 253 DNA Enzymes with Foreign Functionalities 254 Outlook 256 References 257

Part 4

Applications and Outlook

11

In Vivo and In Vitro Target Validation with Nucleic Acid Aptamers as Pharmacological Probes 265 P. Shannon Pendergrast and David M. Epstein Introduction 265

11.1 11.2 11.3

Target Validation with Aptamers as Pharmacological Probes 265 Limitations of Target Validation by Gene or mRNA Knockout 268

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Contents

11.4 11.4.1 11.4.2 11.5

12

12.1 12.2 12.3 12.4

13

13.1 13.1.1 13.1.2 13.2 13.2.1 13.2.2 13.3 13.4 13.5 13.5.1 13.5.2 13.5.3 13.5.4

14

14.1 14.2 14.3 14.4

Target Validation Using Nucleic Acid Aptamers 270 In Vitro Target Validation with Aptamers against Intracellular Targets 271 In Vivo Target Validation with Aptamers against Intracellular and Extracellular Targets 272 Summary 277 References 277 Intramers for Protein Function Analysis and Drug Discovery Michael Famulok and Gnter Mayer Introduction 280 Intramers: Intracellular Aptamers 281 Aptamers as Probes for Inhibitor Screening 284 Summary 287 Acknowledgments 287 References 287

280

Aptazymes: Allosteric Ribozymes and Deoxyribozymes as Biosensors Scott M. Knudsen and Andrew D. Ellington Introduction 290 Oligonucleotide-dependent Aptazymes 291 Activation by Non-nucleic Acid Effectors 291

Creating Aptazymes via Rational Design and In Vitro Selection Methodologies 292 Rational Design of Aptazymes 292 In Vitro Aptazyme Selection 293 Effector Activation 295 Aptazyme Structural and Functional Diversity 299 Uses of Aptazymes in Biology and Biotechnology 303 Aptazymes as Biosensors 303 Aptazymes as Molecular Logic Gates 306 Aptazyme Arrays 306 Aptazyme Use In Vivo 308 Acknowledgments 309 References 309 Conversion of Aptamers into Small-Molecule Lead Compounds 311 Andreas Jenne Introduction 311 Rational Drug Design 312 Biochemical Screening 313 Summary and Outlook 320 Acknowledgments 322 References 322

290

Contents

15

15.1 15.2 15.2.1 15.2.2 15.2.3 15.2.4 15.3 15.3.1 15.3.2 15.4

16

16.1 16.1.1 16.1.2 16.2 16.2.1 16.2.2 16.2.3 16.3 16.3.1 16.3.2 16.4 16.4.1 16.4.2 16.4.3 16.5

17

17.1 17.2 17.2.1 17.2.2 17.2.3 17.3 17.3.1

Aptamers as Ligands for Affinity Chromatography and Capillary Electrophoresis Applications 324 Eric Peyrin Introduction 324

Aptamers as Ligands in Affinity Liquid Chromatography (and Electrochromatography) 325 General Principles of Affinity Chromatography 325 Separation/Purification of Proteins 326 Separation of Small Molecules 329 Target-specific Chiral Separation 332 Aptamers as Ligands in Affinity Capillary Electrophoresis 335 General Principles of Affinity Capillary Electrophoresis 335 Affinity Capillary Electrophoresis for Target (Protein) Quantification 336 Concluding Remarks 340 References 341 Aptamers for In Vivo Imaging 343 Sandra Borkowski and Ludger M. Dinkelborg In Vivo Imaging: Modalities and Requirements 343 Imaging Modalities 343 Requirements for Imaging 345 Aptamers for In Vivo Imaging 346 Oligonucleotide Properties for In Vivo Applications 346 Comparison of Different Classes of Targeting Agents 348 Aptamer Targets for Imaging 349 Labeling of Aptamers 351 SPECT Isotopes 351 PET Isotopes 353 Oligonucleotides in SPECT and PET Imaging 354 Non-targeting Aptamers 354 Antisense Oligonucleotides 356 Targeting Aptamers 359 Outlook 361 References 361 Properties of Therapeutic Aptamers 363 Sharon T. Cload, Thomas G. McCauley, Anthony D. Keefe, Judith M. Healy, and Charles Wilson Introduction 363 Aptamer Targets 363 Cell Surface Targets 366 Intracellular Targets 367 Extracellular Targets 368 Aptamer Binding Characteristics 370 Aptamer Affinity 370

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17.3.2 17.3.3 17.3.4 17.4 17.4.1 17.4.2 17.4.3 17.4.4 17.4.5 17.4.6 17.4.7 17.4.8 17.5 17.5.1 17.5.2 17.6 17.6.1 17.6.2 17.6.3 17.6.4 17.6.5 17.7 17.7.1 17.7.2 17.7.3 17.7.4 17.7.5 17.8 17.9 17.10 17.10.1 17.10.2 17.10.3 17.11 17.11.1 17.11.2 17.11.3 17.11.4 17.12

Aptamer Specificity 372 Aptamer Binding Kinetics 373 Binding versus Function 375 Chemical Modification of Aptamers 376 2l-Modifications 376 Capping the 3l End 378 Capping the 5l End 379 Phosphate Substitutions 379 Base Modifications 380 Polyethylene Glycol 381 Lipid Tags 381 Peptide Tags 382 Routes of Administration of Aptamers 382 Parenteral Administration 382 Comparison to Biologics 383 Opportunities for Alternative Aptamer Formulations 383 Depot 384 Topical 384 Oral 385 Pulmonary 386 Ocular 387 Aptamer Pharmacokinetics and Biodistribution 387 Key Pharmacokinetic and Biodistribution Parameters 388 Factors Governing Pharmacokinetics and Metabolic Stability of Aptamers 389 Biodistribution of Aptamers 392 Bioanalytical Methods for Aptamer Quantification 395 Summary of Pharmacokinetic and Biodistribution Properties of Aptamers 396 Toxicity Profile of Aptamers 397 Immunogenicity of Aptamers 398 Aptamer Manufacture 398 Contributions to the Costs of Aptamer Synthesis 398 Manufacturing Infrastructure 399 Advantages of Chemical versus Biological Synthesis 399 Examples of Therapeutic Aptamers in Development 400 Antithrombin Aptamer ARC183 400 Anticomplement C5 Aptamer ARC187 401 Anti-L-Selectin Aptamer 402 Anti-PDGF-BB Aptamer ARC127 403 Future Prospects for Aptamer Therapeutics 405 References 406

Contents

18

18.1 18.2 18.2.1 18.2.2 18.2.3 18.3 18.3.1 18.3.2 18.3.2.1 18.3.2.2 18.3.2.3 18.3.2.4 18.3.2.5 18.3.2.6 18.3.2.7 18.4

19

19.1 19.2 19.3 19.3.1 19.3.2 19.3.3 19.4 19.4.1 19.4.2 19.5 19.6 19.6.1 19.6.2 19.7

Spiegelmers for Therapeutic Applications – Use of Chiral Principles in Evolutionary Selection Techniques 417 Dirk Eulberg, Florian Jarosch, Stefan Vonhoff, and Sven Klussmann Evolutionary Selection Techniques 417 Chirality 419 Discovery and Consequences of Nature’s Handedness 419 Mirror-Image Proteins 420 Mirror-Image Nucleic Acids 421 Mirror-Image Evolutionary Techniques: Selection–Reflection 422 d-Peptide Aptamers 424 Functional Mirror-Image Oligonucleotides: Spiegelmers 424 Proof of Principle 426 Catalytically Active Spiegelmers: Spiegelzymes 427 Domain Approach 428 Bioactive Spiegelmers 429 Spiegelmer Activity in Vivo 431 Pharmacological Properties of Spiegelmers 432 Production of Spiegelmers 434 Summary 437 Acknowledgments 439 References 439 Applications in the Clinic: The Anti-VEGF Aptamer 443 Tony Realini, Eugene W.M. Ng, and Anthony P. Adamis Introduction 443 Rationale for Targeting VEGF 443 VEGF and Human Disease 445 Cancer 445 Age-Related Macular Degeneration 446 Diabetic Retinopathy 448 The VEGF Therapeutic Dilemma 449 VEGF and Human Physiology 449 Overcoming the Dilemma 450 VEGF Inhibition 450 Enter Macugen 452 Preclinical Studies 452 Macugen Clinical Trials 453 The Future 456 References 457

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Epilogue A Personal Perspecitve: Aptamers after 15 Years 461 Larry Gold The Beginning 461 The First Patent 462 Creation of NeXagen and NeXstar 462 Diagnostic Imaging 463 Aptamer Therapeutics 464 Aptamer-based Diagnostics at SomaLogic 465 Do Natural Aptamers Exist? 465

Conclusions – SELEX Lessons for Drug Discovery 466 Acknowledgments 468 References 468 Index 471

Preface

Preface Why a book about aptamers? This question may be raised by all readers who are already familiar with aptamers, since quite a few reviews of outstanding quality have been published in the scientific literature. But the very same question may be asked by any reader who is – although scientifically interested – not familiar with aptamers at all. When I was asked by the publisher whether I would be interested in editing a book on aptamers I asked myself a different question: Is it really true that there is not a single textbook on aptamers so far – more than 15 years after they had first been described? Indeed, I realized that this was the case! Although the interested reader will find more than 110 000 hits when typing the term “aptamer” into the internet search machine Google, although hundreds and hundreds of publications are listed in the scientific databases, although many patents have been filed and granted, although several companies worked and still work on and with aptamers, and although last but not least a viable aptamerbased drug (Macugen) has been clinically developed, entered the market, and now helps many patients to manage the devastating disease of age-related macular degeneration, to date there has been no textbook that summarizes the great opportunities associated with aptamers. The idea of The Aptamer Handbook is to present a detailed view on the many facets of aptamers and especially their applications. It is conceptually designed for a very broad audience that not only comprises the pure scientific disciplines of molecular biology, biochemistry, and chemistry. It also addresses fields that are usually more application-oriented such as pharmacology and medicine, but it may also be helpful for managers of the pharma and biotech industries who (should) consider new and innovative technologies to be used or established more broadly. The huge amount of work invested by many brilliant scientists and the money that has been spent through either public or commercial funding so far, has helped to create an extensive, broad, and solid basis of knowledge about and around aptamers that should be recognized by an increasing number of people. Even though considerable preliminary work had been carried out earlier, the starting shot was set in 1990 when in vitro evolutionary selection techniques were used in the groups of Joyce, Gold, and Szostak to identify unique RNAbased structures that displayed new or altered functionalities: binding to a target The Aptamer Handbook. Edited by S. Klussmann Copyright c 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-31059-2

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molecule and enzymatic activity, respectively. In the Nature publication of Ellington and Szostak the target-binding RNA molecules were named “aptamers”, while in their Science publication Tuerck and Gold termed the process used to identify aptamers “SELEX” (Systematic Evolution of Ligands by EXponential enrichment). In subsequent years aptamers were raised against almost any type of target imaginable: small molecules, peptides, proteins or even ribosomal and viral particles. While many people believed that only antibodies could bind to targets with high affinity and specificity it had to be recognized that aptamers – oligonucleotide structures – could do these jobs as well, sometimes even better. The first chapter by Carothers and Szostak gives a general overview of aptamers and places their discovery into the context of discussion about the origins of life and the RNA world. The following two chapters provide the reader insight into (in vitro) evolution and fitness landscapes from a theoretical point of view. The second part of the book comprises five chapters, each dealing with aptamers that bind to certain types of targets such as small molecules, antibiotics, proteins, and nucleic acid structures. The last in the row introduces aptamer motifs called “riboswitches” that were evolved by nature itself. These “natural aptamers” are embedded in messenger RNAs and can directly sense small molecules and are therefore able to serve as regulatory elements in gene control. In the smaller, third part of the book two chapters describe the catalytic functionalities of RNA and DNA oligonucleotides. Although these so-called ribozymes or deoxyribozymes are not classified as aptamers, they can be obtained by in vitro evolutionary selection methods and exert their functions through the selected three-dimensional structures. The fourth part of the book presents an overview of the many applications of aptamers. These range from aptamers as in vitro tools for target validation outside the cell and within the cell (intramers) over so-called aptazymes, which can be used as biosensors and are made of a combination of aptamers and (deoxy)ribozymes, to aptamers as lead structures for small molecule development, and as ligands in affinity chromatography applications. Furthermore, these versatile molecules can exert their function in vivo as well. Due to their ability to bind and thereby block important disease targets, aptamers can be and are used as in vivo imaging agents and therapeutics. For these applications aptamers usually have to be chemically modified in order to render them biostable; aptamers that are built from mirror-image nucleotides (so-called spiegelmers) already display a native biostability and do not need further stabilization. The last chapter introduces Macugen, the antivascular endothelial growth factor aptamer that was approved by the US regulatory authorities for the treatment of age-related macular degeneration in December 2004. Finally, Larry Gold, who was the first to transfer SELEX from an academic lab into the environment of a biotech company, summarizes his personal view on aptamers in an epilogue. I hope that this book will help the interested reader to get a comprehensive impression of the fascinating field of aptamers. The different topics were selected to light up as many different areas of aptamer research as possible, knowing that

Preface

completeness is very difficult to achieve if possible at all. Further, I hope to attract the best and brightest to join the field to push the limits further. I am very grateful to all who made this book possible. Many thanks to the authors for their excellent chapters, to Jerry Joyce for his superb foreword, to Christian Mihm for the cover artwork, and last but not least thank you to the staff at Wiley-VCH, especially Frank Otmar Weinreich and Steffen Pauly, for their continuous support. Sven Klussmann Berlin, December 2005

XVII

Foreword

Foreword It has been 15 years since the term “aptamer” and the acronym “SELEX” were coined. With the field of directed molecular evolution now transitioning from its adolescence to young adulthood, it is an appropriate time to take stock of what aptamer science has to offer, both now and for the future. In this monograph, the first ever completely devoted to the subject of aptamers, you will find a well-chosen set of contributions from leading investigators in the field, describing the methods and applications of aptamer technology. This is not a laboratory manual, but neither is it a collection of review articles; it is a handbook that is meant to give you an appreciation for the principles and practice of in vitro selection as applied to functional nucleic acids. Whether you already are or will be a practitioner yourself, or simply want to know what all the fuss is about, this book is something that you will want to attack with a highlighter pen and scratch paper on the side. Evolution is a very powerful process, but it is surprisingly easy to carry out in a modern laboratory. You too can evolve molecules for fun and profit. The first aptamer, although it was not referred to as such, actually was created almost 40 years ago, before the advent of recombinant DNA technology (“B.C., before cloning”, as Sydney Brenner likes to say). In the late 1960s, Sol Spiegelman realized that the three fundamental processes of Darwinian evolution – amplification, mutation, and selection – could be applied to a population of RNA molecules in vitro. Amplification of RNA was achieved by employing an RNA-dependent RNA polymerase, the replicase protein of Qb bacteriophage. Mutation occurred as a result of the intrinsic error rate of the polymerase in copying variants of Qb genomic RNA. Selection was based on the ability of particular RNAs to serve as efficient templates for the production of complementary RNAs and, in turn, for the production of additional copies of themselves. “(Go forth and) multiply, with the biological proviso that (you) do so as rapidly as possible,” Spiegelman famously declared. The result, following multiple rounds of selective amplification and mutation, was a population of evolved RNA molecules that were amplified much more efficiently by the replicase compared with their ancestors. Discussion of Spiegelman’s pioneering work usually focuses on the perhaps unsurprising result that the evolved RNAs were truncated variants of Qb genomic The Aptamer Handbook. Edited by S. Klussmann Copyright c 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-31059-2

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RNA that, by virtue of their smaller size, could be copied more rapidly than the wild type. A more subtle point, however, is that the evolved RNAs also were selected to be efficient ligands for the replicase protein, which recognizes particular features of RNA secondary and tertiary structure in both the positive- and negative-stranded RNA. Thus the evolved RNAs were both an aptamer for the replicase protein and a substrate for the protein, leading to the production of progeny RNAs. One of the great advances in the history of life on Earth was the transition from an “RNA world,” in which both genetic and functional properties resided within RNA, to a DNA and protein world, in which genotype and phenotype were relegated to separate macromolecules. Another critical advance in directed molecular evolution was the development of techniques that decoupled amplification of nucleic acid molecules from selection based on their functional properties. This made it possible to select RNAs that are a ligand for any protein, for example, T4 DNA polymerase, as demonstrated by Craig Tuerk and Larry Gold. RNAs could even be selected that bound to small molecules, as shown by Andrew Ellington and Jack Szostak. In the early 1980s, following the discovery of catalytic RNA by Thomas Cech and Sidney Altman, one wondered what it would take to coax Qb replicase to amplify RNA molecules that included a ribozyme or some other functional motif. Fred Kramer and colleagues had shown that it was possible to sneak exogenous nucleotides into variants of Qb genomic RNA that could be amplified in vitro. Those familiar with the details of the system knew, however, that it was only a matter a time – and usually not much time – before the insert would be trimmed or spit out entirely, resulting in a more efficient amplicon. What was needed was a general-purpose RNA amplification method that would be indifferent to the sequence being amplified. Then came polymerase chain reaction (PCR), soon followed by reverse transcriptase PCR (RT-PCR), and everything changed. A population of nucleic acid molecules could be asked to do anything the investigator had the nerve to ask them to do: bind a target molecule, bind a target molecule but not some closely related molecule, catalyze a reaction, catalyze a reaction only after binding to some other target molecule, and so on. In retrospect, most of the early efforts were rather timid, but soon the gloves came off and it seemed that nearly everything was fair game. Literally, of course, the gloves were kept on a bit longer because RNA molecules are highly susceptible to degradation by biological nucleases, limiting their potential applications. This limitation was overcome by carrying out directed evolution with RNA analogs that are nuclease resistant, yet can be amplified by RT-PCR. Particularly intriguing in this regard are “Spiegelmers,” which first are selected as natural RNAs that bind the enantiomer of the desired target, then are prepared as the corresponding non-natural enantiomer of RNA for binding to the actual target. These reverse aptamers are aptly named because they are the mirror (Spiegel) of their biological counterparts, and in recognition of Spiegelman’s contributions to initiating the practice of in vitro Darwinian evolution.

Foreword

Aptamer science has now reached maturity, not just as a result of its longevity and accumulated knowledge, but through its growing impact on biology and medicine. In December 2004 the first aptamer compound was approved for clinical use. As discussed in the chapter by Anthony Adamis and colleagues, Macugen (pegaptanib) is a chemically modified RNA aptamer that binds tightly and specifically to vascular endothelial growth factor. It has become a preferred treatment for the neovascular form of age-related macular degeneration. Other chapters describe aptamers that are being developed for various therapeutic applications, medical imaging, clinical diagnostics, drug target validation, biosensor applications, and process chemistry. All this and more awaits you on the pages that follow. Darwinian evolution in nature has provided a bounty of functional macromolecules. However, just as synthetic organic chemistry has taken us beyond the small molecules that can be harvested as natural products, directed evolution has expanded upon the set of macromolecules to include compounds that have been tailored for our own purposes. This is not intelligent design – quite the opposite in fact – but in this book you will see how the vision and skill of the experimenter, combined with the power of an evolutionary search, can lead to some remarkable discoveries. Gerald F. Joyce Departments of Chemistry and Molecular Biology and The Skaggs Institute for Chemical Biology The Scripps Research Institute La Jolla, California, USA

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List of Contributors

List of Contributors Anthony P. Adamis Eyetech Pharmaceuticals, Inc. 3 Times Square, 12th Floor New York New York 10036 USA

Sharon T. Cload Archemix Corp, 300 Third Street Cambridge Massachusetts 02139 USA

Sandra Borkowski Research Laboratories of Schering AG Mllerstrasse 178 13342 Berlin Germany

Eric Dausse INSERM U386 European Institute of Chemistry and Biology Universit Victor Segalen 146, rue Lo-Saignat 33076 Bordeaux cedex France

Ronald R. Breaker Department of Molecular, Cellular and Developmental Biology Yale University P.O. Box 208103 New Haven Connecticut 06520-8103 USA James M. Carothers Dept. of Molecular Biology, and Center for Computational and Integrative Biology 7215 Simches Research Center Massachusetts General Hospital 185 Cambridge Street Boston Massachusetts 02114 USA

Fabien Darfeuille INSERM U386 European Institute of Chemistry and Biology Universit Victor Segalen 146, rue Lo-Saignat 33076 Bordeaux cedex France Ludger M. Dinkelborg Research Laboratories of Schering AG Muellerstrasse 178 13342 Berlin Germany


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Carmelo Di Primo INSERM U386 European Institute of Chemistry and Biology Universit Victor Segalen 146, rue Lo-Saignat 33076 Bordeaux cedex France Andrew D. Ellington University of Texas at Austin 2500 Speedway MBB 3.424 Austin Texas 78712 USA David M. Epstein Archemix Corp, 300 Third Street Cambridge Massachusetts 02139 USA Dirk Eulberg NOXXON Pharma AG Max-Dohrn-Strasse 8–10 10589 Berlin Germany Michael Famulok Rheinische Friedrich-Wilhelms Universitt Bonn Kekul-Institut fr Organische Chemie und Biochemie Gerhard-Domagk-Strasse 1 53121 Bonn Germany Heiko Fickert Universitt Hamburg Martin-Luther-King-Platz 6 20146 Hamburg Germany

Iris G. Fransson Universitt Hamburg Martin-Luther-King-Platz 6 20146 Hamburg Germany Larry Gold SomaLogic Inc. 1745 38th Street Boulder Colorado 80301 USA Ulrich Hahn Universitt Hamburg Martin-Luther-King-Platz 6 20146 Hamburg Germany Judith M. Healy Archemix Corp, 300 Third Street Cambridge Massachusetts 02139 USA Mark Helm Institut fr Pharmazie und Molekulare Biotechnologie Ruprecht-Karls-Universitt Heidelberg Im Neuenheimer Feld 364 69120 Heidelberg Germany Florian Jarosch NOXXON Pharma AG Max-Dohrn-Strasse 8–10 10589 Berlin Germany


Andres Jschke Institut fr Pharmazie und Molekulare Biotechnologie Ruprecht-Karls-Universitt Heidelberg Im Neuenheimer Feld 364 69120 Heidelberg Germany Andreas Jenne NascaCell IP GmbH Max-Lebsche-Platz 31 81377 Munich Germany Anthony D. Keefe Archemix Corp, 300 Third Street Cambridge Massachusetts 02139 USA Sven Klussmann NOXXON Pharma AG Max-Dohrn-Strasse 8–10 10589 Berlin Germany Scott M. Knudsen University of Texas at Austin 2500 Speedway MBB 3.424 Austin Texas 78712 USA dm Kun Collegium Budapest Institute for Advanced Study Szenthromsg u. 2 1014 Budapest Hungary and Department of Plant Taxonomy and Ecology Etvs University Pzmny Pter stny 1/c 1117 Budapest Hungary

Yingfu Li Department of Biochemistry and Biomedical Sciences and Department of Chemistry McMaster University 1200 Main Street West Hamilton Ontario L8N 3Z5 Canada Christina Lorenz Max F Perutz Laboratories University Departments of the Vienna Biocenter Department of Biochemistry Dr. Bohrgasse 9/5 1030 Vienna Austria Marie-Christine Maurel Institute Jacques Monod – University Paris VI Biochemistry of Evolution and Molecular Adaptability 2, place Jussieu 75251 Paris Cedex 05 France Gnter Mayer Rheinische Friedrich-Wilhelms Universitt Bonn Kekul-Institut fr Organische Chemie und Biochemie Gerhard-Domagk-Strasse 1 53121 Bonn Germany Thomas G. McCauley Archemix Corp, 300 Third Street Cambridge Massachusetts 02139 USA

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Simon A. McManus Department of Biochemistry and Biomedical Sciences and Department of Chemistry McMaster University 1200 Main Street West Hamilton Ontario L8N 3Z5 Canada Eugene W.M. Ng Eyetech Pharmaceuticals, Inc. 3 Times Square, 12th Floor New York New York 10036 USA Shahid M. Nimjee University Program of Genetics Duke University Medical Center Box 2601, DUMC Durham North Carolina 27710 USA P. Shannon Pendergrast Archemix Corp, 300 Third Street Cambridge Massachusetts 02139 USA Eric Peyrin Dpartement de Pharmacochimie Molculaire UMR 5063 CNRS Institut de Chimie Molculaire de Grenoble FR 2607 Universit Joseph Fourier UFR de Pharmacie de Grenoble Avenue de Verdun 38240 Meylan France

Adam Roth Department of Molecular, Cellular and Developmental Biology Yale University P.O. Box 208103 New Haven Connecticut 06520-8103 USA Tony Realini West Virginia University Eye Institute 1 Stadium Drive P.O. Box 9193 Morgantown West Virginia 26505 USA Christopher P. Rusconi Regado Biosciences, Inc. P.O. Box 14688 Research Triangle Park North Carolina 27709 USA Mauro Santos Collegium Budapest Institute for Advanced Study Szenthromsg u. 2 1014 Budapest Hungary and Departament de Gentica i de Microbiologia Grup de Biologia Evolutiva Universitat Autnoma de Barcelona 08193 Bellaterra (Barcelona) Spain Jrg C. Schlatterer Institut fr Pharmazie und Molekulare Biotechnologie Ruprecht-Karls-Universitt Heidelberg Im Neuenheimer Feld 364 69120 Heidelberg Germany


Kenny Schlosser Department of Biochemistry and Biomedical Sciences and Department of Chemistry McMaster University 1200 Main Street West Hamilton Ontario L8N 3Z5 Canada Rene Schroeder Max F Perutz Laboratories University Departments of the Vienna Biocenter Department of Biochemistry Dr. Bohrgasse 9/5 1030 Vienna Austria Peter Schuster Institut fr Theoretische Chemie der Universitt Wien Whringerstrasse 17 1090 Vienna Austria Bruce A. Sullenger Department of Surgery Division of Experimental Surgery Duke University Medical Center Box 2601, DUMC Durham North Carolina 27710 USA

Ers Szathmry Collegium Budapest Institute for Advanced Study Szenthromsg u. 2 1014 Budapest Hungary and Research Group of Theoretical Biology and Ecology Etvs University Pzmny Pter stny 1/c 1117 Budapest Hungary Jack W. Szostak Dept. of Molecular Biology, and Center for Computational and Integrative Biology 7215 Simches Research Center Massachusetts General Hospital 185 Cambridge Street Boston Massachusetts 02114 USA Jean-Jacques Toulm INSERM U386 European Institute of Chemistry and Biology Universit Victor Segalen 146, rue Lo-Saignat 33076 Bordeaux cedex France Stefan Vonhoff NOXXON Pharma AG Max-Dohrn-Strasse 8–10 10589 Berlin Germany

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Barbara-Sylvia Weigand Institut fr Pharmazie und Molekulare Biotechnologie Ruprecht-Karls-Universitt Heidelberg Im Neuenheimer Feld 364 69120 Heidelberg Germany Rdiger Welz Department of Molecular, Cellular and Developmental Biology Yale University P.O. Box 208103 New Haven Connecticut 06520-8103 USA Charles Wilson Archemix Corp, 300 Third Street Cambridge Massachusetts 02139 USA Andreas Zerressen Institut fr Pharmazie und Molekulare Biotechnologie Ruprecht-Karls-Universitt Heidelberg Im Neuenheimer Feld 364 69120 Heidelberg Germany

Part 1 History and Theoretical Background


1.1 Introduction

1 In Vitro Selection of Functional Oligonucleotides and the Origins of Biochemical Activity James M. Carothers and Jack W. Szostak

1.1 Introduction

In vitro selection is an experimental method for searching oligonucleotide sequence spaces for synthetic structures and activities. Oligonucleotide sequence spaces are very large – they contain the ensemble of all possible sequences of a given length separated by point mutations (Maynard Smith, 1970). For example, the sequence space of an RNA the length of a small tRNA (74 nucleotides) encompasses 1043 different molecules. The largest libraries typically synthesized in the laboratory, approximately 1016 different sequences, represent only a minute fraction of the total number of possible sequences for any nucleic acid molecule of even modest size (Wilson and Szostak, 1999). How can such necessarily sparse samplings of sequence space produce so many different aptamers, ribozymes, and deoxyribozymes? In this chapter, we focus on the technology of in vitro selection and what its application teaches us about the quantity and quality of functional structures in nucleic acid sequence spaces. We begin with a basic introduction to in vitro selection in Section 1.2, presenting a brief history of the technology growing out of the discovery of naturally occurring ribozymes. Section 1.3 deals with the synthetic aptamers, ribozymes, and deoxyribozymes generated in the last 15 years. Rather than enumerating all the successes (see Wilson and Szostak, 1999; other chapters this volume), we condense the results into a set of general lessons about the capabilities and potential limitations of functional nucleic acids. We pay particular attention to the distribution of activity in sequence space. In the next section (Section 1.4) we consider the origins of natural biochemical activity. We elaborate on some of the basic questions about the composition of simple life forms and describe how synthetic approaches make these problems accessible to experimentation. In the last section (Section 1.5) we highlight recent technological developments and research ambitions.


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1 In Vitro Selection of Functional Oligonucleotides and the Origins of Biochemical Activity

1.2 A Brief History of In Vitro Selection

Suggestions came as early as the 1960s that in addition to their templating functions, nucleic acids could fold into complex three-dimensional shapes and perform biochemical activities (Benner et al., 1999). By the 1980s, work from the Cech, Altman, and Pace groups demonstrated that RNA can catalyze at least two different kinds of chemical reactions (reviewed in Cech, 2002). Cech (Kruger et al., 1982) identified the Tetrahymena thermophila group I intron as a naturally occurring RNA that catalyzes a transesterification reaction. Likewise, the RNA portion of RNase P was shown to be both necessary and sufficient for the catalysis of the hydrolysis of the correct phosphodiester bond of its pre-tRNA substrates (Gardiner and Pace, 1980; Guerrier-Takada et al., 1983). The discovery of ribozymes renewed speculation about the functional capabilities of RNA (Cech, 1986; Orgel, 1986). Gilbert coined the phrase “RNA world” (Gilbert, 1986) to refer to an imagined stage in the early evolution of life where RNA carried out most of the biochemical functions in the cell. In this model, ribozymes with RNA polymerase activity replicated all of the functional RNA structures in the cell. The RNA world hypothesis offered a conceptual solution to the problem of how the original functional biochemical structures could have been copied, but initiated a host of other questions. Chief among them, how did the self-replicating RNAs arise? At the time, estimates of the probability of finding an RNA in a pool of random molecules with any binding or catalytic activity, much less polymerase function, ranged from the relatively likely 1 in 105 to the practically impossible 1 in 1050 (Lorsch and Szostak, 1996). Another important unanswered question was whether the functional capabilities of RNA are diverse and robust enough for RNA to have performed all of the functions necessary in a simple cell. Rational design showed that natural ribozymes can be remodeled to perform their chemistries in different contexts. Been and Cech (1988) achieved primer extension by a modified Tetrahymena group I intron; Doudna and Szostak (1989) successfully altered the group I intron to splice together strands of RNA on an exogenous template; Pace and co-workers used phylogenetic analysis to guide the creation of a minimized RNase P catalytic subunit (Waugh et al., 1989). Although those early engineering efforts were successful, it became clear that strategies based on laboratory evolution would be better suited to address questions about the range of functions that RNA can perform and the abundance of active sequences in collections of random sequences (Szostak, 1988; Joyce, 1989). In the classic test tube evolution experiments conducted by Sol Spiegelman, superior variants were isolated from populations of sequences undergoing mutation at the rate inherent to the Qb RNA polymerase (Saffhill et al., 1970; Wilson and Szostak, 1999). Modern in vitro selection experiments take advantage of the ability to direct the chemical synthesis of large combinatorial libraries. Oligonucleotide pools containing as many as 1016 different sequences can be created with nearly any design. The basic scheme for in vitro selection is outlined in Fig. 1.1.

1.2 A Brief History of In Vitro Selection

Fig. 1.1 Schemes for in vitro selection. (a) General scheme for in vitro selection. In vitro selection is an iterative method for searching libraries of partially random or completely random sequences for molecules that can perform a given function. Sequences from an input library are subjected to a selective step in which active molecules are separated from inactive molecules. Sequences that survive the selective step are amplified by polymerase chain reaction (PCR). The cycle is repeated until

functional molecules dominate the population, at which point they are cloned and sequenced. (b) Scheme for an RNA aptamer selection. Transcribing a library of DNA molecules produces RNA. In this example, RNAs that do not bind to the ligand immobilized on the column matrix are washed away. RNAs that bind the column and specifically elute are collected. Reverse transcription and PCR generate a pool of DNA whose corresponding RNA sequences are enriched in ligand-binding activity.

By 1990, in vitro selection was used to isolate rare RNAs from random sequence pools that could bind arbitrarily chosen targets (Ellington and Szostak, 1990; Tuerk and Gold, 1990). Ellington and Szostak termed the resulting motifs “aptamers,” while Tuerk and Gold dubbed the experimental process itself “systematic

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evolution of ligands by exponential amplication” (SELEX). Other applications of the technology soon followed. The same kind of phylogeny-directed analysis conducted on RNase P was demonstrated with the Tetrahymena ribozyme, where functional sequence variants were generated and identified using in vitro selection (Green et al., 1990). Tetrahymena ribozymes that could cleave a DNA substrate were readily isolated (Robertson and Joyce, 1990). Selections for single-stranded DNA molecules that bind to small organic dyes (Ellington and Szostak, 1992) and thrombin (Bock et al., 1992) proved that oligonucleotides other than RNA can also form complex shapes that exhibit non-templating functions. RNA aptamers to adenosine triphosphate (ATP) were isolated from random sequence pools by immobilizing the ligand on a solid support followed by specific elution with ATP in solution (Sassanfar and Szostak, 1993). Yeast tRNAPhe had previously been shown to be a Pb2+-dependent self-cleaving RNA (Brown et al., 1983). An effort to identify molecules with improved lead-dependent self-cleaving activity from a pool of mutagenized tRNAPhe sequences resulted in diverse novel structures (Pan and Uhlenbeck, 1992). The first entirely synthetic ribozymes were molecules with RNA–RNA ligation activity (Bartel and Szostak, 1993). Several different classes of molecules were obtained from a library of 1015 different sequences each with 220 random positions. In a manner reminiscent of the Qb polymerase experiments, error-prone polymerase chain reaction (PCR) was used to generate mutations in later rounds of the ligase selection in order to search areas in sequence space that surrounded the initial isolates. Another technique, DNA shuffling, was created to recombine sequence variations that would otherwise not be likely to be found in the same molecule, further increasing the amount of sequence space that can be searched (Stemmer, 1994; Zhao and Arnold, 1997). Systems of continuous evolution (Breaker et al., 1994) offer a way to perform dozens of rounds of selection in a relatively short time period. Population behaviors such as cooperativity and parasitism could be observed once continuous evolution was applied to ribozyme selections (Wright and Joyce, 1997; reviewed by Robertson and Ellington, 1997).

1.3 Lessons from the Aptamers, Ribozymes, Deoxyribozymes Generated by In Vitro Selection

What have we learned from in vitro selection about the functional capabilities of oligonucleotides? Fast forward to the present and one finds that synthetic RNAs have been identified that can perform many different kinds of reactions including phosphodiester bond formation and cleavage, carbon–carbon bond formation, alkylation, a Diels-Alder condensation and numerous acyl-transfer reactions including amide bond formation, among others (Joyce, 2002). RNA aptamers have been selected to hundreds of small molecule and protein targets including, for example, ATP, GTP, B12, malachite green and caffeine, HIV-1 Rev peptide, MS2 coat protein, and thrombin (reviewed in Wilson and Szostak, 1999 and Hermann


and Patel, 2000). Biochemical and structural characterization (for a review of aptamer structures see Hermann and Patel, 2000 shows that synthetic RNA structures can be stable, intricate, and exhibit very specific binding or catalytic activity. Taken together, these results demonstrate that many different functional RNA structures exist and that these can be accessed through relatively sparse searches of sequence space. In light of all the successes one might ask if there is anything that RNA cannot do. Given the challenge of keeping free radicals localized so they do not cause harm, chemical mechanisms that involve radicals may be difficult for RNA (Joyce, 2002). However, as with naturally occurring proteins, oligonucleotides can use cofactors to introduce structural or chemical characteristics they do not possess (for example, Roth and Breaker, 1998; Tsukiji et al., 2004). With the right cofactor, perhaps B12 or a quinone, a ribozyme or deoxyribozyme may be able to catalyze a radical-dependent reaction such as ribonucleotide reduction (Stubbe, 2000). Because the phosphodiester backbone of oligonucleotides is polyanionic, counterions are required for proper folding (Lilley, 2003). The metal requirements, particularly for divalent cations, can be very specific. For instance, the class I ligase ribozyme, as it was originally selected, requires Mg2+ (Glasner et al., 2002), while other in vitro selected oligonucleotides need Pb2+ (Pan and Uhlenbeck, 1992) or Ca2+ (Okumoto et al., 2003). On the other hand, structures with altered specificity for divalent cations can be selected (Lehman and Joyce, 1993; Riley and Lehman, 2003). And there are examples where divalent cations are dispensable even though the RNA was selected in conditions with a high concentration of the metal (Dieckmann et al., 1996). The widespread success in the selection of functional RNAs raised the issue of whether related nucleic acids could also give rise to functional sequences. Singlestranded DNA aptamers have been selected for many different targets (Wilson and Szostak, 1999). Likewise, deoxyribozymes can catalyze a variety of phosphodiester bond-oriented reactions. In principle, DNA should be able to catalyze as many different kinds of chemistries as RNA, especially with the help of metals or other cofactors (Li and Sen, 1997; Sidorov et al., 2004; Silverman, 2004). With few exceptions, (for example, Lauhon and Szostak, 1995) DNAs made of the same sequence as a functional RNA motif are not active (Huizenga and Szostak, 1995). And yet, the lack of a 2lOH does not seem to diminish the ability of DNA to form aptamers or enzymes. Clearly oligonucleotides such as RNA and DNA can perform many simple biochemical tasks. And, interestingly, they do not have to be made using only, or even all of, the four canonical nucleotides (A, G, C, U/T). Selections with random libraries comprising reduced sets of nucleotides reveal that molecules with even less chemical diversity can form functional structures. Rogers and Joyce (1999) found that ligase ribozymes could be built with only three bases, A, G, and U. Even a ribozyme with only two types of nucleotides, 2,6-diaminopurine and uracil can catalyze a 5l–3l RNA–RNA ligation 36 000 times faster than background (Reader and Joyce, 2002). Note, however, that the rate enhancement observed

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for the ribozyme with two kinds of nucleotides was very slow compared with ligases made of three or four base types (rate enhancements of 105 and i106, respectively) (Reader and Joyce, 2002; Bartel and Szostak, 1993). Selections from random libraries with modified nucleotides demonstrate that the “heteropolymer space” surrounding RNA and DNA also contains active molecules. Vaish et al. (2003) made libraries which incorporated a cationic group, a functionality not usually found in RNA. ATP-binding aptamers were generated whose activity depended on the presence of the modification. Yet, a side by side selection for ATP aptamers from pools with and without a different cationic modification yielded the same weakly binding motif (Battersby et al., 1999). This suggests that at the level of binding stringency required to survive the Battersby selection, sequences possessing the cationic moiety may have been at a disadvantage relative to those made up of the ordinary RNA bases only. Replication bias may have resulted in such a low level of the analog base that the composition of the pool was effectively limited to the three ordinary nucleotides. Synthetic ribozymes whose sugars have 2l-O-Me, 2l-F, or 2l-NH2 modifications are frequently used for therapeutic or technological applications because of their reduced degradation rates (Zinnen et al., 2002). In a similar vein, many naturally occurring RNAs have modified nucleotides such as pseudouridine or 2l-O-Me sugars that improve their folding and stability (Eliceiri, 1999). Selections typically lead to the emergence of the simplest and therefore most common motif capable of surviving the pressure applied (Wilson and Szostak, 1999). The effect is that the same molecule can be discovered repeatedly if the challenges and conditions are the same even though other, more complicated, solutions to the problem may exist. For example, the same “Sassanfar” ATP RNA aptamer (Sassanfar and Szostak, 1993) was isolated in different labs over the course of several years in response to selection against adenosine-containing ligands (Burgstaller and Famulok, 1994; Burke and Gold, 1997). When the selection was repeated with the additional requirement that the aptamers discriminate between binding ATP and AMP, a new motif was recovered (Sazani et al., 2004). Evidently the new aptamer requires more sequence information to specify its structure than the Sassanfar aptamer. A given pool of random molecules should contain more sequences corresponding to the simpler Sassanfar motif than the new, more complex aptamer. Accordingly, without selection pressure against it, the Sassanfar motif dominates the pool of active molecules. The hammerhead ribozyme may be a natural example of the same phenomenon. In vitro selection for cleavage activity in near-physiological conditions produced the same hammerhead motif found in diverse natural phyla (Salehi-Ashtiani and Szostak, 2001). Pragmatism motivates interest in the kinds of library designs most likely to yield functional oligonucleotides (Bartel and Szostak, 1993; Sabeti et al., 1997; Davis and Szostak, 2002). The hypothesis that longer sequence pools are better sources of activity than shorter pools relies on two rationales: (1) Larger oligonucleotides have an inherently greater capacity to fold into functional conformations than shorter molecules. (2) Complex molecules can be split into smaller modules where the likelihood of finding the motifs together in the same sequence in-


creases with the length of the pool. Calculations show that the potential advantages of larger pools depend highly on the number of modules into which the functional structure can be broken (Knight and Yarus, 2003). When sequences in a random library are longer than the functional structure, the functional motif can be present in many different positions, or registers, within a pool molecule. If a functional structure (N nucleotides long) is shorter than the pool molecules (L nucleotides long), the likelihood of finding the functional structure increases by a factor of (L – N). When the functional structure can be assembled from stretches of sequence interspersed with unconstrained regions of variable lengths, further increases in the abundance of active molecules are attained. The theoretical benefits of long random oligonucleotide pools are balanced by the realities that long pools tend to aggregate (Bartel and Szostak, 1993) and that otherwise functional structures may have a propensity to misfold when extraneous stretches of sequence are present (Sabeti et al., 1997). The negative aspects of using pools longer than 70 or 80 random nucleotides imply that they should only be used when the goal is to obtain binding or catalytic activities expected to require very large, very complex structures. Ultimately, the answer to the question of how to best design sequence libraries depends on the goal, the underlying fitness landscapes and the distribution of active structures in sequence space. In some cases there may be simple evolutionary pathways that connect a structure with one activity to a structure with a different activity (Hanczyc and Dorit, 2000; Lehman et al., 2000; Reidys et al., 2001. These structures may lie surprisingly close to one another in sequence space. For example, Schultes and Bartel (2000) found two ribozymes with entirely different secondary structures and very different functions that nonetheless share common sequences that form a neutral mutational “pathway of connectivity” between them (see also Held et al., 2003). In some cases, new functionality can be evolved from an existing structure without the shape of the molecule undergoing substantial rearrangement. The class I ligase from the Bartel and Szostak selection (1993) was converted first into a multiple turnover enzyme (Ekland et al., 1995) and then into a ribozyme with template-directed primer-extension activity (Ekland and Bartel, 1996). Eventually, a new domain was added to the 3l end of the core structure that improves the primer-extension activity to the point that 14 bases can be polymerized in a sequence-dependent manner (Johnston et al., 2001; see Fig. 1.2). The mechanistic similarities between RNA–RNA ligation and RNA polymerization likely facilitated the conversion. The ligase and the polymerase both recognize a double-stranded RNA duplex and position the a-phosphate of a downstream nucleotide for attack by a 3l OH. Even with high-resolution structural data in hand, it can be difficult to predict whether a given functional molecule is a good starting point for the evolution of a new activity. Side by side selections revealed that a library containing an ATP aptamer flanked by random sequence was not a better source of ATP-dependent nucleotide kinase ribozymes than a completely random pool (Urbach, 2000). Because kinases need to position the g-phosphate of ATP in order to activate it

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1.3 Lessons from the Aptamers, Ribozymes, Deoxyribozymes Generated by In Vitro Selection m Fig. 1.2 Stepwise progression from the class I ligase ribozyme to an RNA-dependent RNA polymerase with primer extension activity. (a) The class I ligase was isolated from a library of 1015 different 220 nucleotide-long sequences. It catalyzes the formation of a phosphodiester bond between the 3l hydroxyl of the substrate and its own 5l triphosphate. Base-pairing aligns the substrate with the ribozyme (Bartel and Szostak, 1993). (b) Selection from a mutagenized library based on the class I ligase-identified functional sequence variants. The sequence variation data were used to generate a secondary structure

model and guide the design of a molecule with multiple-turnover, intermolecular ligation activity (Ekland and Bartel, 1995; Ekland et al., 1995). (c) Further engineering converted the ligase into a ribozyme that extends a primer along an internal template by incorporating up to six nucleotides (Ekland and Bartel, 1996). (d) An extra random sequence domain was appended to the catalytic core of the ribozyme. Functional sequences were selected that use an external template to extend a primer up to 14 nucleotides (Johnston et al., 2001).

for nucleophilic attack, the expectation was that library sequences with the embedded ATP aptamer would have a selective advantage relative to completely random molecules. The nuclear magnetic resonance (NMR) structure of the aptamer shows that the ATP is stably bound and that there is good solvent accessibility for the g-phosphate (Jiang et al., 1996; Dieckman et al., 1996). Perhaps the g-phosphate enjoyed too much conformational freedom to provide an advantage to the aptamer-containing library. Or it may be that structures compatible with the geometry of that particular ATP aptamer are, by chance, very rare in sequence space. Several aptamer structures have been shown to be readily evolvable in terms of specificity. Three mutations within the binding loop of an in vitro selected l-citrulline aptamer were enough to change its specificity to l-arginine because the pattern of hydrogen bond donors and acceptors could be flipped in a simple way (Yang et al., 1996). Modifying a single base from C to U alters the specificity of the binding domains of naturally occurring riboswitches from guanine to adenine (Mandal and Breaker, 2004). In other instances, the evolution of new binding specificity has resulted in an entirely different fold. Structures with completely new secondary (and presumably) tertiary configurations were obtained upon evolving the specificity of three aptamers from flavin-adenine diphosphate (FAD) to guanosine monophosphate (GMP), even though the closest overlapping solutions differ by only three sequence mutations (Held et al., 2003). Similarly, selection for GTP binders from a pool of mutagenized ATP aptamers produced molecules with highly diverged sequences and secondary structures (Huang and Szostak, 2003). In a broad sense, it is very difficult to know whether a new function can be evolved from a pre-existing structure. However, there are methods of designing libraries that increase the chances of producing active structures. Oligonucleotides have a tendency to collapse into folded states because of the ease with which Watson–Crick and wobble-type pairings form (Wilson and Szostak, 1999). Not surprisingly, functional oligonucleotides usually contain one or more simple secondary structural motifs such as stems, stable tetraloops, pseudoknots,

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Fig. 1.3 Partially engineered RNA sequence libraries. In one successful example, an RNA library consisting of (a) 2.5 q 1014 random sequences containing a stable internal stem– tetraloop (“partially engineered”) and (b) 2.5 q 1014 completely random sequences

(“undesigned”) was used to select high-affinity aptamers for GTP (Davis and Szostak, 2002). The partially engineered sequences proved to be a much better source of high-affinity aptamers than the undesigned fully random pool (also see Fig. 1.4).

Fig. 1.4 Informational complexity and functional activity. More information is required to specify aptamers that bind a ligand more tightly (Carothers et al., 2004). In this figure, the secondary structure of each of a series of GTP-binding aptamers is placed according to its dissociation constant. Moving to the right (tighter binding), the aptamers have more in-

tricate secondary structures and are more informationally complex. An asterisk indicates structures that originated from the partially engineered sequence library. Notably seven of the 11 aptamers came from the partially engineered portion of the library, including six of the seven aptamers with dissociation constants better than 300 nmol/L.

and hairpins. It may be better to build libraries with random sequence flanking these kinds of elements, than to use completely random molecules or motifs preselected for a particular activity. In one successful example, a library of 2.5 q 1014 molecules was constructed with an internal stem and stable tetraloop flanked by


26 completely random bases on each side (see Fig. 1.3; Davis and Szostak, 2002). The partially designed library was mixed with 2.5 q 1014 completely random sequences of the same length and subjected to selection for high-affinity binding to GTP. Eleven different classes of aptamers with dissociation constants (Kd values) ranging from 8 mmol/L to 9 nmol/L were isolated and characterized (Davis and Szostak, 2002; Carothers et al., 2004). Seven of the 11 aptamers came from the partially designed pool, including, remarkably, six of the seven with affinities better than 300 nmol/L (see Fig. 1.4). To begin to understand how the abundance of functional structures in sequence space varies with the difficulty of the biochemical problem, we determined experimentally the amount of information (Schneider et al., 1986; Adami and Cerf, 2000; Adami, 2004) required to specify each of the 11 GTP aptamer structures in the conditions used in the Davis and Szostak selection (Carothers et al., 2004). In vitro selection was used to identify functional sequence variants for each of the aptamers. Roughly speaking, the information required to specify a structure increases as the number of functional sequence variants decreases (in other words, a structure that is rarer in sequence space requires more information to specify than a simpler structure that is more abundant). Based on the way that informational complexity varied with activity, each tenfold increase in binding affinity was shown to require an RNA structure that is about 1000 times less frequent in a pool of random sequences. Because the informational cost of the internal stem–tetraloop was “pre-paid,” the biased design probably increased the likelihood of finding high-activity aptamers by 250- to 1000-fold. Despite the presence of the engineered stem–tetraloop, several of the GTP aptamers are so informationally complex that they are extremely unlikely to be present in a pool with the length and composition used in the selection (Davis and Szostak, 2002; Carothers et al., 2004). One way to explain their presence, previously invoked to justify the discovery of the class I ligase (Ekland and Bartel, 1995), is that there are a very large number of different complex functional structures. Therefore, although the probability of finding any one particular complex structure may be very low, the number of different possibilities is large enough that a few informationally complex structures can be present in a diverse, welldesigned pool. In the case of the GTP aptamers, about ten more bits of information content, enough to specify five more conserved positions in an RNA molecule, are needed for each tenfold improvement in activity. Interestingly, the same quantitative relationship between activity and information was observed for a pair of ligase ribozymes (Carothers et al., 2004). Perhaps over the range of activities studied, the same types of structural changes can improve binding constants or rate constants. In both cases, increasing the thermodynamic stability of the active structures by forming more specific contacts within the RNA itself (Bergman et al., 2000) could lead to improvements in activity. If so, the informational cost of building structures with better activity could be comparable, regardless of the function. Analysis of the GTP aptamer binding specificities supports this hypothesis. Aptamers that exhibit tighter binding to GTP do not make better or more specific contacts with

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the ligand (Oestreich, Carothers and Szostak, unpublished observations). Instead, tighter-binding aptamers have more intricate and stable secondary structures and greater propensities to fold into active conformations than weaker-binding molecules. These results demonstrate that, contrary to previous suggestions (Eaton et al., 1995), selection for high-affinity binding does not automatically produce aptamers with high specificity for the ligand.

1.4 Synthetic Approaches to Understanding the Natural Origins of Function

Biochemistry began on earth more than 3.5 billion years ago when chemical systems gave rise to simple biological systems that could grow, divide, and undergo Darwinian evolution (Mojzsis et al., 1999; Joyce, 2002). Understanding the origins of life is difficult, if for no other reason than because the distinction between living and non-living matter can be blurry (Szostak et al., 2001; Ruiz-Mirazo et al., 2004; McKay, 2004). At their simplest, living systems need an active metabolism to process inputs of mass, energy, and entropy (or information). And, to enable Darwinian evolution, they must inherit differences in survivability, growth, and reproduction. We imagine that a system needs to be relatively sophisticated in order to be considered alive, but much much simpler than any extant form of life. Assuming the existence of the right precursors, was life likely to happen? The historical record does not provide many details about how early life forms worked; it only hints at what their compositions might have been (Simoneit, 2002). Deconstructing and minimizing existing organisms such as Mycoplasma (Smith et al., 2003; Check, 2002) will only tell us about cells containing DNA, RNA, and protein that are many times more complicated than the first were likely to have been. An alternative approach is to “build up” simple synthetic structures and systems (Szostak et al., 2001, Luisi, 2002; Monnard, 2003; Hanczyc and Szostak, 2004) that are not burdened by the “non-functional” selective pressures in natural organisms (for example Zarrinpar et al., 2003). Synthetic methods can render otherwise intractable questions about the origins of biochemical function accessible to experimentation (Szostak et al., 2001; Luisi, 2002; Monnard and Deamer, 2002; Joyce, 2002; Benner, 2003). For example, (1) How easily can functional structures arise? (2) How many different biochemical structures does a simple organism need? (3) How and when do complexity and systems behavior emerge? (4) How does the nature of the chemical constituents affect evolutionary trajectories? Life is a dissipative, open thermodynamic process. To some extent, the physical chemistry of life makes it similar to other systems, such as BZ chemical oscillators (reviewed in Schneider, 1985), or even fire (McKay, 2004). In all three, inputs of matter and/or energy enable the system to persist in a dynamic state far from equilibrium. But, among open thermodynamic systems, only life has the capacity for Darwinian evolution. Among the early, but not necessarily earliest, ancestors of life on earth were discrete cell-like organisms with simple physical boundaries (Szostak et al., 2001;

1.4 Synthetic Approaches to Understanding the Natural Origins of Function

Luisi, 2002; Ruiz-Mirazo et al., 2004). Physical boundaries such as membranes help maintain the concentration of other components and partially insulate the system from external fluctuations and parasites (Martin and Russel, 2003; Chen et al., 2004). There is much disagreement about whether metabolism, replicators, or some “emergent” network arose first (Kauffman, 1986; Martin and Russel, 2003; Chen et al., 2004; reviewed in Anet, 2004). For the purposes of this discussion, we are agnostic on that point and maintain that all of the models implicitly invoke complicated sets of reactions that eventually would have been catalyzed by heteropolymers. Many chemical reactions are catalyzed efficiently without heteropolymers, or even macromolecules. For example, small non-polymeric biomimetic catalysts have been generated that selectively form asymmetric carbon–carbon bonds (reviewed in Cordova, 2004). However, to achieve selective reactivity in the presence of a complex mixture of potential reactants macromolecular catalysts are usually necessary (Breslow, 2001). Heteropolymeric macromolecules can provide extended complementarity not readily available in simpler catalysts, and the potential to generate new functionality from heteropolymers is certainly more openended than for other kinds of macromolecules (van Hest and Tirrell, 2001). What were the earliest heteropolymers? What were the substrates for their synthesis? We focus attention not on determining the identity of those heteropolymers, but rather on the qualities that those molecules would have possessed (Fig. 1.5). To be biologically significant, a potential primordial heteropolymer Fig. 1.5 Characteristics of the first biopolymers. To evaluate whether a given type of heteropolymer could have been the original biopolymer, we must consider at least four different properties. (Counterclockwise): There must be a simple mechanism for the heteropolymer to template its own synthesis (replicability). Functional structures that can bind to ligands or catalyze chemical reactions must be relatively abundant in the sequence space of the heteropolymer (functional activities). To synthesize the heteropolymer itself, there should be a simple, prebiotic route for generating its subunits (monomer availability). Finally, the conditions required for the functional heteropolymers to be stable and active should be compatible with other components likely to have been part of the system, such as membranes.

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must have the following properties: (1) there must be a simple mechanism for templating the synthesis of additional heteropolymers from pre-existing ones, (2) there should be a chemically plausible supply of substrates necessary to synthesize the heteropolymer, (3) functional molecules must be accessible through relatively small searches of the sequence space of the heteropolymer, and (4) the heteropolymer and membrane components must be compatible with one another. The first biopolymers are generally thought to more closely resemble RNA than protein because the base-pairing potential of oligonucleotides enables them to more readily serve as templates for their own synthesis (Joyce, 2002). With highly activated substrates, the templated synthesis of simple RNA sequences can be achieved even without enzymes (Joyce et al., 1984, 1987). Although occasionally invoked in thought experiments (for example, GodfreySmith, 2002), no simple physical mechanism for templating the open-ended synthesis of polypeptides from another polypeptide has been proposed or achieved (Cane and Walsh, 1999; Halpin et al., 2004). A number of different oligonucleotides show base-pairing ability between complementary strands according to simple physical rules (reviewed in Eschenmoser 1999, 2004). Discerning a prebiotic, or even simple biogenic, route for the synthesis of the various monomer precursors can be complicated. Noteworthy in this regard, plausible prebiotic conditions leading to the synthesis and stabilization of pentose sugars, including ribose, were recently demonstrated (Ricardo et al., 2004). RNA has the characteristics of a good model for the kind of heteropolymer the first life forms might have used even if it was not actually the original biopolymer (Benner et al., 1999). The capacity of RNA for highly developed function is exemplified by its presence at the heart of modern ribosomes (reviewed in Steitz and Moore, 2003) and spliceosomes (reviewed in Doudna and Cech, 2002). From the discussion in Section 1.3 (also see Joyce, 2002), we know that comparatively simple RNAs obtained from samplings of only 1012 –1015 different sequences, can exhibit many of the kinds of diverse functionalities a simple organism would have needed for its metabolism. In vitro selected ribozymes as small as 71 nucleotides can catalyze the synthesis of ribonucleotide monomers from activated ribose (pRpp) and pyrimidine (Unrau and Bartel, 1998; Chapple et al., 2003). Favorable interactions have been shown to occur between clays, liposomes, and RNAs (Hanczyc et al., 2003). And model experiments suggest that emergent, systemslevel behavior could be present in fatty acid vesicles bearing ribozymes where the only function is RNA replication (Chen et al., 2004). It is not always appreciated that in addition to metabolic functions, even very simple cells would need active regulatory mechanisms, particularly to temper stochastic effects resulting from small concentrations of constituents (McAdams and Arkin, 1999). Reassuringly, there are many natural examples of RNA aptamerregulated mRNA translation in both prokaryotes (Barrick et al., 2004) and eukaryotes (reviewed in Kaempfer, 2003; also see Chapter 2.5). Likewise, in vitro selected “aptazymes” that transduce aptamer ligand binding to the control of

1.5 Recent Technological Developments and Future Directions

another activity indicate that RNAs capable of mediating responses to changing conditions can be readily isolated (Koizumi et al., 1999). Finally, a simple RNA organism needs an efficient mechanism for catalyzing the replication of its biopolymeric structures. The class I ligase ribozyme has been engineered to polymerize 14 bases along an RNA template using ribonucleoside triphosphates as substrates (Johnston et al., 2001). However, the ribozyme itself is more than 200 nucleotides long. With such poor activity, molecules that large could never copy other polymerase structures, much less other functional sequences, before the polymerases themselves would chemically degrade. Perhaps polymerases made from more stable heteropolymers, such as DNA or threose nucleic acids (TNA) (reviewed in Eschenmoser, 1999, 2004), can be isolated, which, because of their better resistance to degradation, would have a better chance of exhibiting replicase behavior. Self-replicating ligase structures have been created (Paul and Joyce, 2002) though their capacity for open-ended evolution is limited because the “monomer” units are dozens of nucleotides long.


Breaker and colleagues (Koizumi et al., 1999) demonstrated a method for isolating aptazymes, or aptamer-controlled ribozymes, whereby an obligatory internal stem of a hammerhead ribozyme motif is replaced with a random sequence library (Fig. 1.6). Ligand binding to the random sequence domain induces a conformational shift, after which the ribozyme adopts a reaction-competent state. Aptazymes have been selected that are responsive to a wide variety of ligands, including small molecule metabolites (Seetharaman et al., 2001; Hesselberth et al., 2003) and peptides (Robertson et al., 2004). The relative ease with which aptazymes can be generated is related to the fact that many nucleic acid aptamers are relatively unstructured in the absence of ligand (Hermann and Patel, 2000). The binding loops tend to be rather floppy. And the flanking stems, if short enough, are energetically unstable until the loop becomes structured upon ligand binding. The aptamer domain does not have to be explicitly selected as an aptazyme in order to function properly in that context. Active aptazyme constructs can be designed through simple secondary structure modeling. For example aptazyme constructs made with a pre-existing theophylline aptamer (Jenison et al., 1994) showed regulated group I intron splicing in vivo (Thompson et al., 2002). Significantly, allosteric selections for aptamers do not require the ligand to have a linker or other chemical modification (Wilson and Szostak, 1999). Ordinarily, small molecule ligands have to be immobilized on a solid support to partition functional aptamer sequences away from inactive ones. This aspect of the method permits aptamers that completely enclose the ligand to be isolated, potentially increasing the maximum binding affinity for which an aptamer selection can be performed.

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Fig. 1.6 A simple aptazyme. There are many different ways to couple aptamer ligand binding to the activation of a ribozyme (also see Chapters 9 and 16). This figure illustrates a simple self-cleaving aptazyme (after Koizumi et al., 1999). The structure consists of a selfcleaving hammerhead ribozyme with an inter-

nal stem replaced by an aptamer sequence (“A”). Upon binding the ligand (“L”), the aptamer becomes well-ordered, reconstituting the disrupted hammerhead stem. Once the stem forms, the ribozyme can adopt an active conformation and undergo self-cleavage.

When designing an in vitro selection protocol, it is important to make the activity needed to survive the selective step resemble the desired function as closely as possible. Most successful attempts to engineer multiple-turnover catalysts from random sequence libraries have actually begun with molecules isolated because they could perform a single-turnover reaction on a covalently attached substrate (for example see Bartel and Szostak, 1993; Seelig and Jaschke, 1999; reviewed in Griffiths and Tawfik, 2000). After the selection, the molecules must be reengineered to function as true multiple-turnover enzymes (for example Ekland et al., 1995). In contrast, compartmentalizing each of the library molecules in its own reaction chamber allows for the direct selection of multiple-turnover catalysts (reviewed in Griffiths and Tawfik, 2000; Fig. 1.7). In a typical protocol for selection by compartmentalization, water-in-oil emulsions are prepared by mixing mineral oil and a cocktail of detergents with a water phase containing DNA, protein polymerases, and buffer and substrates for transcription and/or translation. The emulsion compartments are stable over a range of temperature and buffer conditions. Catalysts can be selected on the basis of their ability to modify an exogenous substrate attached to a library DNA molecule enclosed in the same compartment (Tawfik and Griffiths, 1998). Fluorescenceactivated cell sorting (FACS) can be used to enrich active molecules if the reaction can produce, directly or indirectly, a fluorescent signal. FACS has been employed to isolate active library sequences attached to beads (Sepp et al., 2002; Griffiths and Tawfik, 2003). Alternatively, water-in-oil compartments can be emulsified in


Fig. 1.7 Compartmentalized selections with emulsions, beads, and flow cytometry. (a) Fluorescence from Oregon Green dye marks the aqueous droplet of a water-in-oil-inwater emulsion (also see Griffiths and Tawfik, 2003). The droplet in the center of the image contains RNA transcribed from a single DNA template (courtesy of Luptak and Szostak, unpublished). Water-in-oil droplets containing RNAs with a desired activity can be selected on the basis of a spectroscopic signal using flow cytometry. This approach also allows for the

direct selection of aptamers or ribozymes that elicit a particular change in the emission spectrum of a fluorescent dye. (b) In much the same way, flow cytometry can be used to sort microbeads displaying a spectroscopic signal. In the example depicted, RNA is immobilized on beads in an emulsified solution containing a fluorophore (“F”). After breaking the emulsions, beads displaying aptamers that induce changes in fluorescence can be sorted (after Luptak and Szostak, unpublished).

water to produce stable water-in-oil-in-water double emulsion compartments that can be sorted (Bernath et al., 2004). Liposomes also have properties that make them attractive for use as encapsulated reaction chambers (reviewed in Monnard, 2003). However, the emulsion techniques are much farther along in development because liposomes typically exhibit low encapsulation efficiency. To produce reaction chambers 1 mm in diameter, approximately 1 mL of waterin-oil emulsion is needed for every 1012 library molecules (Tawfik and Griffiths, 1998) unless more than one library sequence is put into the same chamber. This limits efficient selections to a library size of about 1014 different molecules. Because FACS is a serial screening process, selections that employ it are further limited; 107 beads or compartments can be easily sorted but approaching even 1010 molecules would be nearly impossible. Bead panning (Coomber, 2002) is an alternative sorting process that may allow for higher throughput. However, until the sorting of larger libraries can be routinely accomplished, the best applications of compartmentalized selection are (1) selecting for simple activities that are very abundant in random sequence pools and (2) searching the sequence space surrounding pre-existing structures for variants with altered, or superior, activity. Compartmentalized selection of protein enzymes has been used to improve a phosphotriesterase (Griffiths and Tawfik, 2003), alter the sequence specificity of

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a methyltransferase (Cohen et al., 2004) and enhance the temperature stability and heparin resistance of Taq polymerase (Ghadessy et al., 2001). However, there is nothing that prevents compartmentalized selections from being used for the in vitro selection of functional nucleic acids. Generating ribozymes with replicase activity remains a significant hurdle to the goal of creating simple chemical systems capable of Darwinian evolution (Szostak, 2001 et al., 2001; Luisi, 2002; Benner, 2003). In principle it should be possible to build replicases through iterations of in vitro selection and rational design. If there are class I ligase-based polymerase variants with improved affinity for the primer–template complex (Lawrence and Bartel, 2003), these could be isolated in a compartmentalized selection based directly on their ability to polymerize longer strands of RNA. Alternatively, compartmentalized selections for completely novel ribozymes with efficient and processive polymerase activity could be undertaken. In either case, these efforts might benefit from the use of substrates that are more highly activated than nucleotide triphosphates, or from the addition of other metal ions and cofactors (Szostak et al., 2001; for example Joyce et al., 1984, 1987). Compartmentalization can also be used to isolate aptamers or ribozymes where a spectroscopic signal is the desired function, not just the readout. Two aptamers have been shown to elicit fortuitous changes in the spectroscopic properties of fluorescent small molecule ligands upon binding (Babendure et al., 2003, references therein). A FACS-based selection could be used to isolate aptamers that induce specific binding-dependent changes in quantum yield or spectroscopic resonance (Fig. 1.7) (Luptak and Szostak, unpublished results). We briefly considered the issue of whether functional molecules can be built from oligonucleotides that are chemically distinct from RNA in Section 1.3. Functional nucleic acids that are more resistant to degradation than RNA have biotechnological applications. It is also possible that increasing the chemical diversity of the constituents used to build a library may improve the quality of the functional molecules that can be obtained (see Section 1.3). In Section 1.4 we alluded to the fact that while RNA is a good model for the first biopolymer, a wide range of possibilities should be explored. Chemical alternatives to the ribose sugar backbone have been examined in some detail (reviewed in Eschenmoser, 1999, 2004). Among these, threose nucleic acid (TNA), with a four-carbon sugar backbone, is the most noteworthy. Because TNA is chemically simpler than RNA but retains many of its other properties, it is considered a plausible alternative to RNA as one of the earliest biopolymers (Eschenmoser, 2004). A DNA duplex containing a single TNA nucleotide maintains good helical geometry (Pallan et al., 2003), and single-stranded TNA base pairs with both RNA and DNA (Schoning et al., 2000). Protein polymerases can incorporate TNA monomers by primer extension on DNA templates (Chaput and Szostak, 2003), and can also use TNA oligos to template DNA synthesis (Chaput et al., 2003). A system for selecting functional TNA structures from libraries of random molecules was recently devised (Fig. 1.8) (Ichida et al., 2005). Briefly, TNA sequences are displayed on DNA by primer-


Fig. 1.8 In vitro selection for functional nonstandard oligonucleotides. Any molecule whose synthesis can be templated on DNA is amenable to selection using DNA display. For instance, libraries of sequences with a fourcarbon sugar backbone, threose nucleic acid (TNA) (reviewed in Eschenmoser, 1999, 2004) can be transcribed using the “therminator” DNA polymerase (Ichida et al., 2005).

The TNA sequence is displaced from the template by extending a DNA primer in the opposite direction. Following the selection step, PCR amplifies the DNA template. Note that this method does not require enzymes to directly copy TNA because of the covalent linkage between the displayed sequence and its DNA template.

extending hairpins at the 3l ends of library molecules. DNA sequences displaying functional TNA structures are amplified using PCR. The TNA selection system is conceptually similar to nucleic acid display technologies for selecting proteins from random sequence libraries (Roberts and Szostak, 1997; Keefe and Szostak, 2001; Bertschinger and Neri, 2004; reviewed in Frankel et al., 2003) and directing the organic synthesis of diverse classes of molecules (Rosenbaum et al., 2003; Gartner et al., 2004; Kanan et al., 2004; Halpin and Harbury, 2004; reviewed in Calderone and Liu, 2004). Experimental results demonstrate that a large number of modified nucleobase-pairing systems can be constructed as long as rules for stacking, charge, geometry, and hydrogen bonding are followed (Geyer et al., 2003; Liu et al., 2003). Recent work has identified protein polymerases that can synthesize strands of other modified oligonucleotides (Ghadessy et al., 2004; Chelliserrykattil and Ellington, 2004; reviewed in Benner, 2004). These, or other novel enzymes, could be utilized to select functional molecules from any type of heteropolymer library displayed on DNA. It will be interesting to see if functional activities, in addition to base-pairing ability, can be obtained from libraries of alternative oligonucleotides such as TNA. Experimentally determining the abundance and distribution of function in various oligonucleotide sequence spaces may shed light on why RNA, as opposed to TNA, or something else, is found in extant biology. By quantifying the number of functional sequences (Szostak, 2003) in sequence space or the diffi-

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culty of specifying functional structures (Carothers et al., 2004), it will be possible to rigorously compare the functional capabilities of different kinds of oligonucleotides. Among the most chemically plausible prebiotic or early-biotic nucleic acids, RNA may turn out to be the most likely to exhibit functional activities. Alternatively, RNA may have become the evolutionary winner because of historically contingent events or contexts.

1.6 Conclusion

In vitro selection was developed by the Gold, Joyce and Szostak laboratories in the early 1990s to search nucleic acid sequence spaces for functional molecules. Since then, in vitro selection experiments have been performed by researchers around the world to isolate, optimize and characterize hundreds of different synthetic aptamers and catalysts. In vitro selection has helped us understand the functional capabilities of oligonucleotides. Work to describe the distribution of functional structures in molecular sequence spaces continues to inform the design of sequence libraries with a greater likelihood of yielding highly active molecules. Study of the relationships between complexity and activity is teaching us how to isolate molecules of higher activity, and is also beginning to improve our comprehension of the origins of biochemistry. Heteropolymers would have been required at a very early stage in the history of life to carry out the necessary biochemical functions. While RNA may not have been the first biopolymer, it serves as a useful model because it possesses many of the qualities that the initial biopolymers would have needed. Recent work strongly suggests that the functional capabilities of other oligonucleotides can be examined in the near future. New techniques and applications for in vitro selection continue to be developed. For example, encapsulated selections will enable direct enrichment for multiple turnover catalysts such as ribozymes with RNA polymerase activity, or for aptamers that elicit changes in the spectroscopic properties of the ligand. Once selections for functional structures made up of alternative nucleic acids such as TNA have been successful, it will be interesting to compare their functional properties with those of RNA and DNA. Is the number of functional structures in an oligonucleotide sequence space relatively constant for different types of molecules, or does it depend heavily on chemical composition? Are there general rules that describe how the number of functional structures or sequences varies with activity, or is every case unique? There are many fundamental questions about the origins of biochemical function and life on Earth that remain unanswered; some may even be unanswerable. We are hopeful that through the careful application of in vitro selection and other synthetic methods, we will come to understand the range of possible pathways that lead from chemical systems to biological ones.

References

Acknowledgments

We thank A. Luptak for sharing unpublished results and providing the image for Fig. 1.7a. We thank A. Luptak and J.K. Ichida for helpful discussions. Work in the authors’ laboratory is supported by a grant from the National Institutes of Health (USA) (GM53936 to J.W. Szostak). J.M. Carothers was supported by a Graduate Fellowship from the National Science Foundation (USA). J.W. Szostak is an investigator of the Howard Hughes Medical Institute.

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2.1 From Early Experiments and Theories to the Concept of Neutral Networks

2 Mathematical Models on RNA Evolution, Simulations In Silico, and Concepts for In Vitro Selection Peter Schuster

2.1 From Early Experiments and Theories to the Concept of Neutral Networks 2.1.1 Evolution in the Test Tube

Sol Spiegelman and his group carried out the first successful evolution experiments in vitro in the 1960s (for a review see, for example, Spiegelman, 1971). These experiments were based on replication of single-strand RNA through catalysis by a bacteriophage-specific enzyme, mostly Qb replicase. A sample of RNA molecules carrying the recognition site for the replicase is introduced into a buffer solution that contains the four nucleotide triphosphates (ATP, CTP, GTP, and UTP) as well as the replicase. Consumed materials are replenished through transfer of small samples into fresh stock solution. After a sufficient number of repeated transfers RNA molecules are isolated that are adapted to the stock solution, in the sense that they replicate most efficiently. Although the molecular details of the replication process and the nature of the optimization process were largely unknown in the early days, the interpretation of the serial transfer experiments as Darwinian evolution in the test was essentially correct. The Qb RNA evolution experiments were repeated and put on a firm molecular basis later on, and finally, they were applied to the design of RNA molecules with predefined properties (for a collection of reviews see Watts and Schwarz, 1997). The mechanism of RNA replication by virus-specific enzymes like Qb replicase consists of two multistep RNA polymerization cycles, each synthesizing the complementary strand of an RNA template molecule, plus-I or minus-I, respectively (Biebricher and Eigen, 1988): plus-I + activated monomers p minus-I + plus-I minus-I + activated monomers p plus-I + minus-I


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After an initial period the plus-minus ensemble grows like a single entity with a rate parameter k = (k+ q k–), where k+ and k– are the parameters for the two reactions shown above. Depending on relative amounts of template and enzyme three different regimes of RNA replication are distinguished: (1) exponential growth of RNA concentration at excess concentration of the replicase, (2) linear growth of RNA concentration at moderate excess of template, and (3) saturation or approach to constant RNA concentration at large excess of template leading to product inhibition of the reaction. Darwinian selection occurs in phase (1) and in phase (2) but the selection criteria are different. Only the kinetic parameters, k+ and k–, are relevant in the exponential growth regime, whereas both the kinetic parameters and the binding equilibrium of template and replicase determine the reproductive success of molecules in the linear phase. Two results of these studies turned out to be central for understanding the mechanisms of evolutionary optimization: (1) the Darwinian mechanism of optimization through variation and selection is not bound to the existence of cellular life, and (2) populations of RNA molecules under conditions that sustain plus–minus replication are optimized in test tube experiments provided RNA replication operates either in the exponential or the linear growth regime. Another important feature of RNA evolution in the test tube is that it is more system specific and cannot be readily extended to more complex evolving assays: the dichotomy of genotype and phenotype (variation occurs on genotypes being nucleic acid sequences and selection operates on phenotypes determining fitness) is confined to a single RNA molecule with the sequence as genotype and the structure as phenotype. 2.1.2 Kinetic Theory of the Evolution of Molecules

Almost simultaneously with Spiegelman’s review, Manfred Eigen published a kinetic theory of molecular evolution that combined chemical reaction kinetics with knowledge from molecular biology (Eigen, 1971). The kinetic model treats evolution at the population level and can be seen as a generalization of population genetics by a straightforward account for molecular data: Correct replication and mutation are modeled as parallel chemical reactions and accordingly low and high mutation frequency cases can be handled in the same way. The model is likewise applicable to in vitro RNA optimization, evolution of viruses, bacteria, and, in principle, higher organisms. Further developments of the theory led to the concepts of molecular quasi-species and error thresholds (Eigen and Schuster, 1977; Swetina and Schuster, 1982): The quasi-species is a stationary mutant distribution centered around a most efficiently replicating master sequence. In formal terms the quasi-species is the largest eigenvector of the replication–mutation matrix (Eigen et al., 1989). The frequencies of individual mutants in this distribution are determined by the mutation rates and by the differences in replication parameters between master sequence and mutant. An increase in the error rate per site and replication, p, leads to broadening of the quasi-species in the sense

2.1 From Early Experiments and Theories to the Concept of Neutral Networks

that the frequencies of the mutants become higher. At a defined and computable error threshold, pcr O ln s/n, 1). the mutant distribution changes abruptly and becomes uniform: All mutants have the same probability of occurrence and inheritance or conservation of sequences over generations breaks down. Error thresholds were found to set limitations to the genome lengths of organisms (Drake et al., 1998). The error thresholds for RNA viruses of chain length n lie in the range of 1/n per site and replication event, which implies that error rates up to 1/n can be tolerated. In other words, the maximal error frequency is one per genome and replication (implying ln s O 1). The kinetic theory of molecular evolution provides insight into the replication– mutation dynamics in large populations. Like population genetics it focuses on processing genotypes through correct and error prone copying and does not account explicitly for the phenotype. Indeed, the phenotype enters the model exclusively as a set of rate and equilibrium parameters. In the 1980s the first attempts were made to incorporate RNA structures or phenotypes as explicit entities into the evolutionary process. Unfolding the phenotype from the genotype then becomes tantamount to the formation of RNA minimum free energy structures and the relation between genotypes and phenotypes boils down to a mapping from a space of sequences onto a space of structures (for review see Schuster, 2003). 2.1.3 Sequence Space and Shape Space

The notion of sequence space was found to be indispensable for a deeper understanding of adaptive evolution and optimization. Every sequence corresponds to a distinct point in sequence space. A metric on sequence space is the Hamming distance between two sequences, the number of positions in which the sequences differ. For example, the distance of the two sequences I1 = AGUCAAGCCUUGUACG ... CCGUAU and I2 = AGUUAAGCCUUCUACG ... CCGUGU

is dH(I1, I2) = 3. Sequence spaces are rather simple objects in n dimensions but they appear complicated when they are projected on two dimensions. Evolution is now considered as migration of populations in sequence space together with its projection onto a space of structures called shape space. This projection is rather complex since even in the simplest case it is mediated by RNA folding. Using RNA as a model for a mapping from sequence space onto shape space requires precise definitions in order to make the concept applicable: (1) It must be guaranteed that one and only one structure is assigned to every sequence, and (2) structures must be discrete objects in order to allow for straightforward compar1) The quantity s is the so-called superiority of the master sequence. In simple cases it represents the ratio between the replication rate parameter of the master sequence and the

average in rate parameters of all molecules except the master sequence Im: P P s = km (1–xm )/ j, j0m kj xj for i xi = 1.

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Fig. 2.1 Sketch of the mapping from sequence space into a space of structures and further into real numbers measuring function. The mapping from sequences into secondary structures of minimal free energy is many to one since there are many more sequences than structures. The number of sequences forming the same structure S is reflected by the degree of neutrality, lS. Depending on the resolution at which the molecular property is recorded the

mapping from shapes into function shows neutrality as well. Distance in sequence space is the Hamming metric, dH. Similarly, the Hamming distance between symbolic notations of structures (Fig. 2.2), e.g. S1 = x x x( ( ( ( ( x x x x ) ) x x ) ) )x x S2 = x x x( ( ( ( ( x x x x x x x x ) ) ) ) ) with dS(S1,S2) = 4, serves as an example for a metric in shape space.

ison and handling. Both conditions are fulfilled by RNA secondary structures of minimal free energies (see Section 2.2). Fundamental to the application of the sequence space concept is the general assumption that function can be derived from structure, whereas the structure is the unique result of a mapping from sequences onto structures (Fig. 2.1): sequence x structure x function

The first computer studies on RNA sequence–structure mappings revealed a high degree of neutrality for all common structures (Fontana et al., 1993; Schuster et al., 1994). By “common” we mean here that the probability of obtaining such a structure through folding a randomly chosen sequence is sufficiently high. (A quantitative expression for “common” is given in Schuster, 1995 and Section 2.3.) The set of sequences folding into the same secondary structure, the neutral set, forms a neutral network in sequence space. 1) The connectivity of neutral net1) In precise terms the neutral network is a graph defined by taking all sequences in the neutral set as nodes and connecting all pairs of sequences with Hamming distance one, dH(Ij,Ik) = 1, by an edge. Neutrality is defined

here with respect to (secondary) structure. Recently, the notion ’structurally constrained neutral (CNS) model’ has been introduced for our definition of neutrality (Bastolla et al., 2003a,b; see Section 2.3).

2.2 RNA Structures, Thermodynamics and Kinetic Folding

works is determined by the (mean) degree of neutrality, lS for the network of structure S, which is calculated by averaging the fraction of neutral neighbors in Hamming distance one or one-error neighborhoods over all sequences belonging to the network: PmS (S) k=1 nk lS = (k –1)mS Here, the number of sequences forming structure S is denoted by mS, the number of neutral sequences forming S in the one-error neighborhood of the sequence Ik (S) is nk , and the number of nucleotide classes in the alphabet is k (e.g. for GC we have k = 2 and k = 4 for AUGC). Neutral networks with a degree of neutrality below a threshold, lS I lcr, are split into several unconnected components representing islands in sequence space, whereas the neutral network is connected if the degree on neutrality exceeds the threshold, lS i lcr. As a kind of zero order approximation we make the assumption that neutral networks are represented well by random graphs. Then, the threshold value is given by the expression lcr = 1 – k–1/(1–k)

Interestingly, lcr depends exclusively on the size of the nucleotide alphabet: lcr = 0.5, 0.42, and 0.37 for k = 2, 3, and 4, respectively. As shown in Section 2.3, connectedness of neutral networks plays an important role in evolutionary optimization. The definition of neutrality used here is worth repeating since it means neutrality with respect to secondary structure and differs substantially from neutrality with respect to full spatial structure or neutrality with respect to function. We shall come back to this notion and discuss it in more detail in Section 2.3.

2.2 RNA Structures, Thermodynamics and Kinetic Folding 2.2.1 Secondary Structures of Minimum Free Energies

All nucleic acids share a universal force of structure formation: the stacking of hydrogen-bonded base pairs. Single- and double-stranded RNA and DNA are shaped likewise by base pairing and base pair stacking. Single-stranded molecules have coarse-grained structures, so-called secondary structures (Fig. 2.2), which are physically meaningful as folding intermediates (Thirumalai et al., 2001) and at the same time accessible to analysis by means of mathematical tools. This contrasts the situation in protein structure formation, where secondary structures play a much less important role. Base pairing, indeed, turns structure prediction into a problem of discrete mathematics (Schuster and Stadler, 2004): Two nucleotides either do or do not form a base pair. This fact makes the calculation and dis-

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Fig. 2.2 Structure prediction for tRNAPhe. Prediction is a mapping from sequence space into shape space that is done in two distinct steps: (a) from sequence to secondary structure and then (b) from secondary structure to the full three-dimensional structure. The secondary structure, in essence, is a listing of base pairs in a planar structure graph, which is free of knots and pseudo-knots. The secondary structure can be represented by an equivalent

symbolic parenthesis notation (shown below the secondary structure) that allows for mathematical analysis by means of combinatorics and other techniques of discrete mathematics. RNA secondary structures were identified as folding intermediates of RNA molecules (Thirumalai et al., 2001) and can be seen as analogs to molten globules in protein folding (Brion and Westhof, 1997).

cussion of RNA folds fairly easy. Counting problems, for example the determination of the number of secondary structures sharing one or more structural elements, can be solved exactly by means of recursion formulae (Hofacker et al., 1998). For long sequences asymptotic expressions are available (an example is given in Section 2.3). Computational prediction of RNA minimum free energy secondary structures is an old problem that was solved by means of dynamic programming in the 1980s (Zuker and Stiegler, 1981). The secondary structure is built from substructures, which are assumed to contribute additively to the free energy of the molecule. The free energies are computed from extensive tables of parameters that are derived from the results of thermodynamic and kinetic investigations of model compounds. Steady update of the empirical parameters for this approach improves the quality of the predictions (Matthews et al., 1999, 2004). 2.2.2 Inverse Folding

The inversion of the folding problem consists in the search for sequences that fold into a predefined secondary structure of minimum free energy (Fig. 2.3),


Fig. 2.3 Folding and inverse folding. Conventional RNA folding is dealing with the prediction of the secondary structure with minimum free energy for a given sequence. The inverse problem, finding a sequence folding into a

predefined secondary structure is commonly not unique: many sequences can be found that form the same structure under the minimum free energy criterion.

and it represents the core issue of rational design of RNA structures (Hofacker et al., 1994; Dirks et al., 2004). 1) A heuristic algorithm for inverse folding of RNA that handles the search for sequences as an optimization problem is part of the Vienna RNA package (Hofacker et al., 1994; for a recent algorithm, RNA-SSD, see Andronescu et al., 2004). The search is confined to the set of sequences that are compatible with the target structure. 2) The procedure starts from a random compatible sequence and proceeds iteratively by modifying the trial sequence in the space of compatible sequences under the condition of decreasing distance between the target structure and the minimum free energy structure of the trial sequence. Several definitions of distances between secondary structures may be used – one example is the Hamming distance between the two 1) It is straightforward to distinguish between rational and evolutionary design of molecules. In the first case the aim is to engineer an RNA sequence for a molecules with predefined properties like structure, folding kinetics, or spectral properties in order to obtain the desired function, whereas the second approach makes use of Darwin’s principle to create molecules for given function. Accordingly, only the function has to be known and testable in the evolutionary approach while the structure of the molecule results by itself from the optimization process.

2) Compatibility means that the sequence allows for the formation of a base pair wherever one is required in the structure: For every base pair in the structure one of the six possible pairing combinations, AU, CG, GC, GU, UA, UG, has to be presented by the two base pairforming positions. At all single-stranded positions of the structure each nucleotide, A, C, G or U, is admitted. Hence, the number of compatible sequences for a structure of chain length n with nbp base pairs and nsn single nucleotides (n = p 2 ffiffinffi bp + nsn) is ncomp = 6nhp q 4nsn and ( 6)n J ncomp J 4n

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symbolic notations – but the choice of a particular distance measure has little influence on the results. The iterations are stopped either when a sequence is obtained that fulfils the search criterion by forming the desired structure or when the search is without success after a predefined maximum number of iterations. In detail, the inverse folding algorithm proceeds by hierarchical design (Hofacker et al., 1994; Dirks and Pierce, 2003, 2004): Modification of the trial sequence begins by trying to match small structural motifs like hairpins and continues by successive attempts to match larger and larger parts of the structure and, eventually, the entire structure is refined as a whole. 2.2.3 Suboptimal Conformations and Kinetic Folding

With very few exceptions all RNA molecules form a large number of suboptimal conformations (Fig. 2.4). At the secondary structure level these suboptimal foldings can be obtained by straightforward computations (Zuker, 1989; Wuchty et al., 1999). 1) An alternative approach to account for suboptimal states computes the partition function of the equilibrium ensemble directly (McCaskill, 1990; a version of the partition function algorithm is included in the Vienna RNA package, Hofacker et al., 1994). Results of the partition function calculations are probabilities of base pair formation between pairs of positions rather than distinct base pairs. 2) A pairing probability close to one implies that this pair is almost certainly observed in the minimum free energy structure. Even knowledge of all suboptimal structures does not tell time-related issues of the folding process: How long does it take to form the minimum free energy structure? Are suboptimal conformations formed, and when is the equilibrium distribution of states reached? Answers to these questions and others can be obtained only by kinetic folding. In addition, kinetic effects were found to be important in the structures of large RNA molecules (Morgan and Higgs, 1996). Several algorithms for kinetic folding that form complete stacks cooperatively in one step have been published (Mironov and Lebedev, 1993; Tacker et al., 1994; Gultyaev et al., 1995; Isambert and Siggia, 2000; Zhang and Chen, 2002; and for DNA sequences Bonnet et al., 1998). For a detailed description of folding kinetics, however, a resolution down to single base pairs is desirable (see, for example, Flamm et al., 2000). Then, however, the computation of kinetic folding is extremely time 1) The major difference between the two algorithms concerns the set of suboptimal states that is computed: Zuker’s (1989) algorithm misses suboptimal conformations of a certain class whereas the algorithm by Wuchty et al. (1999) obtains all conformations within a predefined energy band above the minimum free energy.

2) A base pairing probability of one (or almost one) between two positions implies either that only the minimum free energy structure contributes to the partition function, because all suboptimal conformations have very high free energies, or that the minimum free energy structure and all relevant suboptimal structures have a base pair between these two positions. Hence the prediction of the occurrence of this particular base pair in the structure of the molecule is of high reliability.


Fig. 2.4 Suboptimal conformations and kinetic folding. The free energy levels of the minimum free energy conformation S0 and the 10 suboptimal conformations of lowest energies, Sk, k = 1,…,10, of the sequence GGCCCCUUUGGGGGCCAGACCCCUAAAAAGGGUC are plotted on the left-hand side. On the right we show the barrier tree for

the same molecule, which forms a long-living metastable conformation S1. Both conformations S0 and S1 are situated at the bottom of broad basins of the free energy hypersurface, which both contain a large number of suboptimal conformations. The numbers in vertical direction on the barrier tree represent free energy differences in kcal/mol.

consuming, since it requires averaging over a very large number of individual folding trajectories, and can be applied only to rather small RNA molecules. Algorithms that allow for kinetic folding of larger molecules have to use simplifications. The efforts are reduced, for example, through searching for the rate-limiting steps (Zhang and Chen, 2003, 2004) or through coarse-graining of the conformational landscape by considering barrier trees (Fig. 2.4; Flamm et al., 2000) consisting only of local minima and lowest free energy saddle points connecting them. On such barrier trees Arrhenius-type kinetics based on ordinary differential equations is able to describe the folding process fairly accurately (Wolfinger et al., 2004).

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2.2.4 Cofolding and DNA Parameters

Folding of a single RNA molecule can be readily extended to simultaneous folding of two or more RNA molecules (Hofacker et al., 1994; Andronescu et al., 2003). Algorithms for cofolding make use of dynamic programming and are fast and efficient. It is important also to compute suboptimal conformations for cofolded ensembles in order to be able to distinguish stable complexes from associations of marginal stability (for review see, e.g., Schuster, 2005). DNA resembles RNA in many aspects and the algorithms used for the prediction of RNA secondary structures can be applied successfully to DNA too. The differences are: DNA as a molecule in solution is more stable than RNA and less easily degraded. DNA secondary structures, however, are less stable because of weaker stacking energies. In detail the empirical parameters for DNA folding differ in many aspects from their RNA counterparts (SantaLucia, 1998; SantaLucia and Hicks, 2004). Recent interest in DNA folding as well as RNA–DNA cofolding is caused by the need to design molecules that are suitable primers for PCR as well as candidates for forming stable RNA–DNA hybrids. The discovery of small interfering RNA molecules (McManus and Sharp, 2002) turned the study of RNA–DNA interactions into a topic of high actuality and importance. The digression into RNA structures is completed through mentioning other approaches of secondary structure prediction. Homology modeling is based on the comparison of sequences from different organisms (Brimacombe, 1984; Michel and Westhof, 1990; Schnare et al., 1996; for reviews see Woese and Pace, 1993, Westhof and Michel, 1994), which form the same structure. It is highly successful but appears unsuitable for designing RNA structure and studying RNA evolution (Section 2.3) because it is restricted to natural RNA molecules. An interactive prediction algorithm for secondary structures (Gaspin and Westhof, 1995) applies folding constraints dynamically and allows for intervention by the researcher. By not elaborating on full three-dimensional structures of RNA and DNA molecules here we do not intend to belittle their role in structural biology and nucleic acid catalysis. On the contrary, it is the spatial structure that determines the molecular properties. The forthcoming discussion of RNA in vitro evolution requires, however, some insight into sequence–structure relations that can currently be achieved only for secondary structures and therefore we shall not further discuss tertiary structures of nucleic acids here.

2.3 Neutral Networks and In Silico Evolution of Molecules

2.3 Neutral Networks and In Silico Evolution of Molecules 2.3.1 Neutral Networks in Sequence Space

The relation between RNA sequences and RNA secondary structures of minimal free energies has been used as an example for a simple genotype–phenotype map that provides insight into evolution of phenotypes (Schuster, 2003). This mapping is constrained by three now well-established results: (1) There are orders of magnitude more sequences than structures, (2) relatively few common structures are contrasted by a very large number of rare structures, and (3) sequences forming the same structures are scattered all over sequence space. First, very large numbers of secondary structures can be drawn on paper. Many of them, however, have to be discarded because they lack stability. In particular, (1) hairpin loops of two or one nucleotide lengths are unstable because of steric hindrance and (2) single base pairs are unlikely to occur since the stabilization of double helices results primarily from base pair stacking. The numbers of secondary structures that can be formed under these two constraints, nhploop j 3 and nstack j 2, can be obtained readily from straightforward combinatorics (Hofacker et al., 1998). They fulfill an asymptotic relation for sufficiently long polynucleotide sequences: Nstruct(n) = 1.4848 q n–3/2(1.84892)n. Compared with the numbers of sequences in the different genetic alphabets, 2n, 3n, or 4n in two-letter, three-letter, or in the natural four-letter alphabet, respectively, we observe less steep increase with n of the number of structures. Computed exact values for the maximal numbers of structures (Grner et al., 1996a,b; Schuster, 2003) compared with those realized in different alphabets and with the numbers of binary sequences are shown in Fig. 2.5. Sequences over the AUG or AU alphabet form fewer structures because of the weakness of the A=U base pair. These results are interesting in the light of experiments performed in the laboratory of Gerald Joyce: Ribozymes with perfect catalytic functions were obtained by means of variation and selection for the AUG alphabet (Rogers and Joyce, 1999) and for the two-letter alphabet DU (Reader and Joyce, 2002). 1) Second, an empirical finding revealed that the distributions of frequencies for the occurrence of structures in whole sequence space are very broad: Few common structures of high frequency are opposed by a large number of rare structures. A simple but straightforward definition of common structures is (Schuster, 1995): m (Scommon )i

kn Nstruct (n)

1) In order to overcome the weakness of the AU pair Reader and Joyce (2002) applied 2,6-diamino purine (D) rather than adenine (A) because the DU pair forms more stable stacks.

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Fig. 2.5 The numbers of stable secondary structures realized in different genetic alphabets. In the figure we compare the numbers of structures fulfilling the physical stability criteria (black triangles, solid line), nhploop j 3 and nstack j 2, to those realized as minimum free energy

structures in different alphabets (AUGC: black squares, dotted line; UGC: gray plus signs, dotted line; GC: gray times signs, solid line; AUG: gray diamonds, dotted line; AU: gray squares, solid line). For comparison the numbers of binary sequences (2n) is shown as well.

All structures that are more frequent than the average structure are common where Nstruct(n) is the total number of structures formed by sequences of chain length n. Since the very rare structures are formed only by one sequence, by two or by very few sequences, they play practically no role in artificial or natural evolution because the chances of finding them by a random search process are close to zero and because the numbers of possible sequences are prohibitive for systematic searches. The bias in the frequency distribution increases with chain lengths and thus for long sequences we are dealing with relatively few common structures compared with a large number of rare and unimportant structures. Third, in zero-th order the sequences forming a given neutral network are randomly distributed in sequence space. This assumption leads to the concept of sequence space covering (Schuster et al., 1994; Schuster, 1995; Reidys et al., 1997; Schuster and Stadler, 2004): For all common structures holds that sequences, which form them as minimum free energy structures, are found in the neighborhood of any arbitrarily chosen reference sequence. These neighborhoods are very small compared with the entire sequence space and are represented by an n-dimensional sphere. For structure S the shape space covering radius is b Rcov (S) = 1– 2 nbp (S) k


41

Fig. 2.6 Four cloverleaf RNA structures used tures in natural tRNAPhe with stack lengths in the application of inverse folding to study 6/4/5/5. (b)–(d) Successively longer stacks, RNA shape space. The structures differ in the 6/5/5/5, 6/5/6/6, and 6/6/6/6, respectively. lengths of stacks and hairpin loops. (a) Struc-

Herein nbp(S) is the number of base pairs in structure S and b is the number of allowed base pairs of the alphabet. In the natural alphabet, AUGC, we have b = 6 and k = 4, and hence Rcov(S) = 0.625 nbp(S). Evolutionary studies on aptamers and ribozymes essentially confirmed the concept of shape space covering in any sufficiently large part of sequence space (Huang and Szostak, 2003; Hughes et al., 2004). The distribution of RNA sequences belonging to a neutral network is not, of course, completely random and deviations from the predictions of the random graph model can be derived from details of the secondary structure (Reidys et al., 1997; Schuster and Stadler, 2004). In order to illustrate the influence of structure on the frequency of occurrence and the degree of neutrality we consider in Table 2.1 the example of the four cloverleaf structures shown in Fig. 2.6. The frequency of occurrence is approximated by the probability of finding a sequence for the structure by means of inverse folding (pinv). The degree of neutrality, l, has been computed by consecutive summation over the one-error neighborhoods of individual sequences calculated by inTable 2.1 Degrees of neutrality and probabilities to find a sequence through a successful inverse folding trial are shown for four cloverleaf structures (Fig. 2.6) in five different nucleotide alphabets Alphabet Structure a l

pinv

Structure b l

pinv

Structure c l

pinv

Structure d l

AU

–

–

–

–

–

–

0.073 e 0.032

0.051

AUG

–

–

0.217 e 0.063

0.003

0.0207 e 0.055 0.026

0.201 e 0.056

0.374

AUGC

0.275 e 0.064

0.794

0.279 e 0.063

0.884

0.289 e 0.062

0.934

0.313 e 0.058

0.982

UGC

0.263 e 0.071

0.584

0.257 e 0.070

0.628

0.251 e 0.068

0.697

0.250 e 0.064

0.818

GC

0.052 e 0.033

0.067

0.057 e 0.034

0.086

0.060 e 0.033

0.087

0.068 e 0.034

0.127

pinv

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verse folding until convergence was obtained. The four cloverleaf structures differ by the lengths of individual stacks. Since A=U base pairs are too weak to form stable short stacks, structures a, b, and c are rarely formed by sequences from AU and AUG alphabets. The degree of neutrality, one the other hand, is strongly influenced by the cardinality of the alphabet (k). Although sequences forming cloverleaves with structure d are 2.5 times more likely to be found in the GC than in the AU alphabet, the degree of neutrality is approximately the same. 2.3.2 RNA Evolution In Silico

The internal structures of sequence and shape space, in particular the connectedness of neutral networks, set the stage for evolutionary optimization of RNA molecules. Starting in the 1980s we constructed a model system that allows for simulation of RNA structure optimization in ensembles of up to 100 000 molecules (Fontana and Schuster, 1987, 1998; Schuster, 2003, 2005). The simulations mimic a flow reactor, which is a continuous version of serial transfer experiments. Two kinds of runs were performed: (1) computer experiments aiming at optimizing properties or functions, and (2) attempts to optimize structure with the goal of matching a predefined pattern, called the target structure St, as closely as possible. The latter problem is related to the evolutionary design of aptamers, where molecules are optimized to bind to a predefined shape, and therefore we discuss it in more detail here. Populations search sequence space through replication, mutation and selection. Mutation operates on genotypes, being the individual RNA sequences, whereas selection is based on differences in replication rates, which are purely phenotypic quantities. Replication rates are indeed defined by molecular structures only, since rate parameters are modeled as a function of the distance between the structures of the molecule (Sk) and the target structure (St), dS(Sk,St). About a thousand individual trajectories were recorded for one set of conditions. All were continued for a sufficiently long time such that the populations either died out or reached the target structure. A typical run is shown in Fig. 2.7. In order to facilitate comparisons, series of computer experiments were conducted for identical initial populations and target structures. Accordingly, variations were restricted to the nucleotide alphabet and to the population size. As seen in Fig. 2.7 the population approaches the target in steps: Short adaptive phases of fast decrease of the distance to the target are interrupted by long quasi-stationary epochs, in which the average distance between the structures in the population and the target structure stays apparently constant. Inspection of the sequences, however, shows that the evolution of genotypes continues during the periods of phenotypic stasis. Indeed, the numbers of mutations recorded in the genotypes of the population is almost constant in time, no matter whether the population approaches the target structure or not. This finding matches precisely the conjecture of a molecular clock in neutral evolution (Kimura, 1968; King and Jukes, 1969).


Fig. 2.7 Recording of a typical in silico RNA evolution experiment. A simulated population of N = 1000 RNA molecules is subjected to replication, mutation, and selection in a flow reactor. The initial population is derived from a random sequence and chosen to be homogeneous (i.e. all molecules have identical structures and sequences). The chain length, n = 76, matches that of the target structure tRNAPhe and a mutation rate of p = 0.001 per nucleotide and replication is chosen. The structure difference between the initial structure and the target structure is DdS = 50. The replication rate parameter becomes the larger the closer a given molecule matches the target structure. The upper curve (black) shows the stepwise decrease of the mean distance between population and target. The two lower curves illus-

trate the evolution of molecular populations on quasi-stationary plateaus. The gray curve in the middle presents the width of the population in sequence space, it increases whenever the population spreads on a neutral network through mutation. Spreading continues until the population happens to find a faster replicating variant in the neighborhood of the neutral network. Then, by selection the population width decreases very quickly, the population passes a new adaptive phase and the distance to target decreases. The lowest curve represents the velocity with which the center of the population migrates in sequence space. This speed is almost always small but has spikes at the ends of quasi-stationary epochs that correspond to a jump-like motion initiating the next period of progress in optimization.

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The in silico RNA model of the evolution revealed the following properties for the optimization process (Fontana and Schuster, 1998; Stadler et al., 2001; Schuster, 2005): 1. Fluctuations in the course of evolution are very large: Despite more than 1000 repeats with the same initial conditions 1) no case of two identical optimization trajectories was observed. The scatter in (real) time or in the number of replications required to reach the target is also very large. Nevertheless, some quantities can be predicted with reasonable accuracy. For example, the number of quasi-stationary plateaus fluctuates much less strongly than the number of replications. 2. Evolutionary optimization is successful at relatively small population sizes, N j 20. Reducing the population below this critical size causes the probability for the entire population to die out to approach one. The population size, however, is important for the time that is required to reach the target. The larger the value of N the shorter the expected time for successful optimization. If, on the other hand, materials or energies are limited, small populations fare better since they consume fewer resources. Optimal population sizes can be derived therefore for known constraints only. 3. Fitness or value landscapes of biopolymers were found to be very rugged (Stadler, 1996) and therefore optimization in populations by mutation and selection would soon get stuck in local optima. The way out of these evolutionary traps is provided by the fact that neutral variants exist in the neighborhoods of almost all sequences forming evolutionarily relevant structures. On the quasi-stationary plateaus the populations search sequence space for better target-matching structures through spreading on neutral networks in a diffusion-like process (Huynen et al., 1996), which is the molecular analog of neutral evolution in the sense of Motoo Kimura (Kimura, 1983). In this diffusion process the small populations we were applying here split into individual subclones whose sequences are related by replication and mutation (Schuster, 2003). This observation is similar to results derived from a stochastic model of species evolution (Higgs and Derrida, 1992). 4. Through comparison of optimizations performed with sequences over different alphabets a striking influence of the 1) The same initial conditions mean that all input quantities of the flow reactor chosen were identical except the seeds of the random number generator.


alphabet on the success of the evolutionary search became evident: The expected time to reach a tRNA target structure with GC sequences is about six times as long as with AUGC sequences. The concept of neutral networks provides a straightforward interpretation: Cloverleaf structures have a mean degree of neutrality in GC sequence space of 0.05 I l I 0.07 that is way below the critical value of lcr = 0.5 compared with 0.22 I l I 0.32 in the AUGC alphabet where we have lcr = 0.37. Neutral networks over the natural alphabet are thus close to being connected in sequence space, whereas they are highly fragmented for GC-only sequence space. Fragmented networks make optimization difficult because the diffusion process of populations in sequence space is restricted to rather small islands. Choosing the optimal search space still presents a great challenge for the design of in vitro evolution experiments. Since working ribozymes built from sequences over three- and two-letter alphabets are known, it is conceivable that biased nucleotide compositions for sequences may lead to better results than the commonly used uniform composition (25%, 25%, 25%, 25%). RNA folding can be used to compute preferences for classes of structures in certain alphabets. This problem has an inversion: Which is the most promising nucleotide distribution for the creation of a given structure? An extension of the inverse folding problem might well be applicable to finding the answers. 2.3.3 Lessons from Evolution In Silico

The role of neutrality in evolution has nowhere been recognized so clearly as with the in silico evolution experiments. While the possibility of neutrality with respect to selection was correctly stated by Charles Darwin in the nineteenth century (Darwin, 1859), molecular knowledge about the evolution of biopolymer sequences was required in order to be able to derive evidence for genetic changes without phenotypic consequences, called neutral evolution by Motoo Kimura (Kimura, 1983). The basis for the concept of neutrality was a rich collection of sequence data provided by a large number of molecular biologists. In Kimura’s theory neutral evolution is an inevitable byproduct of mutation and it creates variability through steadily producing new variants. The current view makes use of the concept of sequence space and its application to the design of molecules exploring chemical space by means of target binding RNA or DNA aptamers (Breaker, 2004). These studies provided strong evidence for the existence of connected neutral regions in sequence space (see, for example, Schultes and Bartel, 2000; Held et al., 2003; Huang and Szostak, 2003 and the review by Hughes et al., 2004). Diffusion of populations on such neutral networks prevents populations from getting stuck in local optima of the fitness landscape. The search for aptamers or

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2 Mathematical Models on RNA Evolution Fig. 2.8 Evolution of molecules in vitro. The sketch indicates the sequence of the three major phases in evolutionary design on molecules with predetermined functions: amplification through replication, diversification through mutation or random synthesis, and selection as well as testing.

other RNA molecules with predefined functions through in vitro selection (Fig. 2.8) cannot be successful unless individual structures and functions do not appear over and over again in sequence space. Shape space covering facilitates the search for suitable molecules enormously. It is important to consider different notions of neutrality in more detail. Neutrality was understood here with respect to RNA secondary structures (Schuster et al., 1994; Bastolla et al., 2003a,b). Apparently this is a weak condition and consequently the numbers of neutral sequences are very large. For certain ribozyme functions it is sufficient to provide an appropriate molecular scaffold and, indeed, the Schultes and Bartel (2000) experiment revealed a high degree of neutrality that comes close to the neutral networks derived from secondary structure prediction. Sequences that are neutral with respect to secondary structure differ not only in their full three-dimensional structures, they also differ in the spectra of suboptimal structures and with respect to kinetic folding behaviors. Whenever details of the 3D structures, suboptimal conformations, or folding kinetics matter for the

2.4 Designed and Natural RNA Switches

function of the molecule, the degree of neutrality will be reduced. An excellent example of very stringent multiple constraints on molecular functions is provided by the tRNAs: Here we would expect to find few sequences that are neutral with respect to the various tRNA functions and this is precisely what we find with the natural molecules.

2.4 Designed and Natural RNA Switches

The intersection theorem states that for any two base pairing patterns there exists at least one sequence that can form both structures. The proof (Reidys et al., 1997) does not require the pseudoknot exclusion condition for secondary structure and therefore holds also for structures with pseudoknots. 1) Intersection means here the intersection of the two sets of sequences C1 and C2 that are compatible with the two structures S1 and S2, respectively: C1 \ C2 0 H, or in other words, the intersection of the two compatible sets is non-empty. The proof cannot be extended to three structures and this means that for three structures a sequence forming all three may, but need not, exist. Indeed examples of three structures with empty intersections, C1 \ C2 \ C3 = H, can be easily constructed. The theorem, of course, says nothing about the stability of the two structures. Many examples of RNA molecules with two or more (meta)stable conformations are known. Commonly, but not necessarily, one of the intersecting conformations represents the minimum free energy structure of the sequence, whereas the others are metastable structures corresponding to local free energy minima in conformation space. As can be seen from the example in Fig. 2.4 RNA molecules may exist in two or more long-lived conformations that are separated by a sufficiently high free-energy barrier. Natural RNA molecules existing in two conformations were characterized as self-induced RNA switches. The first examples were reported over 10 years ago (LeCuyer and Crothers, 1993). Since then many examples have been found in nature (Nagel and Pleij, 2002) and switching small RNAs (25 J n J 50) have been rationally designed (Micura and Hbartner, 2003). Algorithms for the design have also been developed (Flamm et al., 2001; Voss et al., 2004) and NMR spectroscopy has been applied to record the conformational changes (Hbartner and Micura, 2003). Here two examples for switching RNAs are mentioned: 1. Conformational equilibria can be designed by proper choice of sequences. Thereby energetic influences of stacking regions and loops are important (Fig. 2.9). 2. A particularly interesting case is represented by an RNA molecule with a chain length n = 88 that has two catalytic 1) Base pairing is here also not restricted to Watson–Crick and wobble pairs. The theorem requires only that all nucleotides can be uniquely assigned to either the set of single

bases or the set of base pairs. Base triplets, therefore, are not allowed and the intersection theorem does not hold for structures with triplets.

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Fig. 2.9 Two examples of self-induced small RNA switches (Nagel et al., 2005). The numbers below the structures represent free energies relative to the unfolded conformation. Both cases demonstrate how stabilities of folds can be engineered by proper choices of parts of sequences. (a) The conformers differ in sequences base pairs in the middle part of the stacking region, 3 AU + 2 GU + 1 GC versus 3 AU + 1 GU + 2 GC. The replacement of a GU by a GC, apart from other minor differ-

ences in the orientation of base pairs, makes the stack in the structure on the right hand side more stable. (b) Here we show the influence of the mutation ApG in position 21 of the sequence destabilizes the double hairpin structure by replacing an AU pair by a GU pair. At the same time the single hairpin structure becomes more stable because the mutation allows for the formation of an especially stable tetraloop of the GNRA class.

2.5 Outlook on Future Problems in RNA Design

functions (Schultes and Bartel, 2000): It acts as a specific RNA ligase and as a specific RNA cleavage ribozyme. This molecule with a sequence situated at the intersection of two completely different RNA folds shows both activities but with reduced catalytic activity. Introduction of a few proper point mutations, however, yields RNA molecules that show catalytic efficiencies, which are essentially as high as those of the reference molecules, an artificial ligase and a natural ribozyme. By single nucleotide and base pair exchanges Schultes and Bartel were able to find neutral paths bridging Hamming distances of dH i 40. These neutral paths reach from the highly active molecules near the intersection without interruption to the reference.

2.5 Outlook on Future Problems in RNA Design

Evolution in vitro can be subdivided into two subclasses of processes that are analogs of natural and artificial selection: (1) RNA evolution in replication assays without intervention and (2) optimization according to predefined external selection criteria in cycles as shown in Fig. 2.1. The latter case can be viewed as “breeding of molecules.” In biotechnology evolutionary design stands in competition with rational design as done, for example, by inverse folding. The advantage of the evolutionary strategy consists in the fact that only the desired function has to be known and testable, whereas no information on structure is required. The success of rational design, on the other hand, depends critically on the state of our knowledge of the relations between structure and function. Admittedly, this knowledge is fragmentary at present but it is rapidly increasing. Function predicted from known structure has the opposite problem: many structures may give rise to the same function. If one day we are in a position to derive structures required for given functions with fair reliability, then the rational design problem will be solvable since we could then invert our initial two-step relation: sequence v structure v function

If the inverse problems were solved, rational design would be superior to its evolutionary counterpart because the effort needed to design structures for functions and to synthesize sequences that are expected to have the required structures is minimal compared with the effort of screening 1015 different molecules or more. For the time being, however, the evolutionary method in most cases is the only one likely to produce the desired outcome. As in other scientific disciplines, inverse methods are coming increasingly into the focus of interest and new algorithms are being developed that provide tools

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for reverse engineering. The inverse folding algorithm described here is presumably the simplest example of such a strategy in the RNA world. Other reverse engineering problems, for example the design of sequences for given spectra of suboptimal conformations or the creation of molecules that show predefined kinetic folding behavior, are much more ambitious and harder to solve. Needless to say, to conceive and implement an algorithm for the universal design of RNA structure for RNA function seems to be a far-fetched problem. Applied mathematicians, on the other hand, have lots of experience in the development of inverse methods and the tasks to be solved invite truly interdisciplinary approaches.

Acknowledgments

The research of the Vienna group reported here is joint work with Professors Peter Stadler (Leipzig), Walter Fontana (Harvard Medical School), Ivo Hofacker and Christoph Flamm (Vienna). Financial support by the Austrian Fonds zur Frderung der wissenschaftlichen Forschung (FWF), Projects No. P-13887 and P-14898, as well as by the Austrian Gen-AU Bioinformatics Integration Network (BIN) sponsored by the Austrian Bundesministerium fr Bildung, Wissenschaft und Kunst (BMBWK) is gratefully acknowledged.

References Andronescu, M., Aguirre-Hernndez, R., Condon, A., Hoos, H.H. (2003). RNAsoft: A suite of RNA secondary structure prediction and design software tools. Nucleic Acids Res 31, 3416–3422. Andronescu, M., Fejes, A.P., Hutter, F., Hoos, H.H., Condon, A. (2004). A new algorithm for RNA secondary structure design. J Mol Biol 336, 607–624. Bastolla, U., Porto, M., Roman, H.E., Vendruscolo, M. (2003a). Connectivity of neutral networks, overdispersion, and structural conservation in protein evolution. J Mol Evol 56, 243–254. Bastolla, U., Porto, M., Roman, H.E., Vendruscolo, M. (2003b). Statistical properties of neutral evolution. J Mol Evol 57, S103– S119. Biebricher, C.K., Eigen, M. (1988) Kinetics of RNA replication by Qb replicase. In: RNA Genetics, Vol. I: RNA Directed Virus Replication, Domingo, E., Holland, J.J., Ahlquist, P., eds. Boca Raton, FL: CRC Press, pp. 1–21.

Bonnet, G., Krichevsky, O., Libchaber, A. (1998). Kinetics of conformational fluctuations in DNA hairpin-loops. Proc Natl Acad Sci USA 95, 8602–8606. Breaker, R.R. (2004). Natural and engineered nucleic acids as tools to explore biology. Nature 432, 838–845. Brimacombe, R. (1984). Conservation of structure in ribosomal RNA. Trends Biochem Sci 9, 273–277. Brion, P., Westhof, E. (1997). Hierarchy and dynamics of RNA folding. Annu Rev Biophys Biomol Struct 26, 113–137. Darwin, C. (1859). The Origin of Species. Source: Everyman’s Library (1928). No. 811. London: J.M. Dent and Sons, p. 81. Dirks, R.M., Pierce, N.A. (2003). A partition function algorithm for nucleic acid secondary structure including pseudoknots. J Comput Chem 24, 1664–1677. Dirks, R.M., Pierce, N.A. (2004). An algorithm for computing nucleic acid base-pairing probabilities including pseudoknots. J Comput Chem 25, 1295–1304.

References Dirks, R.M., Lin, M., Winfree, E., Pierce, N.A. (2004). Paradigms for computational nucleic acid design. Nucleic Acids Res 32, 1392–1403. Drake, J.W., Charlesworth, B., Charlesworth, D., Crow, J.F. (1998). Rates of spontaneous mutation. Genetics 148, 1667–1686. Eigen, M. (1971). Self-organization of matter and the evolution of biological macromolecules. Naturwissenschaften 58, 465–523. Eigen, M., Schuster, P. (1977). The hypyercycle. A principle of natural self-organization. Part A: Emergence of the hypercycle. Naturwissenschaften 64, 541–565. Eigen, M., McCaskill, J., Schuster, P. (1989). The molecular quasispecies. Adv Chem Phys 75, 149–263. Flamm, C., Fontana, W., Hofacker, I.L., Schuster, P. (2000). Elementary step dynamics of RNA folding. RNA 6, 325–338. Flamm, C., Hofacker, I.L., Maurer-Stroh, S., Stadler, P.F., Zehl. M. (2001). Design of multistable RNA molecules. RNA 7, 254–265. Fontana, W., Schuster, P. (1987). A computer model of evolutionary optimization. Biophys Chem 26, 123–147. Fontana, W., Schuster, P. (1998). Continuity in evolution. On the nature of transitions. Science 280, 1451–1455. Fontana, W., Konings, D.A.M., Stadler, P.F., Schuster, P. (1993). Statistics of RNA secondary structures. Biopolymers 33, 1389– 1404. Gaspin, C., Westhof, E. (1995). An interactive framework for RNA secondary structure prediction with a dynamical treatment of constraints. J Mol Biol 254, 163–174. Grner, W., Giegerich, R., Strothmann, D., Reidys, C., Weber, J., Hofacker, I.L., Schuster, P. (1996a). Analysis of RNA sequence structure maps by exhaustive enumeration. I. Neutral networks. Mh Chem 127, 355– 374. Grner, W., Giegerich, R., Strothmann, D., Reidys, C., Weber, J., Hofacker, I.L., Schuster, P. (1996b). Analysis of RNA sequence structure maps by exhaustive enumeration. II. Structures of neutral networks and shape space covering. Mh Chem 127, 375–389. Gultyaev, A.P., van Batenburg, F.H.D., Pleij, C.W.A. (1995). The computer simulation of

RNA folding pathways using a genetic algorithm. J Mol Biol 250, 37–51. Held, D.M., Greathouse, S.T., Agrawal, A., Burke, D.H. (2003). Evolutionary landscapes for the acquisition of new ligand recognition by RNA aptamers. J Mol Evol 57, 299–308. Higgs, P.G., Derrida, B. (1992). Genetic distance and species formation in evolving populations. J Mol Evol 35, 454–465. Hbartner, C., Micura, R. (2003). Bistable secondary structures of small RNAs and their structural probing by comparative imino proton NMR spectroscopy. J Mol Biol 325, 421–431. Hofacker, I.L., Fontana, W., Stadler, P.F., Bonhoeffer, L.S., Tacker, M., Schuster, P. (1994). Fast folding and comparison of RNA secondary structures. Mh Chem 125, 167– 188. The Vienna RNA Package can be downloaded from www.tbi.univie.ac.at/ Zivo/RNA/. Hofacker, I.L., Schuster, P., Stadler, P.F. (1998). Combinatorics of RNA secondary structures. Discr Appl Math 89, 177–207. Huang, Z., Szostak, J.W. (2003). Evolution of aptamers with a new specificity and new secondary structures from an ATP aptamer. RNA 9, 1456–1463. Hughes, R.A., Robertson, M.P., Ellington, A.D., Levy, M. (2004). The importance of prebiotic chemistry in the RNA world. Curr Opin Chem Biol 8, 629–633. Huynen, M.A., Stadler, P.F., Fontana, W. (1996). Smoothness within ruggedness: The role of neutrality in adaptation. Proc Natl Acad Sci USA 93, 397–401. Isambert, H., Siggia, E.D. (2000). Modeling RNA folding paths with pseudoknots: Application to hepatitis delta virus ribozyme. Proc Natl Acad Sci USA 97, 6515–6520. Kimura, M. (1968). Evolutionary rate at the molecular level. Nature 217, 624–626. Kimura, M. (1983). The Neutral Theory of Molecular Evolution. Cambridge: Cambridge University Press. King, J.L., Jukes, T.H. (1969) Non-Darwinian evolution: Random fixation of selectively neutral variants. Science 164, 788–798. LeCuyer, K.A., Crothers, D.M. (1993) The Leptomonas collosoma spliced leader RNA can switch between two alternate structural forms. Biochemistry 32, 5301–5311.

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2 Mathematical Models on RNA Evolution Mathews, D.H., Sabina, J., Zuker, M., Turner, D.H. (1999). Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structures. J Mol Biol 288, 911–940. Mathews, D.H., Disney, M.D., Childs, J.L., Schroeder, S.J., Zuker, M., Turner, D.H. (2004). Incorporation of chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure. Proc Natl Acad Sci USA 101, 7287–7292. McCaskill, J.S. (1990). The equilibrium partition function and base pair binding probabilities for RNA secondary structures. Biopolymers 29, 1105–1190. McManus, M.T., Sharp, P.A. (2002). Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 3, 737–747. Micura, R., Hbartner, C. (2003). On secondary structure rearrangements and equilibria of small RNAs. ChemBioChem 4, 984–990. Michel, F., Westhof, E. (1990). Modelling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis. J Mol Biol 216, 585–610. Mironov, A.A., Lebedev, V.F. (1993). A kinetic model of RNA folding. Biosystems 30, 49–56. Morgan, S.R., Higgs, P.G. (1996). Evidence for kinetic effects in the folding of large RNA molecules. J Chem Phys 105, 7152–7157. Nagel, J.H.A., Pleij, C.W.A. (2002). Self-induced structural switches in RNA. Biochimie 84, 913–923. Nagel, J.H.A., Flamm, C., Hofacker, I.L., Franke, K., de Smit, M.H., Schuster, P., Pleij, C.W.A. (2005). Structural parameters affecting the kinetic competition of RNA hairpin formation.Nucleic Acids Res, in press. Reader, J.S., Joyce, G.F. (2002). A ribozyme composed of only two nucleotides. Nature 420, 841–844. Reidys, C., Stadler, P.F., Schuster, P. (1997). Generic properties of combinatory maps. Neutral networks of RNA secondary structures. Bull Math Biol 59, 339–397. Rogers, J., Joyce, G.F. (1999). A ribozyme that lacks cytidine. Nature 402, 323–325. SantaLucia, Jr., J. (1998). A unified view of polymer, dumbbell and oligonucleotide

DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci USA 95, 1460–1465. SantaLucia, Jr., J., Hicks, D. (2004). The thermodynamics of DNA structural motifs. Annu Rev Biophys Biomol Struct 33, 415–440. Schnare, M.N., Damberger, S.H., Gray, M.W., Gutell, R.R. (1996). Comprehensive comparison of structural characteristics in eukaryotic cytoplasmatic large subunit (23 S-like) ribosomal RNA. J Mol Biol 256, 701–719. Schultes, E.A., Bartel, D.P. (2000). One sequence, two ribozymes: Implications for the emergence of new ribozyme folds. Science 289, 448–452. Schuster, P. (1995). How to search for RNA structures. Theoretical concepts in evolutionary biotechnology. J Biotechnol 41, 239–257. Schuster, P. (2003). Molecular insights into evolution of phenotypes. In: Evolutionary Dynamics – Exploring the Interplay of Selection, Accident, Neutrality, and Function, Crutchfield, J.P., Schuster, P., eds. New York: Oxford University Press, pp. 163–215. Schuster, P. (2005). Prediction of RNA secondary structures: From theory to real molecules. Rep Prog Phys, in press. Schuster, P., Stadler, P.F. (2004). Discrete models of biopolymers. In: Compact Handbook of Computational Biology, Konopka, A.K., Crabbe, M.J.C., eds. New York: Marcel Dekker, pp. 187–222. Schuster, P., Fontana, W., Stadler, P.F., Hofacker, I.L. (1994). From sequences to shapes and back: A case study in RNA secondary structures. Proc R Soc B 255, 279–284. Spiegelman, S. (1971). An approach to the experimental analysis of precellular evolution. Q Rev Biophys 4, 213–253. Stadler, P.F. (1996). Landscapes and their correlation functions. J Math Chem 20, 1–45. Stadler, B.R.M., Stadler, P.F., Wagner, G.P, Fontana, W. (2001). The topology of the possible: Formal spaces underlying patterns of evolutionary change. J Theor Biol 213, 241–274 Swetina, J., Schuster, P. (1982). Self-replication with errors – A model for polynucleotide replication. Biophys Chem 16, 329–345.

References Tacker, M., Fontana, W., Stadler, P.F., Schuster, P. (1994). Statistics of RNA melting kinetics. Eur Biophys J 23, 29–38. Thirumalai, D., Lee, N., Woodson, S.A., Klimov, D.K. (2001). Early events in RNA folding. Annu Rev Phys Chem 52, 751–762. Voss, B., Meyer, C., Giegerich, R. (2004). Evaluating the predictability of conformational switching in RNA. Bioinformatics 20, 1573–1582. Watts, A., Schwarz, G. (1997). Evolutionary biotechnology – From theory to experiment. Biophys Chem 66, 67–290. Westhof, E,. and Michel, F. (1994). Prediction and experimental investigation of RNA secondary structure and tertiary foldings. In: RNA–Protein Interactions: Frontiers in Molecular Biology. London: IRL Press, pp. 25–51. Wolfinger, M.T., Svrcek-Seiler, W.A., Flamm, C., Hofacker, I.L., Stadler, P.F. (2004). Efficient computation of RNA folding dynamics. J Phys A: Math Gen 37, 4731–4741. Woese, C.R., Pace, N.R. (1993). Probing RNA structure, function, and history by comparative analysis. In: The RNA World,

Gesteland, R.F., Atkins, J.F., eds. Plainview, NY: Cold Spring Harbor Laboratory Press, pp. 91–117. Wuchty, S., Fontana, W., Hofacker, I.L., Schuster, P. (1999). Complete suboptimal folding of RNA and the stability of secondary structures. Biopolymers 49, 145–165. Zhang, W., Chen, S.-J. (2002). RNA hairpinfolding kinetics. Proc Natl Acad Sci USA 99, 1931–1936. Zhang, W., Chen, S.-J. (2003). Master equation approach to finding the rate limiting steps in biopolymer folding. J Chem Phys 118, 3413–3420. Zhang, W., Chen, S.-J. (2004). Analyzing the biopolymer folding rates and pathways using kinetic cluster method. J Chem Phys 119, 8716–8729. Zuker, M. (1989). On finding all suboptimal foldings of an RNA molecule. Science 244, 48–52. Zuker, M., Stiegler, P. (1981). Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res 9, 133–148.

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3 Fitness Landscapes, Error Thresholds, and Cofactors in Aptamer Evolution

3 Fitness Landscapes, Error Thresholds, and Cofactors in Aptamer Evolution dam Kun, Marie-Christine Maurel, Mauro Santos, and Ers Szathmry

3.1 Introduction

The idea that RNA was genetic as well as enzymatic material goes back to earlier speculations concerning the possible role of RNA in the origin of life (Woese, 1967; Crick, 1968; Orgel, 1968). Although the idea was clearly expressed, and the molecular nature of RNA – with genetically specified positioning in three dimensions of different chemical building blocks – should have convinced everybody of the potential enzymatic capacity of RNA based almost entirely on first principles, the acceptance of the idea – in line with the deeply non-theoretical nature of molecular biology – came only after the discoveries of RNA self-splicing and the enzymatic activity of RNase P RNA (Kole and Altman, 1981; Zaug and Cech, 1982). The empirical finding that RNA can be a catalyst as well as an information carrier made it strongly plausible that the first genetic systems could indeed have consisted of RNA alone (Pace and Marsh, 1985; Sharp, 1985; Orgel, 1986). White (1976) had formerly argued that the so-called nucleotide coenzymes (like NAD, NADP, FAD, FMN, etc.) are fossils of an earlier metabolic stage when RNAs acted as enzymes. It is interesting to note that whereas in White’s paper the emphasis was on metabolism, in Gilbert’s (1986) manifesto for the “RNA world” interconversions of RNA molecules are the focus. A much lesser known – but very rigorous – attempt was that of Gnti (1979), who put early enzymatic RNAs into his minimal cell model (see Fernando et al., 2005, for a review on models of minimal cells): the so-called chemoton (see Gnti, 2003a,b). In that model it is assumed that replicative ribozymes catalyze steps of an autocatalytic metabolic cycle, surrounded by a growing membrane. This approach was complemented by a thorough confirmation of White’s insights on coenzymes as remnants of early ribozymes (Kornyi and Gnti, 1981). In Gnti’s theoretical world the RNA world was in full bloom by the advent of the experimental demonstration of ribozyme activity in natural systems. Analysis of a “bag of genes” enclosed in compartments led to the stochastic corrector model (Szathmry and Demeter, 1987), demonstrating that selection at the The Aptamer Handbook. Edited by S. Klussmann Copyright c 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-31059-2

3.1 Introduction

Fig. 3.1 The stochastic corrector model (Szathmry and Demeter, 1987). Different templates (open and closed circles) contribute to the well-being of the compartments (protocells) in that they catalyze steps of metabolism, for example. During protocell growth templates replicate at differential expected rates, but stochastically. Upon division (p)

there is chance assortment of templates into offspring compartments. Stochastic replication and reassortment generate variation among protocells, on which natural selection at the compartment level can act and oppose to (correct) internal deterioration due to withincell competition. Such compartmentation selects for efficient ribozyme variants.

compartment (protocell) level is strong enough to oppose (correct) the adverse consequences of within-cell competition among replicative, unlinked genes. The difficulty of internal competition within an early genomic set was first pointed out by Eigen (1971). The stochastic corrector not only solves the competition problem, but is also a theoretical construct to account for selection for ribozyme functions in reproducing compartments (Fig. 3.1), as explicitly stated in the paper.

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However, a burning open question, also realized by Eigen (1971), still remains; namely, the problem of the error threshold of replication (that is, a sharply defined threshold beyond which heredity breaks down and evolutionary adaptation becomes impossible). This concept is the flip side of the coin on which we see “mutational load” on one side. Ever since the pioneering works of Haldane (1937) it was clear to population geneticists that too high a mutational load (the decrease in average fitness of a population due to recurrent deleterious mutations) could kill a population, but Eigen looked at this problem from another angle. If you fix the mutation rate, how long can a replicator grow before it can no longer be maintained by natural selection, despite it being a fast replicator? Early replicators must have been error prone; therefore, they could not have been very long (the size of a tRNA is usually assumed). Does the stochastic corrector model push up the error threshold so that genomes composed of several to many different genes can be maintained by selection? The answer is encouraging, but not sufficient (Zintzaras et al., 2002): the origin of a sizeable genome is still a problem (see also Santos et al., 2004). We outline here a possible resolution to this conundrum. In the first part of this chapter we analyze existing ribozymes to obtain a “function landscape,” which assigns an activity value (ideally) to each mutant sequence. Then we use this function landscape as a proxy for the fitness landscape; the crucial assumption being that ribozyme activity affects protocell fitness, hence protocell fitness translates back to some average ribozyme fitness. In conclusion, we argue that the position of the error threshold was previously estimated to be too severe: neutral and compensatory mutations crucially modify the picture. This result strengthens the possibility of an RNA world in protocells (Szathmry, 1990a). In the late 1908s one of us was interested in putting the important theoretical considerations of an RNA world to experimental test. Contemporary natural ribozymes almost exclusively conform to Gilbert’s vision of an RNA world, rather than lending support to a metabolically complex RNA era, as envisaged by Benner et al. (1987, 1989). It was clear that a proof of the principle of the general enzymatic capability of RNAs should come from novel experiments. It was suggested that a protocol similar to the production of catalytic antibodies should be followed (Szathmry, 1989, 1990b). The suggested method envisaged selection of RNAs by binding to transition state analogs of a given reaction, linked to an affinity chromatography column. Amplification would have happened at the DNA level after reverse transcription of the best-binding RNA molecules. Szathmry (1989) also pointed out that the same protocol could possibly be used to obtain RNA “aptamers” (as we call them now) that could specifically bind amino acids, thus enabling the community to test certain ideas (primarily the stereochemical one) of the origin of the genetic code. Following this line, in the second half of the chapter we summarize experimental work on co-ribozymes (cofactor-assisted ribozymes) and aptazymes (aptamers attached to ribozymes). Finally, we discuss ideas around how aptamers and nucleotides could have been relevant for the origin of the genetic code and translation.

3.2 Functionality Landscapes Inferred from Examples

3.2 Functionality Landscapes Inferred from Examples 3.2.1 Fitness Landscape

Fitness landscapes or adaptive landscapes are often used in evolutionary biology to envisage the relationship between genotypes – or phenotypes – and Darwinian success (fitness). The idea of studying evolution by visualizing the distribution of fitness values as a kind of landscape was introduced by Wright (1932). Maynard Smith (1970) was the first to coin the term “protein space” for a high-dimensional space in which each sequence of length N amino acids (out of 20N possible sequences) represents one point and is next to 19N points representing all the one-mutant neighbors of each other. In order to produce an adaptive landscape in sequence space a fitness value has to be assigned to each sequence, and an evolving population of proteins typically climbs uphill in the fitness landscape. This concept has since been used by a number of authors (for example Eigen, 1985; Schuster, 1986, 1987). The simplest theoretical fitness landscape is the single-peaked fitness landscape used in Eigen’s (1971) study of the error-threshold in a replicating population of RNA sequences. In this landscape one sequence (the “master sequence”) has the highest fitness value, and all other sequences have the same or lower fitness. This biologically rather unrealistic fitness landscape still attracts considerable theoretical interest, mainly because it can be tackled analytically (Drossel, 2001). In evolutionary optimization methods such as genetic algorithms (see references in Flamm et al., 1999), as well as the use of the concept of a potential or energy function in physics (for example spin-glasses; Bonhoeffer and Stadler, 1993), fitness landscapes have also been applied to the study of biological evolution. However, the structure and characteristics of these landscapes are quite unlikely to match with the fitness landscapes of biological systems. The N-K model of Kauffman (Kauffman, 1993) describes a landscape where K out of N elements are involved in some epistatic interaction. The model produces a rugged fitness landscape which was believed to resemble molecular fitness landscapes on the basis of its ruggedness. Mutational additivity usually holds for positions in biological sequences that do not interact, and such mutational additivity has been demonstrated for several proteins (Tekada et al., 1989; Sarai and Tekada, 1989; Sandberg and Terwilliger, 1993; Serrano et al., 1993; Zhang et al., 1995; Skinner and Terwilliger, 1996; Nikolova et al., 1998; Aita et al., 2001, 2002). In these cases, the most realistic of the N-K landscapes is the Mount Fuji type fitness landscapes (also known as multiplicative fitness landscapes), where each element in a sequence individually and independently contributes to the fitness and there is a single fittest sequence.

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3.2.2 Damage Selection Experiments with Ribozymes

A wealth of information has been accumulated on ribozymes since their discovery nearly 20 years ago. Most of the studies can be fitted into one of the three main lines of research: (1) characterization of known ribozymes (that is, inferring the structure and mechanisms of catalysis; Lilley, 1999); (2) modification of natural ribozymes to be used in therapeutics (Sullenger and Gilboa, 2002); and (3) in vitro evolution of novel ribozymes (Joyce, 1998, 2002; Landweber et al., 1998; Spirin, 2002). The characterization of ribozymes frequently involved mutagenesis experiments, where the enzymatic activity of certain mutants was measured in order to get insight into either the structure of the molecule or the mechanism of catalysis. While not directed toward the study of fitness landscapes, these experiments certainly contain a wealth of empirical information necessary for assembling the realistic fitness landscape of the studied ribozyme. Albeit all naturally occurring ribozymes are being studied extensively, there are only a few instances where the realistic fitness landscape can be conveniently investigated. Group I and group II introns, as well as the RNAase P, have to be excluded because of their rather large size. Furthermore, it is inevitable to employ an RNA folding algorithm in any sensible investigation of the fitness landscapes of ribozymes. Therefore, ribozymes with a pseudo-knot in their structure also have to be excluded because most conventional folding algorithms cannot satisfactorily cope with pseudo-knots. This requirement singles out the hepatitis delta virus, which contains such structural elements (Perrotta and Been, 1991). On the other hand, the hammerhead, hairpin, and Neurospora VS self-cleaving ribozymes can be separated into a substrate and a trans-cleaving ribozyme. With respect to these three ribozymes the trans-cleaving enzyme does not contain a pseudo-knot structure. The hammerhead can be separated into a 13-mer enzyme and a 41-mer oligonucleotide substrate (Jeffries and Symons, 1989). The hairpin can be separated into a 50-mer enzyme and a 15-mer substrate (Fedor, 2000). The trans-acting ribozyme is 144 nucleotides long for the Neurospora VS ribozyme (Fig. 3.2), and the substrate is 20 nucleotides long (Guo and Collins, 1995). We restrict our further analysis to the trans-acting ribozyme, and assume that the substrate is the same as the natural one. Unfortunately for our study, many of the mutagenesis experiments have been directed towards the substrate and substrate-binding regions (Joseph et al., 1993; Joseph and Burke, 1993; Nishikawa et al., 1997; Ananvoranich and Perreault, 1998; Prez-Ruiz et al., 1999) in order to produce new RNA- or DNA-cleaving ribozymes to be used in therapeutics (Yu et al., 1998; Andng et al., 1999; Andng et al., 2004; Zhang et al., 2004). Based mainly on experiments with the VS and the hairpin ribozymes the following general conclusions can be derived: Structure is important. From experimental data on the VS ribozyme Lilley and co-workers (Lafontaine et al., 2002c) state that “the secondary structure of the ribozyme is important, but the nature of most individual base pairs is not. Many can be reversed x


x

x

or replaced by a different pair without major loss of activity, so long as a base pair is retained at a given position.” Similarly, in the hairpin ribozyme all base pairs can be altered (except base pair G11:C/U-2) as long as the base pairing is maintained (Fedor, 2000). There are critical regions in the molecule. For the single-stranded regions the structure has to be maintained, but at many such positions the nature of the base located there is also important. For example, most of the bases in the four loops of the hairpin ribozyme are essential, and any change in those positions severely reduces activity (Siwkowski et al., 1997; Shippy et al., 1998). For the VS ribozyme 16 such critical sites were identified (Lafontaine et al., 2002a): these sites are located around the active site, the substrate-binding region, and in the two- or three-way junctions. Structure can be varied slightly. The structure of the naturally occurring ribozymes can be slightly varied, as there are regions that are not crucial to function. For example, the stem–loop IV of the VS ribozyme is virtually completely dispensable, but the junction 3–4–5 must be formed (albeit after the complete removal of stem–loops IV and V the ribozyme still has detectable activity; Sood and Collins, 2002). Similarly, in the hairpin ribozyme the helices H1 and H4 can be shortened and greatly extended without any loss of activity (Fedor, 2000; Sargueil et al., 1995).

Fig. 3.2 Sequence and secondary structure of the enzyme part of the Neurospora VS ribozyme (numbering according to Beattie et al., 1995). Roman numbers indicate the regions of the

ribozyme. Capitalized nucleotides indicate positions for which mutagenesis studies are available. Bold nucleotides indicate critical sites.

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While the previous general conclusions can be easily incorporated into a model of a fitness landscape, one general difficulty stills remain; namely, the combined effect of multiple mutations. Most mutagenesis experiments have investigated only single mutations (or mutations involving a base pair) in the vicinity of the wild type in sequence space and rarely report the activity of double or higher order mutants. In those few instances where the effects of multiple mutations were evaluated, the activities of the single-point mutants were not always included. The only remarkable exception is the study of Lehman and Joyce (1993) from an initial pool of the Tetrahymena ribozyme, where they found that in general the mutational effects were multiplicative (which implies mutational additivity for ribozyme activity). Table 3.1 summarizes the available experimental information on multiple mutational effects for some of the known nucleolytic ribozymes. The plot of the measured enzymatic activities of the double mutants on the estimated activities from the single mutants (Fig. 3.3) clearly suggests that mutational effects are nearly multiplicative, with a slight positive synergy. Such positive synergy was also found for chemical modifications of the hairpin ribozyme (Klosermeier and Millar, 2002). Accordingly, the fitness of a molecule containing n mutations (wn) could be estimated as: multiplicative

wn

wn =

=

n Y

i=1 multiplicative wn p

wi

(3.1)

(ax b )

where wi is the fitness of a single-error variant, wnmultiplicative is the fitness of an n-error variant assuming multiplicative effects, and a and b are parameters to be fitted given the data (Fig. 3.3). We stress, however, that although the data set contains information from three different ribozymes the number of points is still quite small. Therefore, some care should be taken when translating the empirical available information to a fitness function. Besides these synergistic effects there are also examples of mutations that “rescue” enzymatic activity to some extent. Mutations that result in the loss of catalytic activity also exist (Table 3.2). Mutants containing these and other point

Fig. 3.3 Plot of the measured enzymatic activities of the double mutants on the estimated activities from the single mutants for the Neurospora VS ribozyme.

3.2 Functionality Landscapes Inferred from Examples Table 3.1 Available experimental data for the evaluation of multiple mutant effects on fitness Mutant 1

Activity Mutant 2 Activity Activity of the double mutant

Estimated activity of the double mutant

Reference

U39Ca

0.289

A11G

0.32

0.093

0.095

Joseph et al., 1993

G21Ua

0.01

A20U

0.9

0.01

0.009

Sargueil et al., 2000

G21Ua

0.01

A20G

0.2

0.003

0.002


a

G21U

0.01

A20C

0.6

0.15

0.06


G21Ua

0.01

A43G

0.4

I 0.001

0.004


G21Ua

0.075

A43G

0.085

I 0.001

0.0064

Siwkowski et al., 1997; Sargueil et al., 2000

G21Ua

0.075

A43U

0.002

I 0.001

0.00015

Siwkowski et al., 1997; Sargueil et al., 2000

A7Ca

1

A20C

0.81

1.04

0.81

Anderson et al., 1994

A730Cb

0.32

A731C

0.39

0

0.12

Kumar et al., 1992

b

G726A

0

A730C

0.32

0

0

Kumar et al., 1992

U752Cc

0.80

U753C

0.42

0.52

0.336

Lafontaine et al., 2001b

G722C; C763Gc

0.81

C723G;G762C

0.84

0.75

0.680

Beattie et al., 1995

G716Cc

0.21

U717A

0.21d

0.02

0.044


A661U

d

0.06

0.053


C662G a b c d

c

0.23

0.23

Hairpin ribozyme (numbering follows Butcher and Burke, 1994a,b). Hepatitis delta virus (numbering according to Makino et al., 1987). Neurospora VS ribozyme (numbering according to Beattie et al., 1995). No data available. The activity of mutant 2 is assumed to be equal to the activity of mutant 1.

mutations might or might not have detectable activity. To our knowledge these interactions are impossible to predict, thus they can only be incorporated into a definition of a fitness landscape if known from experiments. In conclusion, the easiest way to deal with multiple mutations is to assume mutational independence (multiplicative effects), although it slightly overestimates the decrease in fitness due to multiple mutations. A more realistic assumption can come from taking the synergy into account, albeit more data would be highly welcome. If rescue mutations or other such effects are known of the ribozyme, then they can also be incorporated to increase the realism of the fitness landscape.

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3 Fitness Landscapes, Error Thresholds, and Cofactors in Aptamer Evolution Table 3.2 Single null and multiple mutations that in some cases “rescue” enzymatic activity to some extent Mutation that abolishes activity

Multiple mutant

Relative activity

Ribozyme

Reference

A43C

A43C; G21U

0

Hairpin

Siwkowski et al., 1997a; Sargueil et al., 2000

G726A

G726A; A730C

0

HDV

Kumar et al., 1992

G726A

G726A; G727C; A731C

0.02

HDV

Kumar et al., 1992

G726A

G726A; G728A; C729G

0

HDV

Kumar et al., 1992

G726A

G726A; G727U; C729G

0.01

HDV

Kumar et al., 1992

G726C

G726C; G728U; A730C

0

HDV

Kumar et al., 1992

G726U

G726U; G727U; A731C

0.04

HDV

Kumar et al., 1992

G727C

G726A; G727C; A731C

0.02

HDV

Kumar et al., 1992

G727C

G727C; C729A; A731G

0

HDV

Kumar et al., 1992

G728C

G727A; G728C

0.03

HDV

Kumar et al., 1992

G728C

G728C; A731U

0.01

HDV

Kumar et al., 1992

G728C

G728C; A730G

0.06

HDV

Kumar et al., 1992

G728C

G727A; G728C; C729U

0.04

HDV

Kumar et al., 1992

C763A

C763A; A766G

0

HDV

Kumar et al., 1992

C763A

C763A; G764A

0

HDV

Kumar et al., 1992

C763G

C763G; A765U

0

HDV

Kumar et al., 1992

C763G

C763G; A765U

0

HDV

Kumar et al., 1992

3.2.3 Construction of the Fitness Landscape

Based on the foregoing general conclusions we propose a method to generate a fitness landscape for any ribozyme for which enough mutagenesis data are available. The result of the method is an algorithm which assigns a relative activity to each of the 4N possible RNA sequences of length N. For the sake of simplicity we restrict the sequence space to sequences of a given length; however, the method could be applied if insertions and deletions were also considered. The algorithm consists of four steps: (1) compatible structure, (2) mispairs, (3) critical sites, and (4) predicted structure. In each step we calculate an activity value pertaining to the given step. The relative activity and the fitness of the sequence (Asequence) is the product of the individual activities calculated at each step (that is, Asequence = Astructure q Amispair q Acritical q Aenergy).


3.2.3.1 Compatible Structure In order to have any enzymatic activity the molecule should fold into the structure of the ribozyme. Here a number of alternative structures can also be considered. A sequence is said to be compatible with a structure if for every base pair (i,j) in the structure the bases at the ith and jth positions in the sequence can form one of the allowed base pairs (that is, A:U, U:A, G:C, C:G, U:C, G:U). Enforcing strict compatibility might result in an overestimation of the negative effects of mispair mutations. Some mispair mutants retain relatively high level of activity; for example the C662G mispair mutation in the VS ribozyme decreases activity to 23% of the wild type (Beattie et al., 1995). Thus, even sequences with partial compatibility should be considered compatible in this step. The negative effect of mispairs is taken into account in the next step. If a sequence is not compatible – even when considering the possibility of mispairs – with any of the possible structures, then it has no activity and its fitness is set to 0. The activity factor for this step (Astructure) is the activity associated with the structure to which the sequence can fold. The activities of the various possible structures can be different.

3.2.3.2 Mispairs When a sequence is perfectly compatible with a structure (that is, there are no mispairs in it) then Amispair = 1, otherwise every single allowed mispair decreases activity to some extent. Every mispair has an associated relative enzymatic activity (Amispair,i), and the activity factor for this step is the product of the activities of the individual mispairs: Amispair = PAmispair,i. If synergistic effects are taken into account, then they should be incorporated in this step and/or the next step. Some regions of the ribozyme can show different sensitivity to mispairs, thus the associated relative enzymatic activity (Amispair,i) will be different.

3.2.3.3 Critical Sites The nature of nucleotides at critical sites of the molecule is taken into account in this third step of the algorithm. These sites are well studied so we can nearly assign a measured activity to every possible nucleotide at these positions. In fact, all possible single mutants of the single-stranded regions of the hairpin ribozyme have been analyzed (Siwkowski et al., 1997; Shippy et al., 1998). As before, the product of the individual activities (Acritical,i) gives the activity factor for this step (Acritical = PAcritical,i). If synergistic effects are taken into account, then they should be incorporated in this step and/or the previous step. Furthermore, if other epistatic effects were present, they would likely affect positions involved in this step.

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64


3.2.3.4 Predicted Structure The last step in establishing the fitness of a sequence is to predict the secondary structure the sequence will fold into and contrast it with the structure resolved in the first step. The folding can be done with any available RNA folding routine, as for example the MFold (Zuker et al., 1999) or the Vienna RNA Package (Hofacker et al., 1994). It has to be noted at this stage that the predicted minimum free energy structure of the wild-type ribozyme sequence does not always correspond with the actual secondary structure. In this case that structure can also be accepted as a good structure. Furthermore, if mispairs are allowed then they have to be taken into account during structure comparisons. When the predicted and the target structure are the same Aenergy = 1, otherwise Aenergy = 0. This step is undoubtedly the most costly in terms of CPU time. 3.2.4 Case Study: The Fitness Landscape of the Neurospora VS Ribozyme

The Neurospora VS ribozyme (Saville and Collins, 1990) can be used as a model system to show the usefulness of the algorithm in generating the functional fitness landscape of the molecule (Fig. 3.2). There is a wealth of mutagenesis information available for this ribozyme (Beattie et al., 1995; Lafontaine et al., 2001a,b, 2002a,b,d; Rastogi et al., 1996; Rastogi and Collins, 1998; Sood and Collins, 2002): out of the 144 positions in the ribozyme (stem–loops II–VI; nucleotides 640–783 according to the conventional numbering), 87 positions have documented mutants (excluding deletions and insertions) and the total number of mutants studied so far is 183. The ribozyme has six regions (Fig. 3.2), where stem–loop I contains the cleavage site and is the substrate of the reaction, while regions II–VI perform the catalysis. The naturally occurring VS ribozyme is self-cleaving; however, it can be divided into a trans-acting ribozyme plus a substrate system (Guo and Collins, 1995). Unlike trans-cleaving versions of the hammerhead, hairpin, and HDV ribozymes, the VS ribozyme does not use long stretches of complementary base pairing to associate with its substrate. Nonetheless, the ribozyme–substrate interaction is quite tight (Guo and Collins, 1995; Lafontaine et al., 2001b). The pseudo-knot forming between the loops of the substrate and the stem–loop V of the ribozyme is crucial for substrate biding and orientation (Rastogi et al., 1996; Andersen and Collins, 2001).

3.2.4.1 Compatible Structure of the VS Ribozyme The length of stem–loop IV and the distal part of stem–loop VI can be varied to a great extent. The length of stem–loop V can be varied slightly (Lafontaine et al., 2002b), but the length of stem–loop III cannot be changed (Lafontaine et al., 2002b). There are no data available on stem–loop II. Furthermore, the A718 bulge in region III can be complemented (that is, replaced by a base pair, with the insertion of U after position 660) without significant loss of activity (Beattie et al., 1995; Lafontaine et al., 2002b). These changes constitute the basis of

3.2 Functionality Landscapes Inferred from Examples Table 3.3 Enzymatic activities of different structures of the Neurospora VS ribozyme Structure

Astructure

Original structure

1.000

Deletion of the A652 bulge

0.013


Deletion of the A718 bulge

0.136


Reference

Pairing of the A718 bulge

0.820


Length 8 stem III

0.087


Length 7 stem V

0.045


Length 6 stem IV

0.470


Length 4 stem IV

0.590


Length 8 stem VI

0.970


Length 6 stem VI

1.000


other acceptable structures beside the wild-type structure. Table 3.3 lists various structures and their activities. In order to maintain the wild-type total length N = 144 constant, all structures including deletions or insertions were slightly modified in our study. Singlestranded regions were removed from or added to the beginning and the end of the structure in all possible combinations. We allowed some mispairs in the structure, but a mispair can only be considered if the following criteria are met (in what follows all numbers refer to positions in the wild-type sequence. In the alternative structures these positions change according to the shift due to the insertion or deletion): Two mispairs cannot be adjacent. There are six experimentally tested mutants that have mispairs at adjacent positions. Four of them have absolutely no activity ([G727C, U728A], [G722C, C723G], [G762C, C763G], [A759U, C760G]). In the case of the remaining two mutants ([G716C, U717A], [A661U, C662G]) the activities are 0.02 and 0.06, respectively. The pairs [653:771], [654:770], and [655:769] in stem–loop II have to be paired. The mispair mutants of the region ([G653C], [C771G], [G655C]) do not have measurable activities, except for the mutant [C769G] whose relative enzymatic activity is 0.18. The base pairs adjacent to a junction have to be paired. The stable forming of the junction is critical for enzymatic activity. If the bases next to a junction do not form a base pair, then the structure of the junction changes considerably and enzymatic activity would vanish. Accordingly, the following base pairs have to be paired: [658:721], [663:715], [666:685], [687:709], [722:763]. x

x

x

65

66

3 Fitness Landscapes, Error Thresholds, and Cofactors in Aptamer Evolution x

A stem–loop cannot contain more than two mispairs. Generally, two mispairs in the same region greatly reduce activity (0.78 [G704C, U706A], 0.54 [U670A, C672G], 0.28 [A748U, U750A], 0.25 [A735U, U737], 0.07 [A690U, C692G], 0.06 [A661U, C662G], and 0.02 [G716C, U717A]) or abolish it completely (see the adjacent mispairs mentioned above or the mutant [G679C, A681U]). We assume that if a stem–loop contains more than two mispairs, then the resulting molecule would not have any enzymatic activity.

3.2.4.2 Allowed Mispairs in the VS Ribozyme We have distinguished various kinds of different mispairs according to their position. First, a mispair next to the active site (that is, at positions [731:754] and [729:758]) decreases activity to Amispair,i = 0.025 e 0.011. Second, a mispair next to the loop of stem V (at positions [695:701]) decreases activity to Amispair,i = 0.05. Thus, mutants [U695G] and [A701C] have enzymatic activities of 0.06 and 0.04, respectively. Third, a mispair inside a stem decreases activity to 0.2. The experimentally tested mispairs that do not fall into any of the previous categories have the following activities: 0.29 [C773G], 0.23 [C662G], 0.21 [G716C], 0.12 [G650C], 0.05 [A720U], 0.00 [U659A]. The mean activity of these mutant is 0.15 e 0.095, but it should be stressed that these mispairs are located in important parts of the ribozyme. There are no experimentally tested mutants in the functionally less important parts, and it would be reasonable to assume a slightly less severe decrease of activity. Finally, a mispair next to the loops of stem IV and VI will probably not decrease activity to a great extent. For these mispairs we have assumed Amispair,i = 0.80.

3.2.4.3 Critical Sites in the VS Ribozyme Critical sites in the VS ribozyme are located in the junctions, in the substrate binding region, and in the A730 internal loop (Table 3.4). Thus, the two junctions of the ribozyme (3–4–5 and 2–3–6) play an important role in the formation of the 3D structure of the molecule (Lafontaine et al., 2001a,b). Some critical positions exist in both junctions: positions 656, 657, 665, 686, 710, 712, 713, 767 and 768. The other nucleotides of the junctions can be changed to some other nucleotide with minor loss of activity. The substrate interacts with the enzyme through base pairing with bases 697– 699 located in the loop at the end of region V. Therefore, these positions are also considered as critical for the function of the molecule. Finally, the internal loop of region VI (positions 730, 755, 756, and 757) shows the greatest susceptibility to nucleotide substitution (Lafontaine et al., 2001b; Sood and Collins, 2002) and virtually any change in the sequence leads to severe reduction in cleavage rate. This loop is quite probably the active site of the VS ribozyme. For those positions nucleotide A756 appears to be the most important, particularly the amino group at location 6 of the purine base (Lafontaine et al., 2002d).

3.2 Functionality Landscapes Inferred from Examples Table 3.4 Activities of the critical sites in the Neurospora VS ribozyme Acritical,i A

U

C

656

1.000

0.002

0.003

0.061

657

1.000

0.063

0.063a

0.063a

665

0.014

0.01b

1.000

0.01

686

0.006

1.000

0.006a

0.006a

697

0.000

0.001

0.000

1.000

698

1.000

0.000

0.041

0.000

699

0.006

0.018

1.000

0.000

710

0.002

1.000

0.029

0.002

712

1.000

0.005

0.005b

0.006

713

0.019

1.000

0.136

0.019

730

1.000

0.036

0.055

0.011

755

0.840

0.170

1.000

0.019

756

1.000

0.001

0.002

0.002

757

0.044

0.018

0.014

1.000

767

1.000

0.047

0.047a

0.047a

768

0.573

0.015

0.573c

1.000

Critical site

a

b

c

G

No data available. We assume that enzymatic activity is the same as for the known mutant. No data available. We assume that enzymatic activity is the same as for the known mutant with the lower activity. No data available. We assume that enzymatic activity is the same as for the known mutant with the higher activity.

3.2.4.4 Predicted Structure for the VS Ribozyme The minimum free energy (MFE) structure of the original sequence of the VS ribozyme is not the experimentally determined secondary structure. The two structures differ in only three base pairs in stem–loop VI, and the energy difference between the two is a mere 0.3 kcal. But it has to be noted that the nuclear magnetic resonance structure of the isolated stem–loop VI is the same as in the MFE structure (Flinders and Dieckmann, 2004). Thus both structures might play an important role in the catalysis. Accordingly, we accept the sequence if it folds either to the MFE or the experimentally predicted structure of the wild-type VS ribozyme. We have used the RNA folding algorithm (Hofacker et al., 1994) for secondary structure predictions. As previously indicated, the enzymatic activity of the molecule is simply the product of the activity factors estimated in the above four steps. If the resulting activity was less than the lowest activity that can be reliably measured (that is, Asequence J 10 –3), then it was set to 0.

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3 Fitness Landscapes, Error Thresholds, and Cofactors in Aptamer Evolution Fig. 3.4 Fraction of all possible mutants of the original sequence folding into a perfect ribozyme (circles), and a ribozyme with some activity (square) as function of the number of point-mutations introduced into the sequences.

3.2.4.5 Properties of the Estimated Fitness Landscape for the VS Ribozyme To characterize the fitness landscape some additional analyses were made. All possible one- or two-mutant molecules around the original sequence were generated and their fitness recorded. Molecules containing more than two mutations could not be completely enumerated in reasonable time, thus only one million randomly chosen sequences with 4, 5, 6, 7, 8, 9, and 10 mutations were evaluated (Figure 3.4). Of the 432 possible single mutants, 114 (26.4%) had the same activity as the wild-type ribozyme (that is, they are “selectively neutral” neighbors), 222 (51.4%) had an activity higher than 0.1, and 254 (58.8%) still retained “measurable” enzymatic activity. Of the 92 664 possible double mutants only 6,579 (7.1%) had the same activity as the wild type, whereas 29 096 (31.4%) still retained some enzymatic activity (that is, for 68.6% of the sequences Asequence J 10 –3). In addition, we generated 10 million random sequences (a very tiny fraction of the sequence space, which contains approximately 4.973 q 1086 sequences). None of them had any enzymatic activity.

3.3 Error Thresholds Inferred from Functional Landscapes: The “Realistic” Error Threshold of the Neurospora VS Ribozyme

As previously pointed out, the formal description by Eigen (1971) of the mutation–selection dynamics of a population of biological sequences led to the realization of one of the greatest paradox of prebiotic evolution: the error rate poses a limit to the length of the information that can be selectively maintained within the system. A stable cloud of mutants (that is, a quasi-species) can form around a master sequence as long as the maximum chain length (N) is below the critical error rate per site per replication (m*) as determined by the following expression:

3.3 Error Thresholds Inferred from Functional Landscapes

NI

ln s m

(3.2)

where s is the selective superiority of the master. Very roughly then, assuming as customary that lns z 1, Eq. (2) states that the maximum selectively maintainable amount of information (N) is about the inverse of the mutation rate per base per replication. Experimental evidence suggests that without the aid of peptide enzymes the upper bound of copying fidelity per nucleotide per replication could not be higher than 0.99 (Johnston et al., 2001), and is quite likely significantly lower than this figure. Accordingly, the maximum N would be lower than 100 nucleotides. However, Eigen’s model is based on the assumption that the whole genotype – the master sequence – has to be maintained for functionality. This assumption might be justifiable in a DNA–protein world, but in the RNA world the enzymatic activity of a molecule was mainly based on its three-dimensional structure rather than on the exact order of its building blocks (except for a few critical sites). It is a well-established fact that the number of possible RNA secondary structures is considerably less than the number of possible sequences (Schuster et al., 1994; Stadler and Haslinger, 1999), and it is also possible that two molecules with completely different sequences share the same secondary structure (Huynen, 1996; Huynen et al., 1996; van Nimwegen et al., 1999). In other words, when RNA structure is considered it might be feasible to maintain ribozyme functionality (phenotype) at mutational rates that would not allow the preservation of the master sequence. In order to determine the “realistic” error threshold for the VS ribozyme we have explored the dynamics of a population of RNA molecules with N = 144 at various mutation rates per nucleotide per replication (m). At the replication step a sequence is picked at random according to its fitness. Thus, the probability pj of choosing sequence i with enzymatic activity Ai is: pi =

Ai SAj

(3.3)

where SAj is the sum of activities for all sequences in the population. The next step is to copy the chosen sequence with error rate m (only point mutations were considered). Because a quarter of the times (assuming equal probability for each nucleotide) no effective change will occur in the position even though there is a mutational event, the effective mutation rate is m* = 0.75 m. The new sequence then replaces a randomly chosen sequence, which allows keeping a constant population of molecules and is also equivalent to the assumption that the rate of degradation is the same for all molecules and independent of enzymatic activity (Bonhoeffer and Stadler, 1993). As a final point we should emphasize here that the occurrence of thresholds for error propagation was originally derived as a deterministic kinetic theory that is only valid in the limited case of an infinite number of molecules. Alves and Fontanari (1998) have extended it to finite populations and found that the critical error rate per site per

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70


Fig. 3.5 Error threshold for the VS ribozyme. Maximum (downward triangles), mean (square) and minimal (upward triangles) observed times to extinction are plotted as a function of the error rate. The population size

was 5000 molecules. A generation is defined as a number of replication steps equal to the population size. For each error rate 10 replicates were obtained. A line is fitted to the last four data points (solid line; R = –0.904).

replication decreases linearly with 1/N. In our present case we extrapolated to an infinite population size by recording the time to extinction (that is, the number of generations when no functional ribozymes remained in the population) at various error rates and fitting a straight line to those last few points which still showed a downward trend. The error threshold is then the intersection of the line with the error rate axis. As shown in Fig. 3.5 the “realistic” error threshold for the VS ribozyme was estimated to be m* = 0.052 (this figure refers to the “effective” mutation rate; see above). To compare this figure with the error threshold that would be obtained without considering secondary structure we have considered two different fitness landscapes: Mount Fuji and single-peaked landscapes. In Mount Fuji landscape we have assigned an activity value to all possible nucleotides at a given position, with the wild-type nucleotides at each position having a value Ai = 1 for the enzymatic activity. For those positions where experimental data are available we considered that value, otherwise we either used the value for the same position derived for another nucleotide (if more than one such value were available, we used the lowest of the two) or used a predefined value. In the last case we considered two scenarios: those mutants have uniformly either Ai = 0.8 or Ai = 0.2. Therefore, in the first case we assumed that those positions are not functionally important, whereas in the second case they are. In no case can the enzymatic activity of the molecule be higher than 1. The fitness value of a

3.4 Looking for Catalytic Partners: Cofactors and Aptamers

sequence was then calculated as the product of the individual activities, and the resulting “Mount Fuji error thresholds” were m* = 0.032 (Ai = 0.2) and m* = 0.025 (Ai = 0.8), which are substantially lower than the “realistic” error threshold from the functional landscape. Mount Fuji landscape retains some characteristics of the functional landscape – the fitness effect of the single-stranded regions – but it is no longer possible to have compensatory mutations by changing one base pair into another. In fact, every mutation affecting a helical region counts as a mispair, which causes the error catastrophe to occur at lower value. For the single-peaked landscape we used the same assumption as in Eigen’s model. Thus, the wild-type sequence has Ai = 1 at each position and all other sequences have Ai = 0.217, which is the average activity of all experimentally tested one-point mutants. The “single-peaked error threshold” was found to be m* = 0.023, lower than in either of the previous cases. The reason is that this landscape retains no information about the structure, and no neutral mutations are possible. By using Eq. (2) above and assuming lns = 1, the maximal error rate for the VS ribozyme would be m* z 0.007. This figure is nearly an order of magnitude lower than the one we got by using a realistic fitness landscape. Furthermore, according to the Eigen’s model the error rate of 0.052 would permit a ribozyme of maximum length 20 to be maintained. In summary, it is quite obvious that the inclusion of structural information, as well as information derived from experimental data, crucially alleviates the burden imposed by Eigen’s (1971). This is the first report of a realistic error threshold calculated for an existing ribozyme. To the best of our knowledge there is only one other indication of an error threshold calculated using a structural landscape. Huynen and co-workers (1996) used the secondary structure of the phenylalanine tRNA (N = 76) as their object of investigation, and assumed that the fitness decrease of a mutant is proportional to the difference between the target structure and the structure of the mutant sequence. They reported that the error threshold for the tRNAPhe is 0.0031. This seems to be too low, as even the Eigen’s model predicts a higher error threshold (m* z 0.01). This low value might be an artefact of the folding algorithm, which for tRNAs often predicts a minimum free energy structure unlike the known cloverleaf structures. There is as yet no reported replicase ribozyme. The most promising result thus far is a ribozyme that can extend a sequence by 14 nucleotides according to a template (Johnston et al., 2001). This ribozyme works with a 0.967 copying fidelity. If a functional replicase ribozyme had the same fidelity, then it could replicate the VS ribozyme without the threat of the error catastrophe.


The discovery that present-day living cells use RNA catalysts to hydrolyze RNA molecules or to perform the complex reactions of excision, ligation, and cyclization supporting a limited catalytic diversity, raises the question of the respective domains of RNA and protein catalysis. The recent advances in RNA catalysis

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using the SELEX method make it possible to enhance the catalytic capabilities of RNA with small molecules as catalytic partners. In this way some RNAs may be analogous to protein in catalytic competence. Purine nucleotides, and in particular those containing adenine, participate in a large variety of cellular biochemical processes (Maurel and Dcout, 1999). Their best-known function is that of monomeric precursors of RNAs and DNAs. Nevertheless, derivatives of adenine are universal cofators. They serve in biological systems as source of energy (ATP), allosteric regulators of enzymatic activity and regulation signals (cyclic AMP). They are also found as acceptors during oxidative phosphorylation (ADP), as components of coenzymes (such as in FAD, NAD, NADP, coenzyme A; Fig. 3.6), as transfer agents of methyl groups of S-adenosylmethionine, as possible precursors of polyprenoids in C5 (adenosylhopane) (Neunlist et al., 1987), and – last but not least – adenine 2451 conserved within the large rRNA in the three kingdoms, would be involved in catalysis during the formation of the peptide bond (Muth et al., 2000, 2001; Green and Lorsch, 2002). On the other hand, biosynthesis of the amino acid histidine, which would have appeared late in evolution, begins with 5-phosphoribosyl-1-phosphate (PRPP) that forms Nl-(5-phosphoribosyl)-ATP by condensation with ATP. This reaction is akin to the initial reaction of purine biosynthesis. Finally, the ease with which purine bases are formed in prebiotic conditions (Or, 1960) suggests that these bases were probably essential components of an early genetic system. The nucleotides that by post-transcriptional modification can acquire the majority of functional groups present in amino acids possess a great potential diversity that is expressed in the modified bases of tRNAs and rRNAs and also at the level of ribonucleotide coenzymes (several coenzymes derive from AMP; Fig. 3.7). In particular many coenzymes are nucleotide analogs and the role of these cofactors at all steps of the current metabolism, and their distribution within the three kingdoms, suggests that a great variety of nucleotides were present in the last common ancestor. It as been suggested (White, 1976; Trmolires, 1980) that coenzymes and modified nucleotides that were present before the appearance of the translation machinery may have played a prominent role in primeval catalysis. Proteins would have appeared only at a later stage, coenzymes and ribozymes being fossil traces of past catalysts. Indeed, in the living cell, only a minority of enzymes function without coenzyme; they are mostly hydrolases, and apart from this group, 70% of the enzymes require a coenzyme. If metal coenzymes involved in catalysis are considered, the number of enzymes that depend on coenzymes increases further. Present-day coenzymes, indispensable cofactors for many proteins, would be living fossils of catalysts of primitive metabolism (Maurel and Haenni, 2005). Most coenzymes are nucleotides (NAD, NADP, FAD, coenzyme A, ATP, etc.) or contain heterocyclic nitrogen bases that can originate from nucleotides (thiamine pyrophosphate, tetrahydrofolate, pyridoxal phosphate, etc.). Consequently the efficiency of selected ribozymes can be further expanded if coenzymes and/or nucleotide analogs containing functionalities (thiols, amino groups, imidazolyl


Fig. 3.6 Coenzyme structures.

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Fig. 3.7 List of coenzymes derived from AMP.

moieties, etc.) are used. Modern selections yield various co-assisted dependent ribozymes, justifying an RNA-based metabolism. 3.4.1 Co-ribozymes (cofactor-assisted ribozymes)

Aptamers are capable of recognizing targets as small as metal ions (most RNA enzymes are metallo-ribozymes using metal as cofactors). They can interact with a wide variety of molecules that are important for metabolism, including amino acids, porphyrins, nucleotide factors, coenzymes, small peptides, and short oligonucleotides (Illangasekare and Yarus, 1997; Jadhav and Yarus, 2002a; Joyce, 2002; McGinness et al., 2002; Reader and Joyce, 2002) (Table 3.5). The first aptamer selected for a biological cofactor was an ATP-binding RNA (Sassanfar and Szostak, 1993; Fig. 3.8) showing a change in the conformation of the RNA and number of close contacts between the ATP and RNA. Since the ATP motif also binds adenosine and NAD+, the idea was that it could serve to bind adenosine-derived cofactors as well. The presence of adenosine in many common biological cofactors (ATP, CoA, FAD, NAD+, SAM, coenzyme B12) has been postulated to reflect an evolutionary origin for modern metabolism. It is even possible to consider that catalytic groups that were part of nucleic enzymes were incorporated in specific amino acids rather than being “retained” as coenzymes. This could be the case for imidazole, the functional group of histidine, whose present synthesis in the cell is triggered by a nucleotide (Maurel and Ninio, 1987; Benner et al., 1989; Maurel, 1992). Further, the use of organic cofactors was illustrated by a DNA enzyme that requires histidine as a cofactor during RNA cleavage (Szazani et al., 2004). A small aptamer recognizing anionic moieties has also been obtained by Szostak and co-workers (Szazadni et al., 2004). In this case the significant interactions are with the phosphate of ATP (Kd of 5 mmol/L compared with the AMP Kd of 5.5

3.4 Looking for Catalytic Partners: Cofactors and Aptamers Table 3.5 Small targets of RNA aptamers Target

Minimal size

Kd (mmol/L)

Reference

Adenine

–

10

Meli et al., 2002

Guanine

32

1.3

Kiga et al., 1998

Guanosine

–

32

Connell and Yarus, 1994

l-Adenosine

58

1.7

Klussmann et al., 1996

Theophylline

38

0.1

Jenison et al., 1994

Caffeine

–

3 500


Riboflavin

35

0.5

Burgstaller and Famulok, 1994

NMN/NAD

–

2.5

Lauhon and Szostak, 1995

d-ATP

40

14

Sassanfar et al., 1993

Cyanocobalamin

35

88 nmol/L

Lorsch and Szostak, 1994

l-Valine

–

12 000

Majerfeld and Yarus, 1994

l-Citrulline

44

65

Famulok, 1994

l-Arginine

–

0.33

Geiger et al., 1996

d-Arginine

38

135

Nolte et al., 1996

Biotin

31

6

Wilson et al., 1998

Fig. 3.8 ATP and the corresponding aptamer.

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Fig. 3.9 Self-incorporation of coenzyme (adapted from Breaker and Joyce, 1995).

mmol/L). This is of particular interest as two messenger RNA riboswitches from Escherichia coli are known to discriminate between phosphorylated small molecules. The first self-incorporation of a coenzyme into a ribozyme was performed in 1995 by Breaker and Joyce (1995), substituting neatly alternative coenzymes into the primary structure of group I intron by replacing the guanosine substrate with natural coenzymes or analogs (Fig. 3.9a and b respectively). Looking for significant metabolic reactions and for coenzyme-dependent ribozymes, the primary biological cofactor used in acyltransfer reactions, coenzyme A (CoA), has been the target of RNA pools leading to a 52-nucleotide minimal aptamer (Burke and Hoffmann, 1998) which recognizes the adenosine moiety of CoA and binds others ATP analogs (Fig. 3.10). The selection of coenzyme synthetase ribozymes is of particular interest in an RNA-catalyzed energy metabolism. Yarus, exploring the origin of ribonucleotide coenzymes, demonstrated the RNA-catalyzed formation of three common coenzymes CoA, NAD, and FAD from their precursors, 4l-phosphopantetheine, NMN, and FMN, respectively (Huang et al., 2000). A ribozyme capable of utilizing CoA for the synthesis of acyl-CoA was selected in vitro. The co-ribozyme isolated, that is an acyl-CoA synthetase, produced acetyl-CoA and butyryl-CoA (Jadhav and Yarus, 2002b). The use of organic cofactors has been illustrated by a hairpin ribozyme that requires adenine as a cofactor during RNA reversible self-cleavage reaction (Meli et al., 2003). This may lead to a better understanding of prebiotic cofactors in primeval catalysis. Our working hypothesis is based on the demonstration of

3.4 Looking for Catalytic Partners: Cofactors and Aptamers Fig. 3.10 Secondary structures of coenzyme– RNA aptamers.

esterase activity in a nucleoside analog, the N6 -ribosyladenine (Fuller et al., 1972; Maurel and Ninio, 1987). The activity, due to the presence of an imidazole group that is free and available for catalysis, is comparable to that of histidine placed in the same conditions (Fig. 3.11). We have studied the kinetic behavior of this type of catalyst (Ricard et al., 1996) and have shown that the catalytic effect increases greatly when the catalytic element, the pseudohistidine, is placed in a favorable environment within a macromolecule (Dcout et al., 1995). Moreover, primitive nucleotides were not necessarily restricted to the standard nucleotides encountered today, and because of their replicative and catalytic properties, the N6 and N3 substituted derivatives of purines could have constituted essential links between the nucleic acid world and the protein world. Following this line of investigation we started the selection of ribozymes dependent on adenine. Starting from a heterogeneous population of RNAs with 1015 variants (a population of 1015 different molecules) we have selected five populations of RNAs capable of specifically recognizing adenine after ten generations (Meli et al., 2002). When cloned, sequenced, and modeled, the best one among the individuals of these populations, has a shape reminiscent of a claw capable of grasping adenine. Following this result we have isolated from a degenerated hairpin ribozyme, by in vitro selection, two adenine-dependent ribozymes capable of triggering reversible cleavage reactions (Fig. 3.12). One of them is also active with imidazole alone (Meli et al., 2003). A quarter of classified enzymatic reactions are redox reactions involved in various biological events, such as metabolism of biological molecules, detoxification, energy production, and regulation of protein functions. An RNA molecule binding hemin and exhibiting a peroxidase activity has been reported (Travascio et al., 1999). In this case, RNA and DNA of the same nucleotide sequence are capable of forming comparable cofactor-binding sites promoting catalysis.

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Fig. 3.11 (a) Adenine. (b) Comparison of modified adenosine and histidine. (c) Catalytic activity of adenine residue.

Fig. 3.12 Adenine-dependent hairpin ribozymes (ADHR). Arrowheads: cleavage sites. Gray dots: degenerated (mutated) sites. Vertical bars: separation between the primer binding region and the ribozyme sequence.

Recently, Tsukiji et al. (2004) reported a ribozyme that exhibits activity analogous to alcohol dehydrogenase (ADH). The ADH ribozyme was selected in vitro from a pool of random RNA sequences in the presence of NAD+ and Zn2+, based on the ability to convert a benzyl alcohol derivative to the corresponding aldehyde that was covalently attached to the 5l end of the RNA pool. In fact, the ribozyme oxidizes the benzyl alcohol to benzaldehyde in an NAD+ and Zn2+-dependent manner, with a rate acceleration at least 7 orders of magnitude higher than the uncatalyzed reaction.

3.5 The Use of Coenzymes

3.4.2 Aptazymes

Joining aptamer and ribozyme yields aptazymes. Several examples of new aptazymes have been obtained from random-sequence RNA selections. Breaker et al. merged sequences of ATP aptamers into the hammerhead ribozyme to design allosteric ribozyme controlled by ATP (Tang and Breaker, 1997). The same idea was used by Araki et al. (1998) with the hammerhead ribozyme and a flavin-specific RNA aptamer. FMN binding affects the necessary conformation for catalytic activity. The aptazymes exhibit high activation such as this ATP-dependent ligase showing 830-fold activation and a theophylline-dependent ligase that shows 1600fold activation (Robertson and Ellington, 1999, 2000). These allosteric ribozymes composed of two independent structural domains can activate kinetic reactivity and regulation providing molecular switches made of RNA (Soukup and Breaker, 1999). Furthermore, these kinds of selected ribozymes can have allosteric responses that are orders of magnitude greater than those seen for protein enzymes. The allosteric selection strategy also provides novel RNA molecular switches responding to cNMP targets. For instance Koizumi et al. (1999) demonstrated specific activation of ribozymes cleavage with cGMP and cAMP exhibiting an 5000-fold activation in the presence of the effector compounds. One of the most popular biotech applications of riboswitches is developing in gene therapy, allowing patients to take pills to switch genes on or off. On the other hand, natural aptamers are involved in riboswitch regulation of transcription termination and translation initiation in bacteria. Either FMN or thiamine acts on bacterial riboswitch RNAs located within the 5l-untranslated region (5l-UTR). Binding of the target ligand alters the conformation resulting in a change in gene expression (Winkler and Breaker, 2003). Recently Winkler et al. (2004) demonstrated that glmS ribozyme responds to glucosamine 6-phosphate (GlcN6P), a crucial compound in sugar metabolism in all three kingdoms of life and in cell wall biosynthesis in Gram-positive microorganisms, for genetic purposes. Also in prokaryotes, mRNA binds coenzyme B12 to modulate gene expression involved in cobalamine transport protein. The complexity of riboswitch structures is supported by numerous short helices and conserved sequences elements, allowing a great functional diversity in modern cells as well as possible diversity of ancient RNA functions.

3.5 The Use of Coenzymes: From the RNA World to the Protein World via Translation and the Genetic Code

Although conclusive evidence for an RNA world does not exist, there is a widely shared belief that such a world did exist. The main obstacle to the acceptance of this appealing idea is that we do not understanding the origin of nucleic acid

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replication of long templates (which would give unlimited hereditary potential: Maynard Smith and Szathmry, 1995), despite some excellent results concerning the non-enzymatic replication of short pieces of nucleic acid analogues (von Kiedrowski, 1986), and exponential, non-autonomous amplification of somewhat longer oligonucleotides (the SPREAD procedure; Luther et al., 1998). It is the successful selection for aptamers and ribozymes (see various chapters of this book), more than anything else, that convinces people of the plausibility of an RNA world. In line with an early suggestion (Szathmry, 1989, 1990b), it now seems that transition state stabilization is a crucial component of successful catalysis by ribozymes (Rupert et al., 2002). In the previous section we gave an overview of coenzyme usage by aptamers (co-ribozymes). In this final section we comment on the evolutionary significance of these findings. We think, in the footsteps of White (1976), Kornyi and Gnti (1981), and Gnti (2003a), that coenzymes have played a crucial link between the RNA and RNA–protein worlds. If we assume that coenzymes were already of significance in primordial metabolism in the RNA world, then it is difficult to see how they could have been widely replaced by any other molecules in metabolic evolution. One can replace RNA enzymes by protein enzymes (see Maynard Smith and Szathmry, 1995, for a discussion) through evolution one by one, but any one coenzyme takes part in so many different reactions that unless there is a very strong selective force, replacement is unnecessary and/or unimaginable. The fact that ribozymes can make use of cofactors has been shown in a number of experiments. Roth and Breaker (1998) demonstrated the use of histidine in one DNA enzyme and recently, Tsukiji et al. (2004) showed that a selected ribozyme can catalyze reduction of an aldehyde using the NADH cofactor. The replacement of ribozymes by protein enzymes must have been driven by the higher catalytic versatility of the latter (Benner et al., 1987), given the fact that the 20 amino acids offer significantly more functional groups than the four nucleotides (Table 3.5). Thus if such enzyme takeover indeed has taken place then modern metabolism is a “palimpsest” (Benner et al., 1989). Yet, inspection of Table 3.5 reveals that when complemented by coenzymes, the catalytic versatility of ribozymes approaches that of proteins, at least based on the diversity of functional groups. But of course it makes a big difference how often an enzyme needs such an aid. Presumably proteins in a rich metabolism need coenzymes less often and/or use them more efficiently than ribozymes. It now seems that all critical steps of protein synthesis can be catalyzed by ribozymes, including amino acid binding (Famulok and Szostak, 1992) and activation (Kumar and Yarus, 2001), peptide formation, as it happens in contemporary ribosomes; Noller et al., 1992), or in artificial systems (e.g. Illangasekare and Yarus, 1999; Sun et al., 2002). The next logical question then is how and why amino acids have been introduced into the RNA world in the first place. We cannot review the entire relevant literature here, but rather briefly present our view here that amino acids were among critical coenzymes in the RNA world (Szathmry, 1990c, 1993, 1996, 1999), and that proteins are evolved descendants of amino acid coenzymes.


Before one accepts this line of argument another question must be considered: why did evolution not raise the number of nucleotides in replicable templates? One constraint is replicability. For example, N6 -ribosyl-adenine could be a chemically feasible substitute for the amino acid histidine (Maurel and Ninio, 1987; Dcout and Maurel, 1993), but it could not be inserted into RNA by conventional replication. From the chemical point of view even replicable alternative base pairs do not look unfeasible: Piccirilli et al. (1990) have shown that alternative base pairs can be designed, synthesized, and incorporated into nucleic acids. Further additions to the genetic alphabet have also been proposed (reviewed by Szathmry, 2003). To be sure, the requirement of replicability puts a severe constraint on the diversity of these novel building blocks, but it does seem reasonable to propose that increasing the size of the genetic alphabet in templates would also increase the size of the catalytic alphabet when such molecules are used an enzymes. A theoretical analysis predicts that the increase in catalytic efficiency of ribozyme-like molecules should be slower than exponential with the number of letters in the alphabet (Szathmry, 1991, 1992). But this advance comes at a price: copying fidelity of these molecules should decrease faster than exponential, so there must be an optimal alphabet size, where the product of catalytic efficiency and copying fidelity (a measure of fitness, cf. Eigen, 1971) is maximal (reviewed in Szathmry, 2003). Despite the technical difficulties involved, it would be important to test this prediction in experimental systems. Assuming that the trade-off between replicative fidelity and enzymatic efficiency holds then the canonical solution to the problem of increasing catalytic versatility should come by the addition of coenzymes to ribozymes, rather than by increasing the alphabet size of templates (cf. Szathmry and Maynard Smith, 1997). Once again there are two options. Coenzymes can be either covalently linked to ribozymes or they can be used by reversible binding. Most experimental attempts demonstrated the first option (reviewed by Jadhav and Yarus, 2002b) but we emphasize that this is more the result of the applied selection protocol, where selection on cells is not an option. Note that it is possible to obtain nucleic acid enzymes where cofactors are reversibly rather than covalently bound (Roth and Breaker, 1998; Tsukiji et al., 2004). In general we favor the reversible alternative, for the following reason. Imagine a ribozyme (R) catalyzing a step in metabolism, using a cofactor (C). If the cofactor is bound covalently (R–C), then the following constraints hold: (1) there must be a selective charging activity, either within the ribozyme or performed by another ribozyme, specific to the first ribozyme (R + C p R–C); (2) each cofactor molecule is stuck with its own ribozyme molecule; (3) coenzymes cannot be used as cosubstrates at various points in metabolism, since they are irreversibly linked to particular ribozyme molecules. The last point is perhaps the most important one. First, it holds for the contemporary nucleotide-like coenzymes that in any particular reaction they are not catalysts but cosubstrates: only the coenzyme coupling is catalytic, when the coenzyme is recycled by other reactions restoring its original state (Gnti, 2003a). Schematically it holds that A + C p B + C* and D + C* p E + C, where C and C* are the two states of the same coenzyme (e.g. NAD and NADH), and A, B, D, and E are metabolic intermedi-

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ates. Suppose that a full reaction looks as follows: A + R–C p B + R–C*. Because of the constraints on this ribozyme R, specific for the A p B conversion, it cannot catalyze the reaction D p E that would restore the original state of its coenzyme part C*. Therefore, the only possibility for the coenzyme coupling would be a reaction such as Rl + D + R–C* p Rl + E + R–C, where Rl is a different ribozyme, specific for the D p E reaction! We conclude that efficient use of coenzymes and the realization of coenzyme coupling require in general that coenzymes be reversibly bound by most ribozymes, where the latter are specific to different individual reactions in the network. (Coenzymes in the contemporary protein world work this way.) A good question still is whether we should find coenzymes such as C mostly in free form or bound to a nucleic acid moiety Rll (which, according to the above reasoning, in general need not be a ribozyme). In the scenario of White (1976) and Jadhav and Yarus (2002a,b) the favored alternative is an R–C option. This is consonant with the fact that typically the nucleotide parts act as “handles” (H = Rll) only and do not take part in catalysis (Benner et al., 1987). A good reason for this solution is that RNA handles can be more readily recognized and bound by other RNA molecules than anything else (Szathmry, 1990c, 1993). Thus, rather than forcing all coenzyme-using ribozymes to selfcharge the non-nucleic acid part C, they can bind the H–C molecule reversibly, for which there is only one synthetic reaction per coenzyme: S + H + C p S + H–C, where S is a coenzyme synthetase. Occasionally it may happen that the handle is self-charging (H + C p H–C), but in general one would favor specific but as short handles as possible, which hence could not self-charge, therefore in general one would expect synthetases (S) to catalyze the H + C p H–C reaction. The link between these general considerations on coenzymes and the way out of the RNA world is that, according to our favored scenario, amino acids had been utilized as coenzymes of ribozymes before the genetic code was used in the coded synthesis of proteins in translation. As emphasized before, there is evidence that histidine can complement a nucleic acid enzyme (Roth and Breaker, 1998). Furthermore, various short peptides turn out to be useful in a nucleic acid context (Bergstrom et al., 2001; Viladkar, 2002). We urge for more experiments where the use of various amino acids as coenzymes of ribozymes can be tested. The rationale for amino acids (aa) acting alone or linked to handles (H–aa), favoring the latter, is similar to the one revealed above for coenzymes in general (Szathmry, 1993, 1996, 1999). One can imagine that amino acids are introduced into ribozymes via specific post-replicational modification. Suppose that a ribozyme R uses two different amino acids, aa1 and aa2, at two different points in the molecule. This would need two modifying ribozymes M1 and M2, specific for aa1 and aa2 and for R, similar to tRNA modification after transcription (Bjrk, 1995). Even if another ribozyme Rl uses the same two amino acids aa1 and aa2, a different pair of modifying ribozymes M1l and M2l would be needed to produce the functional form of Rl. Introduction of more and more amino acids into more and more ribozymes by post-replicational modification is therefore an impasse (Szathmry and Maynard Smith, 1997).


A feasible alternative is to bind amino acids reversibly. The simplest known aptamer for isoleucine (Lozupone et al., 2003) is significantly longer than a short RNA hairpin, such as the anticodon stem and loop of tRNA, although the latter would be a specific and stable handle for a linked amino acid (Szathmry, 1999), provided it would not have to self-charge. A small ribozyme self-charging Phe is 29 nucleotides long (Illangasekare and Yarus, 1999), which is also considerably longer than an anticodon stem–loop. Thus amino acids could be bound specifically and reversibly via oligonucleotide handles by the ribozymes using them. Stability and reversible binding of such handles would have favored an RNA hairpin (Szathmry, 1999). A consequence of the above reasoning is that the anticodon hairpin is likely to have been the earliest adapter. A remarkable other benefit is that genetic coding pops out for free: different handles could have been bound safely to the same amino acid, but charging of different amino acids to the same handles would have been forbidden. The origin of the unambiguous but degenerate nature of the genetic code lies in the use of such coding coenzyme handles (CCHs), according to our view (Szathmry, 1990c). The crucial difference between the CCH hypothesis and the similar, in many ways, idea of Wong (1991) about selection for RNA peptidation is that in the latter coding does not naturally emerge due to the fact that oligopeptides rather than amino acids are assumed to be linked to RNA. As discussed above, many aptamers that bind amino acids are self-charging, but this is not necessary, as shown by a series of remarkable experiments performed in Hiroaki Suga’s lab. First, they selected for a ribozyme that self-acylates at the 3l end of its integral tRNA part (Saito et al., 2001a). The leader sequence at the 5l end can be cut off by RNAse P from this pre-tRNA, which in turn is able to aminoacylate the tRNA part in trans (Saito et al., 2001b). The cognate amino acid Phe is recognized by a U/A-rich (codonic/anticodonic!) region, whereas the 3l end of tRNA is recognized by a triplet in the ribozyme. Perhaps even more interesting is the next series of experiments, in which a somewhat similar bifunctional ribozyme charging Gln was selected for (Lee et al., 2000). One domain of this ribozyme recognizes an activated glutaminyl ester and charges it to its own 5l-OH group (thus forming a covalent intermediate). The other domain binds tRNA and transfers the aminoacyl group to its 3l end. The first domain can be separated from the other and can be significantly simplified. The glutamine-binding domain can be reduced to 29 nucleotides and can aminoacylate the other domain in trans (Lee and Suga, 2001). Interestingly, a paired cognate codon:anticodon triplet is essential in this domain for the recognition of Gln. These experiments lend support to the idea that short adapter molecules (handles) could have been specifically charged by synthetase ribozymes (Szathmry, 1993, 1999). Where on the adapter this was carried out is an open question. Woese (1967) and Szathmry (1999) suggested that the primordial site for amino acid linking may have been nucleotide 37, immediately adjacent to the anticodon. Not only is this position universally modified, but several amino acids play a part in the process, in a way that makes sense in the light of scenarios about evolution of the genetic code (cf. Szathmry, 1999). As suggested by

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Wong (1991), primordial amino acids may have been N-linked, because the ester bond is too labile. Surprising suggestive evidence came to light recently. In E. coli the tRNAAsp molecule is modified in its wobble position. The queuosine residue is glutamylated by a truncated version of glutamyl-tRNA synthetase (Blaise et al., 2004; Salazar et al., 2004) that recognizes the anticodon stem and loop of tRNAAsp. This finding raises the possibility of a role of other synthetase-related molecules in tRNA modifications as well as the possibility of similar activity in the RNA world, catalyzed by ribozymes. Testing this idea again calls for further experimentation. We mentioned that, perhaps surprisingly, codons and/or anticodons tend to show up in aptamers selected to bind amino acids. Does nature want to tell us something or is this merely due to chance? The most recent analysis (Caporaso et al., 2005) favors the latter view. As was noted by Szathmry (1999), it is not only codons but also anticodons that show up more frequently than random in selected aptamers. Indeed now the data set (including aptamers for Gln, Tyr, Leu, Ile, Phe, His, Trp, Arg) seems to be biased towards anticodons, as advocated in the CCH model (Szathmry, 1993). We note that there are still many puzzles around tRNA to be sorted out. Krzyzaniak et al. (1994) found that Phe and Met can be specifically charged to their cognate tRNAs in the absence of enzymes at high pressure. Should this result be confirmed, then it shows that at least these two tRNAs are barophylic aptamers, which raises interesting thoughts in connection with the deep-sea hotspring scenario for the origin of life (reviewed by Hazen et al., 2002) and the need to select for extremophilic aptamers in general. Another odd result is that histidine and its anticodon GpUpG act similarly as catalysts in certain in vitro metabolic reactions (Shimizu, 2004). Clearly, it would be very important to test and possibly generalize these observations. We are content that many surprising results will come to light concerning aptamers and their role in evolution.

3.6 Outlook

Certain stages of evolution can only be reconstructed by inference from results of comparative and experimental analysis. The ultimate goal is to construct scenarios for certain critical transitions that are plausible and preferably make some predictions that were not part of the initial assumptions and that can be tested. Analysis of present-day living systems gives us strong hints that an RNA world in some form existed for a while. Reconstruction of that world can only be partial, and should be rather based on experiments that prove certain principles. Aptamers eminently serve this purpose for the RNA world: the experimental results of their in vitro evolution can be justifiably called breathtaking. In this chapter we have addressed two key issues: how the fitness of RNA replicators is affected by mutations, and how coenzymes could have complemented the RNA world and how they, in a literary sense, could have

References

betrayed it by paving a way out of it into the protein world. None of these issues can be taken as settled, however. Although we have put forward a detailed analysis of the function landscape of an RNA enzyme, and have used this to infer the fitness landscape of RNA replicators, many more data are needed in order to be conclusive. In a similar vein, hypotheses centered on the evolutionary role of coenzymes should be more rigorously tested. More ribozymes utilizing cofactors, including various amino acids, should be selected for. Ribozymes that charge other RNA molecules with amino acids should be analyzed to see whether codonic or anticodonic triplets predominate in the critical binding sites. Ultimately, some crucial steps in the takeover of ribozymes by protein enzymes need to be re-enacted in experiments and detailed models.

Acknowledgments

.K. is supported by a postdoctoral fellowship from the Hungarian Scientific Research Fund (OTKA D048406). M.S. is supported by Fundacin Ramn Areces (Spain). E.Sz. is supported by the Hungarian Scientific Research Fund (OTKA). The sponsorship from COST-European Cooperation in the field of Scientific and Technical Research, Internal Project number D27/0003/02 “Emergence and Selection of Networks of Catalytic Species” (URL: http://cost.cordis.lu/src/ extranet/publish/D27WGP/d27–0003–02.cfm), and from COST D27 working group (D27/0005/03) entitled “Etiology, Replication, Activity and Persistence of RNA” (URL: http://cost.cordis.lu/src/extranet/publish/D27WGP/d27–0005– 03.cfm), is very much appreciated.

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Part 2 In Vitro Selection of Target-binding Oligonucleotides


4.2 Aptamers to Nucleotides/Nucleosides/Nucleobases

4 Aptamers to Small Molecules Heiko Fickert, Iris G. Fransson, and Ulrich Hahn

4.1 Introduction

The RNA world hypothesis led to the idea that aptamers are capable of binding small molecules. According to this theory RNA molecules with catalytic properties, so called ribozymes, should also be able to bind cofactors. Moreover ribozymes might be regulated allosterically by small cellular components. Therefore, aptamers should have the capability of binding small molecules such as ATP, NAD(P), FMN, amino acids, or carbohydrates. Thus several different aptamers have been selected towards these targets. Aptamers to cellular components have also been used to mask target molecules upon binding, thereby eliminating them from further biological processes (e.g. the inhibition of cell adhesion upon binding sialyl Lewis X). Finally, aptamers have been selected to artificial small molecules. Some of these can be used as “tags” for purifying or for labeling. Consequently aptamers were selected to matrices like sephadex or to fluorescent dyes.

4.2 Aptamers to Nucleotides/Nucleosides/Nucleobases

The first aptamer to a nucleotide was isolated in 1993 by Sassanfar and Szostak (1993). The selection of this RNA aptamer to adenosine was performed with an ATP-agarose column; bound RNA was eluted with free ATP. A 40-nucleotide truncated aptamer bound ATP with a Kd of 0.7 mmol/L. Elution experiments with ATP analogs revealed that the base and the sugar of the ATP were recognized by the aptamer while the 5l phosphates did not take part in binding. To determine whether a DNA aptamer can also bind adenosine/ATP, Huizenga and Szostak (1995) selected DNA aptamers for ATP. One characterized aptamer bound adenosine with a Kd of 6 mmol/L. Just like the RNA aptamer to ATP, this DNA bound ATP via the base and the sugar and the 5l phosphate had no inThe Aptamer Handbook. Edited by S. Klussmann Copyright c 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-31059-2

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4 Aptamers to Small Molecules Fig. 4.1 Xanthine and guanine. The RNA aptamer selected for xanthine also binds guanine, while adenine, cytosine, and uracil are not bound.

fluence on binding. Furthermore, the sizes of the RNA and DNA aptamers to ATP were similar (40 versus 42, respectively), so one could draw the conclusion that aptamers (of similar complexity) for one target molecule can be isolated from both RNA and DNA pools. However, the selected RNA and DNA aptamers had significantly different sequences and fold into different structures because the DNA lacks the 2l-hydroxyl group of the RNA. This was demonstrated when the DNA version of the RNA aptamer for ATP and the RNA version of the DNA aptamer for ATP were tested for binding, respectively, and neither recognized ATP. A difference between the RNA and DNA aptamers was detected by nuclear magnetic resonance (NMR) studies of the aptamers, revealing that the DNA aptamer binds two ATP molecules, while the RNA aptamer binds just one (Lin and Patel, 1997). Kiga et al. (1998) isolated an RNA aptamer binding xanthine and guanine (Fig. 4.1). The aptamers were selected via affinity chromatography with xanthine agarose; bound RNA was eluted with free xanthine. A representative RNA aptamer was truncated to 32 nucleotides and turned out to bind xanthine with a Kd of 3.3 mmol/L. Guanine was bound by the aptamer as well, while adenenine, cytosine, and uracil were not bound. RNA aptamers that exhibited a new mode of purine recognition were published by Meli et al. (2002). The aptamer contained two relatively independent secondary structure elements, which formed a bipartite RNA structure that bound adenine with a Kd of 10 mmol/L. In this aptamer the imidazole moiety of adenine is not trapped in the binding side, as it is in other adenine/ATP-binding aptamers. Hence this aptamer could be supposed to be a cofactor-binding site in a complex ribozyme. In 2004 RNA aptamers that recognized the triphosphate of ATP were selected in the laboratory of Jack W. Szostak (Sazani et al., 2004). By employing a restrictive selection protocol containing AMP–agarose for counterselection and washing the selection column (ATP–agarose) with free AMP prior to elution of ATP-bound RNA, they could isolate aptamers that strongly interacted with the b and g phosphate. The Kd-value of a 57-nucleotide minimized aptamer was 11 mmol/L for ATP and 1700 mmol/L for AMP. In contrast to other ATP aptamers, this aptamer showed binding to GTP, UTP, and CTP, also. The aptamer was not isolated in an earlier selection for ATP aptamers, although it had an affinity comparable to other ATP aptamers. That might be due to its more complex structure. Therefore, this triphosphate-binding aptamer could first be isolated after counterselection for the simpler ATP-binding molecules.

4.3 Aptamers to Cofactors

Koizumi and Breaker (2000) published the selection of RNA aptamers for cAMP. For this selection cAMP was attached at the C8 position of agarose via a nine-atom spacer. Elution of bound RNA was achieved by washing the column with buffer containing cAMP. After four rounds of selection the enriched pool contained two classes of aptamers. Class II aptamers exhibited Kd-values of about 10 mmol/L for cAMP. These aptamers did not bind to 5l or 3l phosphorylated adenosine like ATP, 5l-AMP, or 3l-AMP. However, adenine and adenosine were bound with the same affinity as cAMP. Hence it is assumed that the aptamer recognizes the target via the adenosine and the (groups on the) ribose can influence the specificity via steric interactions. Remarkable is the fact that the aptamers seemed to interact with the part of the target molecule that faced the matrix, and did not strongly interact with the part that had greatest accessibility.

4.3 Aptamers to Cofactors

To explore a possibly more complex role of RNA in catalysis in a hypothetical RNA world several researchers have attempted to isolate aptamers for different cofactors. By the use of these cofactors, “RNA world ribozymes” containing the appropriate binding sites may have catalyzed complex cofactor-dependent reactions that are nowadays facilitated by protein enzymes. Burke and Gold (1997) reported the selection of RNA aptamers to S-adenosyl methionine (SAM). The isolated aptamers were similar to previously selected ATP (and NAD) aptamers. SAM is solely bound by the aptamer via the adenosine component with the 5l-group reaching out of the binding pocket. The authors draw the conclusion that this aptamer might be a useful starting point for the selection of cofactor-dependent ribozymes as it comprises a general binding site for adenosine-containing cofactors. In 2000 Gebhardt et al. isolated RNA aptamers to S-adenosyl homocysteine (SAH) (Gebhardt et al., 2000). They isolated different molecules including a known ATP aptamer. The best aptamer bound SAH with Kd-values of 200– 800 nmol/L and ATP with 2- to 3-fold lower affinity. The authors could show that SAH was mainly bound via the adenosine moiety with contact of the aptamer to the 5l-group. RNA aptamers to coenzyme A were first reported by Burke and Hoffman (1998). The most abundant aptamers were bound mainly to the adenosine of CoA with Kd-values for ATP of 2.4 mmol/L under selection conditions and 0.5 mmol/L under optimized conditions. Employing a counterselection with 3l AMP in a secondary selection CoA-specific aptamers could be enriched. These aptamers could not be eluted from a CoA column by AMP alone. In 2003 new aptamers for CoA were published by the same group (Saran et al., 2003). This time CoA was linked via its sulfhydryl group to the resin; in the previous selection CoA had been linked via the N6. The isolated aptamers showed no sequence or structural relation to the CoA aptamers described above, but did also

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mainly recognize the adenosine moiety. Two aptamers also specifically interacted with the 3l-phosphate of CoA. However, none of the aptamers interacted with the pantotheine group, even though it offers different hydrogen-bonding opportunities. RNA aptamers to cyanocobalamin were selected by Lorsch and Szostak (1994). A truncated aptamer bound cyanocobalamin with a Kd of 90 nmol/L and cobinamide dicyanide with a 200-fold lower affinity; adenosylcobalamin was not bound by the aptamer. The primary selection was done with selection buffer containing 1 M LiCl and 5 mmol/L MgCl2. Thus, although aptamers from the first selection were dependent on high Li+ concentrations, they did not require Mg2+ for binding. The sequence of one representative aptamer was mutagenized with a frequency of 30% to yield a pool for a secondary selection. This selection was done under the same conditions as the first one. After five cycles aptamers were enriched, then cloned and sequenced. Sequence analysis revealed a 31-nucleotide consensus sequence. Another secondary selection replacing LiCl by NaCl yielded aptamers after six selection cycles. These aptamers differed from the previously isolated ones in that they were not Li+ dependent but were strongly dependent on Mg2+. They also interacted differently with their target, as they only weakly bound to the cobinamide moiety of the molecule, and probably interacted more strongly with the dimethylbenzimidazole ribonucleotide. Furthermore the aptamers from the NaCl selection also weakly bound adenosylcobalamin. Roychowdhury-Saha et al. (2002) isolated flavin-binding RNA aptamers in a selection targeting FAD. The approximate Kd for FAD was 50 mmol/L. The aptamers recognized the isoalloxazine nucleus, hence also binding to FMN, riboflavin, and lumiflavin. The adenosine moiety was not recognized, as the aptamers could not be eluted from a FAD column with AMP alone. This was different to other selections targeted to nucleotide-containing cofactors like NAD, SAM, CoA, and FAD, which yielded “ATP aptamers.” The flavin-binding aptamers did not distinguish between the two redox states and bound over a wide pH range (4.5–8.9) making them good candidates for the selection of ribozymes utilizing flavin cofactors. Lauhon and Szostak (1995) reported the isolation of RNA aptamers that bind flavin and nicotinamide cofactors. The selection of molecules that bind riboflavin yielded aptamers after nine cycles of selection. The aptamers were proposed to form a G-quartet structure and to bind the ligands with low micromolar affinity. Also “the DNA version” of the aptamer bound the riboflavin with a somewhat lower affinity. This was the first report of a DNA aptamer and an RNA aptamer with identical sequences binding one target (containing U in the RNA instead of T in the DNA, of course). A mutagenized pool of one of these RNA aptamers was used in a secondary selection, which aimed at the isolation of redox state-specific aptamers. The pool was loaded onto a NAD column and washed with free NMNH for counterselection. Bound RNA was eluted with free NMN. After seven rounds of selection a pool was enriched which bound four times better to the oxidized form of nicotinamide. Sequence comparisons reveal that only some mutations in the stem region of a stem–loop caused the NMN specificity.

4.4 Aptamers to Amino Acids

Burgstaller and Famulok (1994) isolated RNA aptamers specific for the flavin moiety of FMN and FAD. In two separate selections targeting either FMN or FAD, the same binding motif was enriched. A truncated version of the aptamer had Kd-values of 0.5 mmol/L for FMN, 0.7 mmol/L for FAD, and 0.3 mmol/L for 7,8-dimethylalloxazin. Araki et al. (1998) fused this FMN aptamer motif to the hammerhead ribozyme to construct an allosterically regulated ribozyme. FMN binding assisted the folding of the catalytic core by stabilization of the aptamer domain. In the case of short substrates (which cannot contribute sufficient binding energy for core folding) FMN can substantially enhance the catalytic activity of the allosteric ribozyme. Wilson and Szostak (1995) isolated aptamers binding to biotin with the intention of further selecting the aptamer pool for a self-alkylating ribozyme that uses N-biotinoyl-Nl-iodoacetyl-ethylenediamine as N-alkylating substrate. One dominating aptamer and some minor aptamers were found, all of them showing remarkable folding: a pseudo-knot, which closely resembles retroviral frameshift elements (Wilson and Szostak, 1998). Compared with other aptamers, which generally have an internal bulge or a hairpin loop that is responsible for binding, the extraordinary folding of the biotin aptamers is likely due to the nature of the biotin. It has neither planar aromatic rings, nor charged functional groups, which are known moieties for aptamer binding. Further crystallographic studies done by the group confirmed the pseudo-knot folding (Nix et al., 2000).


The first aptamers that bound a free amino acid were selected in the laboratory of Michael Yarus in 1993 (Connell et al., 1993). Connell and colleagues isolated RNA aptamers for l-arginine via affinity chromatography by using l-arginyl-l-cysteine linked to thiopropyl sepharose as selection matrix. The elution of bound RNA was accomplished by washing with a solution containing free l-arginine. With Kdvalues of 0.2–0.4 mmol/L for the immobilized target and 1.0 mmol/L for free l-arginine, the aptamers exhibited affinities comparable to the two natural l-arginine-binding RNAs, Tetrahymena group I intron and HIV TAR RNA, respectively. Like the group I intron the selected aptamers also bound guanosine 5l-monophosphate, probably due to the structural similarities between arginine and guanine. Famulok and co-workers published the selection of other RNA aptamers that bind l-arginine. In 1994 first a l-citrulline-binding RNA motif was isolated which was subsequently evolved into an l-arginine binder (Famulok, 1994). The citrulline aptamers bound l-citrulline with Kd-values in the range of 62– 68 mmol/L, d-citrulline with a Kd of 180 mmol/L, and showed no affinity for citrulline analogs like arginine (Fig. 4.2). One citrulline-binding sequence was resynthesized at the DNA level with a mutation rate of 30% per base, and this doped pool was then used in several selections targeting different l-citrulline ana-

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4 Aptamers to Small Molecules Fig. 4.2 Structures of l-citrulline and l-arginine. An l-citrulline binding RNA aptamer (Kd 62 mmol/L) was evolved into an l-arginine aptamer (Kd of 60 mmol/L), which had no affinity to l-citrulline.

logs, i.e. l-arginine, l-albizziin, l-lysine, and l-glutamine. Only the selection for l-arginine and the reselection for l-citrulline succeeded. One half of the selected aptamers from the l-arginine selection showed similarity to the “parental” l-citrulline aptamer. The consensus sequence showed three changes in comparison to the starting sequence. The Kd-value for l-arginine was 60 mmol/L and that for d-arginine was 410 mmol/L; l-citrulline was not bound by the aptamer (due to counterselection with l-citrulline). This work showed that it is possible to reselect aptamers for molecules closely related to the original target as three mutations were sufficient to turn an l-citrulline aptamer into an l-arginine aptamer. Structural probing and NMR titration experiments could show for both aptamers that binding of the ligand changes the conformation of the RNA and stabilizes non-canonical base pairs (Burgstaller et al., 1995). Furthermore it was pointed out that the difference in citrulline and arginine caused the “mutation” of a uridine in the citrulline aptamer into a cytosine in the arginine aptamer. While the oxygen of the urea moiety of citrulline acts as a hydrogen bond acceptor in the interaction with the N3 of the uridine, the corresponding amino group in the arginine acts as hydrogen bond donor in the interaction with acceptor cytosine. In 1996 the structure of both aptamer–amino acid complexes could be solved by NMR spectroscopy (Yang et al., 1996). It was shown that the RNA folds around the amino acid, recognizing the target via several hydrogen bonds. With a modified selection protocol RNA aptamers that bind l-arginine with a Kd of 330 nmol/L were enriched (Geiger et al., 1996). This time a heat denaturation step in the presence of l-citrulline was included to remove RNA molecules with lower specificity and molecules, which interacted with the matrix. Also a washing step with free l-arginine was carried out prior to elution of tightly bound RNAs to eliminate aptamers with fast dissociation rates (which could be assumed to be weak binders). As several unrelated aptamers were enriched that tightly bound l-arginine, it was thus demonstrated that different RNAs can recognize the same molecule with high specificity and selectivity. A remarkable fact is the capacity of one aptamer to discriminate between the d- and l-enantiomer of arginine by 12 000-fold. Arginine-binding RNA molecules that resemble TAR of HIV were isolated by Tao and Frankel (1996). Encouraged by the fact that TAR RNA can be eluted from an arginine column with 200 mmol/L NaCl, the authors made use of an


NaCl concentration gradient to enrich arginine aptamers. Forty per cent of the selected sequences contained TAR-like arginine-binding sites, demonstrating that a SELEX (“systematic evolution of ligands by exponential amplication”) experiment can yield aptamers similar to naturally occurring RNA motifs. Also from the laboratory of Alan D. Frankel single-stranded DNA aptamers for l-arginine have been published (Harada and Frankel, 1995). The selection was performed with an NaCl concentration gradient, just like for the RNA aptamers described above, yielding aptamers that bind arginine with a Kd of 2.5 mmol/L and argininamide with a Kd of 100 mmol/L. The lower affinity for arginine resulted from the negative charge of the carboxyl group of arginine. As expected, none of the enriched single-stranded DNA molecules showed sequence similarities to known arginine-binding RNAs. The structures of the two most frequent aptamers could be resolved by NMR spectroscopy (Lin and Patel, 1996; Lin et al., 1998; Robertson et al., 2000), giving insights into DNA–amino acid (protein) interactions. Majerfeld and Yarus (1994) selected RNA sequences specifically binding aliphatic amino acids. Aptamers for l-valine bind with a Kd of 12 mmol/L, and do have at least a 10-fold lower affinity for analogous amino acids like d-valine, l-leucine, l-alanine, or l-threonine. This specificity indicates that the aptamers can discriminate for size, shape, and orientation of the aliphatic amino acid side chain. Aptamers that bind l-isoleucine (Majerfeld and Yarus, 1998) with Kd-values of 200–500 mmol/L are also highly specific, as they bind their target 5–10 times better than alanine or valine, and favor l- over d-isoleucine ninefold. It is worth noting that these aptamers require Zn2+ for binding, possibly because either the aptamer contains a “Zn2+ RNA structure” or Zn2+ isoleucine is the bound species. In a follow-up publication Lozupone and colleagues (2003) searched for the simplest RNA binding isoleucine by shortening the length of the randomized region of the SELEX pool. They started with 26, 22, and 16 randomized positions, ending up with RNA aptamers for l-isoleucine only from the 26N and 22N pool. The consensus sequence of most of the aptamers was the one already isolated in the first isoleucine aptamer selection described above (starting from a 50N pool). The size of the enriched RNA motif was 27 nucleotides, which needed 20 sequence-specific nucleotides (7 nucleotides could derive from paired constant regions). However, as no isoleucine-specific aptamers could be isolated from the 16N pool, the 27-nucleotide motif is probably the smallest isoleucine binding RNA known so far. Larger binding motifs were not isolated from the 50N pool, which can be explained by the fact that smaller motifs are more common. In 2000, the evolution of dopamine-binding RNA into a tyrosine aptamer was published by the group of Tocchini-Valentini (Mannironi et al., 2000). An aptamer that binds free dopamine with a Kd of 1.6 mmol/L (Mannironi et al., 1997) was used as the parental molecule for the selection. This sequence was mutagenized at a level of 30%. The obtained pool was selected on l-tyrosine agarose; elution of bound RNA was realized with free l-tyrosine. The best binding aptamer had a Kd of 35 mmol/L for free l-tyrosine and bound l-tryptophan and l-dopa equally well (Fig. 4.3). However, d-tyrosine, l-phenylalanine, and dopamine were bound

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Fig. 4.3 Structures of l-tyrosine, l-dopa and dopamine. An RNA aptamer for dopamine (Kd 1.6 mmol/L) was evolved into an l-tyrosine aptamer (Kd 35 mmol/L), which also binds l-dopa and l-tryptophan equally well, while dopamine is not bound by the l-tyrosine aptamer.

with an about 10-fold lower affinity, showing that this aptamer was not specific for the aromatic moiety. With the aim of selecting a recognition motif for tyrosine, which is essential for the catalytic activity of several distinct enzymes, Vianini and colleagues (2001) selected single-stranded DNA that bound l-tyrosinamide. The best binding aptamer had a Kd of 45 mmol/L for l-tyrosinamide and also bound l-phenylalaninamide with a Kd of 80 mmol/L, which is surprising as counterselection with l-phenylalaninamide was employed. The application of the aptamer against any tyrosinedependent enzyme has not yet been published. Since some RNA aptamers for amino acids (arginine, isoleucine, and tyrosine) contain coding triplets of their cognate target in the binding site, it is appropriate to discuss this fact in the context of the origin of the genetic code and the “RNA world” theory. For a detailed overview of this topic the interested reader is recommended to the articles by Knight et al. (1999), Knight and Landweber (1998), and Yarus (1998, 2000).

4.5 Aptamers to Carbohydrates

Aptamers to carbohydrates were originally isolated mainly for two different purposes. One was to use an RNA aptamer binding to the sephadex matrix as a purification tag for RNA or ribonucleoparticles (RNPs) from complex RNA mixtures. The other main purpose was to identify and block specific sugars on cell surfaces by using highly specific aptamers. For this purpose, not only the affinity but also the specificity of the aptamer is of great importance. By blocking the sugar on cell surfaces, many important mechanisms such as recognition, cell adhesion, metastasis, or viron infection could be disturbed. The Kd-values of the carbohydrate aptamers are often higher than those obtained for other macromolecules (Gold et al., 1995). Carbohydrates lack charged groups and aromatic ring structures, both motifs known to direct strong aptamer–target interactions. They only have hydroxyl groups, which are likely to form non-covalent bonds. Therefore, it is not surpris-

4.5 Aptamers to Carbohydrates

Fig. 4.4 Cellobiose, lactose, maltose, and gentiobiose. Only cellobiose gets recognized by the selected aptamers, while lactose, maltose and gentiobiose are not. The Kd-value of the best cellulose binding aptamer is 1 mmol/L.

ing that mainly lower affinities were obtained for the carbohydrate aptamers. On the other hand this makes the aptamer very selective. As the interaction relies only on hydrophobic sites and hydrogen bonding to hydroxyl and pyranose ring oxygens in the sugar, the loss of contact due to geometric changes of the target could cause the loss of a large part of the binding energy. Yang et al. (1998) isolated DNA aptamers to cellulose in order to test aptamers as potential tools capable of selective recognition of small differences in oligosaccharide motifs for experimental applications and diagnostics. They generated a set of aptamers, some of which recognized only the polymer cellulose and some of which recognized the polymer and its minimal repeating unit cellobiose. The recognition of the aptamers occurred with high selectivity and related disaccharides like lactose, maltose, and gentiobiose (see Fig. 4.4) were either not recognized at all or were recognized with only very little affinity. The dissociation constants of the aptamers varied between 1 and 100 mmol/L to cellulose. As the aptamers recognized only cellulose and its repeating unit, the authors demonstrated that aptamers to saccharides could be used to recognize a particular glycosylation (e.g. as part of a glycoprotein surface). Thus aptamers are able to discriminate among minor differences in glycoprotein modifications and therefore might be useful tools in diagnostics. A series of aptamers directed to different combinations of oligosaccharides might easily be able to discriminate between a mixture of macromolecular surfaces, including relatively minor differences in glycoprotein modifications.

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Jeong et al. (2001) isolated an RNA aptamer to sialyl Lewis X (sLex), which is a tetrasaccharide glycoconjugate of membrane proteins that acts as a ligand for the selection proteins during cell adhesion of inflammatory processes. Aberrant production of sLex is also a characteristic of cancer cell metastasis. A blocker of sLex would be useful as an anti-inflammatory or anti-metastasis drug. The selected aptamer shows high affinity in the subnanomolar to nanomolar range and higher specificity to its cognate sugar than to similar ones. It also showed a binding affinity to sLex that was similar to or even better than that of the commercially available anti-sLex antibody. The group reasoned that the aptamer could discriminate minor differences in the carbohydrates, though the binding affinity to sLex is only 5–10 times higher than that to similar Lewis groups, but it is 100 times stronger than that to dissimilar sugars such as lactose. Finally the aptamer was used in an in vitro assay showing, that the cell adhesion was inhibited in sLex-presenting HL60 cells (Jeong et al., 2001). A further example of an aptamer to cell surface carbohydrates is the modified DNA aptamer binding to sialyllactose (Masud et al., 2004). The sialyllactose is an essential receptor component of many viruses, and its block could lead to the loss of viron binding and inhibition of the initial step of viral infection. Thus the aptamer could be an antiviral drug and a useful tool for the study of cell adhesion and viral infection. To enhance binding of the aptamer, Masud et al. used modified thymidine residues. These have a cationic protonated amino group at the C5 position, which could enhance binding to sialyllactose, which has an anionic carboxyl group. The selected aptamer with the highest affinity to sialyllactose had a Kd of 5 mmol/L. The aptamer was predicted to form a three-way junction structure with a stem region containing the modified thymidines. The aptamer with the modified bases was compared with its natural twin, comprising the same sequence but with no modified thymidines. The modified aptamer showed a stronger affinity to sialyllactose than the unmodified counterpart, suggesting that the positively charged amino group of the modified thymidine residues did indeed enhance the binding ability. Finally, Srisawat et al. (2001) selected an aptamer towards sephadex G-100. Sephadex mainly consists of repeating units of glucose linked via a-1,6 glucosidic bonds. One of the selected aptamers was further characterized and showed binding to dextran B512, the soluble raw material of sephadex. However, no binding could be observed to isomaltose, isomaltotriose, or isomaltotetraose, suggesting that the minimal recognition site might consist of more than four glucose residues linked via a-1,6 linkages. The binding was very specific: other matrices like sepharose, sephacryl, cellulose, or pustulan were not recognized by the aptamer. The purpose of this RNA aptamer was to use it as an affinity tag for RNAs or RNA subunits of ribonucleoparticles (RNPs) to allow rapid purification from complex mixtures using only sephadex. The group gave primary proof of this application by purifying the selected RNA aptamer with sephadex beads from a complex mixture of human cellular RNA.

4.6 Aptamers to Natural Products


Jenison et al. (1994) selected an RNA aptamer that binds to the alkaloid theophylline with high sensitivity and high specificity. Theophylline is used as bronchodilator in the treatment of asthma, bronchitis, and emphysema, but because of its narrow therapeutic index, serum levels must be monitored carefully to avoid serious poisoning. Theophylline is also chemically similar to theobromine and caffeine (Fig. 4.5), which may be present in serum samples due to consumption of coffee or chocolate. Thus, diagnostic methods must discriminate efficiently among these compounds. A counterselection with caffeine was performed during the SELEX process. The dissociation constant of the selected aptamer was 100 nmol/L and its affinity to theophylline was remarkably 10 000 times higher than to caffeine, which differs only from theophylline by a single methyl group at nitrogen atom N7. Antibodies against theophylline show a discrimination factor of only 1000. This demonstrates that RNA molecules can exhibit an extremely high degree of ligand recognition and discrimination. Solving the structure of the theophylline aptamer by NMR spectroscopy (Zimmermann et al., 1997) yielded an explanation of the high specificity and demonstrated an unusual folding motif, including an S-turn of the backbone, a novel base zipper, and a 1-3-2 stacking motif. The theophylline is nearly completely enclosed within the aptamer and the many contacts between the ligand and the RNA direct the specificity. In particular, the presence of hydrogen bonds between the N7 position of the theophylline and the aptamer account for the high discrimination factor compared with caffeine. The methyl group at position N7 of caffeine disrupts the hydrogen bonds and steric repulsion may lead to a loss of further hydrogen bonds and therefore a loss of binding affinity. The structure is deposited at the RCSB protein databank under the code 1EHT.

Fig. 4.5 Theophylline and its closely related compounds caffeine, theobromine and 3-methylxanthine. The selected aptamer recognizes theophyllin 10 000 times better than caffeine with a Kd of 100 nmol/L for theophylline. A single C to A mutation in position 27 switches the specificity of the aptamer from theophylline to 3-methylxanthine.

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Recently Anderson and Mecozzi (2005) have reduced the theophylline aptamer from an original 33-mer to a remarkable 13-mer by using iterative computational deletion and molecular dynamics simulations. The resulting 13-mer still shows good binding affinity and specificity to theophylline by discriminating the structurally close caffeine. The theophylline aptamer has been used to allosterically regulate ribozymes. The allosteric ribozyme can either be activated or inhibited by effectors binding to the appended aptamer domain. Soukup et al. (2000) selected allosteric hammerhead ribozymes that were activated more than 3000-fold upon theophylline binding. In addition they isolated an allosteric ribozyme variant that exhibited an effector specificity change from theophylline to 3-methylxanthine by carrying only a single C to A replacement in position 27 (C27A). Robertson and Ellington (2000) designed a ribozyme ligase whose activity was enhanced up to 1600-fold in the presence of theophylline. Frauendorf and Jschke (2001) used the allosteric hammerhead ribozyme in combination with a molecular beacon for the detection and quantification of theophylline. The allosteric hammerhead ribozyme cleaves only in the presence of the effector theophylline with a regulation factor of 100. Here they used an intermolecular system by combining the ribozyme with a substrate oligonucleotide, which carried the hammerhead cleavage site, a fluorophore, and a quenching dye as terminal labels. With this system the fluorescence signal of the ribozyme cleavage rate could be correlated to the theophylline concentration in a dynamic range of 0.01–2 mmol/L theophylline. The presence of caffeine showed no influence on the cleavage reaction (Frauendorf and Jaschke, 2001). The theophylline aptamer has also been used to regulate gene expression. Harvey et al. (2002) showed inhibition of translation in the presence of theophylline by having introduced the theophylline aptamer into the 5l-untranslated region (UTR) of the target mRNA. The translation was inhibited efficiently, when the aptamer was introduced in triplicate within the 5l-UTR. Introduction within the coding region or in the 3l-UTR did not show any inhibition (Harvey et al., 2002). Additionally they demonstrated that the translation inhibition was due to the inhibition of the interaction of the small ribosomal subunit with the mRNA, thus inhibiting the formation of the 80S initiation complex. Another example of the use of the theophylline-responsive riboswitch to control gene expression was given by Suess et al. (2004) and Desai and Gallivan (2004). Suess et al. introduced the riboswitch by using the theophylline aptamer as a receptor domain for specific ligand binding and a communication module proposed to perform helix slipping. This was inserted near the ribosome-binding site (RBS) in such a way that the aptamer’s non-bound conformation interfered with the ribosome assembly. Binding of the ligand theophylline induced a structural transition and allowed ribosome binding and thus control of gene expression in Bacillus subtilis (Suess et al., 2004). Desai and Gallivan (2004) used the theophylline aptamer to show that synthetic riboswitches can also operate in the Gram-negative bacterium E. coli. They cloned the theophylline aptamer in the 5l-UTR close to the RBS of a b-galactosidase reporter gene. They could show a theophylline


dose-dependent increase of b-galactosidase activity in E. coli, while the structurally similar caffeine showed no effect. They also approved the change in selectivity for 3-methylxanthine for the previously published point mutation C27A of the theophylline aptamer, showing gene regulation upon addition of 3-methylxanthine, but not theophylline. They further determined that gene regulation occurs on the translation rather than the transcription level (Desai and Gallivan, 2004). Stojanovic et al. (2000) selected a DNA aptamer to cocaine as a fluorescent sensor. The secondary structure of the aptamer was predicted to be a three-way junction with a lipophilic binding pocket at the junction. The authors split the cocaine aptamer at a loop region into two halves, which were allowed to reassemble to form the ligand-binding pocket as shown in Fig. 4.6. By tuning the complementarity of the two strands the equilibrium could be adjusted so that the two individual subunits would be favored over the assembled heterodimer in the absence of the ligand, but shifted to the assembled form upon ligand binding. One subunit was labeled with a fluorophore and the other with a quenching dye. Upon ligand binding, the labeled ends come into close proximity resulting in signal quenching. The authors demonstrated that the sensor could reliably report cocaine concentrations in a range from 10 to 1000 mmol/L. The sensor showed excellent selectivity, as cocaine metabolites such as benzoyl ecgonine or ecgonine methyl ester were not recognized. Figure 4.7 shows the structures of cocaine and its metabolites.

Fig. 4.6 Secondary structure of the cocaine sensor. Self-assembly of the two aptamer halves in the presence of cocaine results in fluorescence quenching. F, fluorescence dye; Q, quenching dye; cocaine, cocaine-binding site. Figure according to Stojanovic et al. (2000).

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Fig. 4.7 Cocaine and its metabolites. The aptamer recognizes only the cocaine, but not its metabolites.

A year later, the same group presented an alternative sensoring system by shortening and thereby destabilizing one of the three stems. Now the stem and the three-way junction can only be formed upon ligand binding. Thus double labeling the ends of the stem with a fluorophore and a quencher would result in a quenching of the fluorescence signal upon ligand binding. The new aptameric sensor had a detection range for cocaine concentrations from 10 mmol/L to 2.5 mmol/L, similar to the previously presented one. Additional features of the new aptameric sensor are its function in magnesium-free buffers and even in serum media. The selectivity of the sensor is still the same in discriminating the cocaine metabolites. The only drawback is that for clinical or forensic applications the sensor must have a sensitivity in the picomolar range (Stojanovic et al., 2001). Another strategy was followed in using the original cocaine aptamer as a colorimetric sensor. For this the authors screened about 35 dyes that would bind to the three-way helical junction of the aptamer, but binding of the ligand cocaine would change the microenvironment of the dye chromophore by displacing it from the aptamer and thus generate a signal. It turned out that the cyanine dye diethylthiotricarbocyanine iodide displayed a significant attenuation of absorbance dependent on the cocaine concentration. By this an aptamer-based colorimetric cocaine sensor was constructed with a detection range from 0.5 to 64 mmol/L (Stojanovic and Landry, 2002). Again the cocaine metabolites were not detected by the sensor. Kato et al. (2000a) have isolated several single-stranded DNA aptamers that bind to the steroid cholic acid. Cholic acid is a metabolite of cholesterol, which belongs to the bile acids. After minimization the selected aptamers all showed a consensus secondary structure of a three-way junction, which probably forms a hydrophobic cavity in which the cholic acid could bind. The dissociation constants of the aptamers ranged from 6.4 to 67.5 mmol/L. In a follow-up publication the same group reported that the cholic acid aptamer binds even stronger to other

4.7 Aptamers to Organic or Fluorescent Dyes

bile acids like chenodeoxycholic acid, lithocholic acid, cholic acid methyl ester, or even trans-androsterone, indicating that the binding action is governed merely by hydrophobic interaction (Kato et al., 2000b). It was suggested that binding depends only on the size or shape of the steroids and not on specific interactions between polar functional groups. This was further confirmed by mutagenesis analyses of the aptamers, suggesting that the binding does not require a sequence-specific tertiary structure but proper folding of the secondary structure. Driven by the publication of Kato et al. (2000b), Stojanovic et al. (2003) used their cocaine aptamer with its three-way junction-binding site with various small modifications to sense other hydrophobic compounds like metabolic steroids characteristic for particular diseases in human urine (Stojanovic et al., 2003). For this they selected a variety of four different aptamers, all based on the cocaine aptamer. A phosphorthioate group was introduced at the three-way junction and functionalized with a thiol-reactive fluorophore. In the absence of the ligand, the fluorophore enters the three-way junction of the aptamer and gets quenched by the guanosine residues. In the present of the ligand, the fluorophore gets displaced by ligand binding, resulting in a threefold increase of fluorescence. The constructed variants of the aptamer have introduced either a mismatch in different positions on the stem and/or the position of the fluorophore was varied. The different aptamers had small differences in response to different metabolites and Stojanovic et al. could demonstrate by using a panel of four different aptamers that unspiked human urine could be distinguished from urine spiked with deoxycorticosterone 21-glucose or with dehydroisoandrosterone-3-sulfate, both metabolites characteristic for diseases (Stojanovic et al., 2003).


Ellington and Szostak (1990, 1992) were the first to demonstrate that singlestranded polynucleotides can have specific ligand-binding properties and can be selected by in vitro selection (SELEX) from a randomly synthesized RNA (Ellington and Szostak, 1990) or DNA pool (Ellington and Szostak, 1992) (Table 4.1). For the proof of principle they chose organic dyes as one of the first targets for aptamers, because these can mimic metabolic cofactors. Cibacron Blue 3GA, for example, binds tightly to the NAD-binding site of many dehydrogenases. First, they demonstrated the selection of RNA aptamers specific for six different but closely related dyes and showed that some of the selected aptamers show high specificity towards the selected dye ligand, but not to the other dyes (Ellington and Szostak, 1990). By repeating the experiment with a DNA pool and towards the same organic dye targets, they demonstrated that single-stranded DNA had the same capacity to bind the ligands selectively and specifically (Ellington and Szostak, 1992). No sequence similarities between RNA and DNA aptamers selected towards the same organic dye could be found. The authors also revealed that the aptamer binding is sequence specific, as binding of the complementary

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110

4 Aptamers to Small Molecules Table 4.1 Aptamers selected to bind organic dyes Ligand

Protein Type Application purpose Data Bank of entry aptamer

Reference

Cibacron Blue 3GA, Reactive Red 120, Reactive Yellow 86, Reactive Brown 10, Reactive Green 19 and Reactive Blue 4

–

RNA

Proof of principle of RNA aptamers

Ellington and Szostak, 1990

–

DNA

Proof of principle of DNA aptamers

Ellington and Szostak, 1992

Sulforhodamine B

–

DNA

Binding and redo reaction of dihydrotetramethyl-rosamine

Wilson and Szostak, 1998

Sulforhodamine B, Fluorescein

–

RNA

Double labeling of RNA

Holemann et al., 1998

Hoechst dye 33258

–

RNA

Control of expression

Werstruck and Green, 1998

RNA

Chromophore assisted laser-mediated inactivation (CALI) of expression

Grate and Wilson, 1999

Control of expression

Grate and Wilson, 2001

Increase of fluorescence quantum yield upon binding

Babendure et al., 2003

Signaling sensor

Stojanovic and Kolpashchikov, 2004

1F1T

X-ray structure

Baugh et al., 2000

1Q8N

NMR-structure

Flinders et al., 2004

Malachite Green

sequence as well as the RNA version of the selected DNA aptamers failed to bind the ligand. In subsequent years several aptamers to fluorescent dyes were selected for different reasons. The groups of Wilson and Szostak isolated a DNA aptamer to sulforhodamine B (Wilson and Szostak, 1998), two RNA aptamers, one to sulforhodamine B and one to fluorescein (Holeman et al., 1998) and an RNA aptamer to malachite green (Grate and Wilson, 1999). Werstuck and Green (1998) isolated an RNA aptamer to the Hoechst dye 33258. The final goal of all the fluorescent dye-specific aptamers isolated was to have a tool for labeling RNA in situ, in vitro, and/or in vivo similar to the green fluorescent protein. The possibilities for the application of the selected aptamers are manifold.


Wilson and Szostak (1998) tried to isolate a DNA aptamer that catalyzes the redox reaction of the colorless compound dihydrotetramethylrosamine to the fluorescent product tetramethylrosamine. A fluorescent signal would thus appear upon ligand binding. They started by isolating small DNA aptamers with high affinity to sulforhodamine B, which closely resembles dihydrotetramethylrosamine and tetramethylrosamine, but does not oxidize spontaneously. Several clones were found within the selected pool but they harbored only weak redox reactivity. In parallel Holeman et al. (1998) isolated RNA aptamers specific for sulforhodamine B and for fluorescein and showed in an in vitro test that these fluorophore aptamers could discriminate between the two fluorescent dyes and specifically localize each fluorophore tagged to immobilized beads (Holeman et al., 1998). This opens the possibility of tagging RNA transcripts with the isolated aptamers for double labeling or fluorescence resonance energy transfer (FRET) studies in vitro and in vivo. Werstuck and Green (1998) demonstrated the control of gene expression in living cells through small molecule–RNA interaction. They used an RNA aptamer specific for the Hoechst dye 33258 to regulate translation of a gene by having the aptamer inserted within the 5l-UTR of mammalian b-galactosidase. On addition of H33342, a closely related drug, which is non-toxic and 10 times more cell permeable than the mentioned dye, the group showed that in Chinese hamster ovary (CHO) cells the expression of b-galactosidase was indeed inhibited, while in the absence of the drug no effect of the aptamer on gene expression or rather the translation could be observed. Grate and Wilson (2001) conducted a similar experiment by insertion of the malachite green aptamer into the 5l-UTR of a cyclin gene transcript in S. cerevisiae. By this they affected the expression of the encoded protein and thus allowed control of the yeast cell cycle, regulated via the presence or absence of the ligand. They even found that the ligand-mediated effects on expression did not involve changes in transcript concentration but rather transcript translatability, as shown by one-dimensional NMR spectroscopy. It is hypothesized that ligand binding causes the aptamer to form a stable complex that may resist association with the small ribosomal subunit and thereby inhibit translation of the message. One of the most extensively analyzed fluorescent dye-specific aptamers is the malachite green RNA aptamer, which was originally isolated by Grate and Wilson (1999) for the use of chromophore-assisted laser-mediated inactivation (CALI) of RNA transcripts. The binding site of the minimal aptamer was identified as an asymmetric internal bulge flanked by RNA duplexes. Introduction of the aptamer bulge into the signal-recognition particle from Canis familiaris showed in in vitro experiments that the RNA was specifically cleaved upon laser radiation between uridine-25 and adenosine-26 of the bulge. Grate and Wilson suggested that a possible in vitro and in vivo application of the aptamer would be the insertion of the bulge into the 5l-UTR of mRNA. Irradiation of the sample leads to the separation of the 5l-cap followed by downregulation of gene expression due to reduction of the transcript stability and translatability.

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Another strategy using fluorophore-binding aptamers is to enhance the quantum yield upon binding of the fluorophore to the aptamer. Triphenylmethane dyes such as malachite green normally have low quantum yields for fluorescence due to easy vibrational de-excitation. Upon aptamer binding the dye could be stabilized in a planar, more fluorescent conformation. Babendure et al. (2003) analyzed the malachite green aptamer complex and indeed observed a significant increase of fluorescence quantum yield up to 2300-fold upon malachite green binding. In an additional experiment they were able to further enhance the fluorescence by cloning the malachite green aptamer in tandem, resulting in increased levels of fluorescence without quenching one another. Stojanovic and Kolpashchikov (2004) constructed sensors that were capable of signaling the presence of small molecules. They used the malachite green aptamer as a “signaling domain” coupled to another aptamer, which binds the analyte as “recognition domain” connected via a “communicating stem” to build an aptameric sensor, which could be used for intracellular imaging. On binding the analyte in the recognition domain, the otherwise unstable connecting stem becomes stable, which in term stabilizes the binding of the malachite green fluorophore of the signaling domain. As recognition domain, the group tested a DNA aptamer that binds ATP, and two further RNA aptamers, one binding FMN and one binding theophylline. All three sensor constructs showed good response. The malachite green–FMN construct showed the most impressive signaling. The latter two constructs, consisting only of RNA, could be expressed in cells and used as sensors for intracellular imaging. All these examples open the way to in situ, in vitro, and in vivo fluorescent tagging and imaging of important messenger, ribosomal, and microRNAs to analyze their metabolism, trafficking, and function within living cells.

4.8 The Chimeric Approach for Aptamer Selection

Manimala et al. (2004) demonstrated an alternative method of selecting an aptamer to small molecules. Instead of isolating the aptamer directly towards the desired ligand, they used a synthetic bis-boronic acid receptor. The receptor binds the ligand, in this case citrate and dl-tartrate. The aptamer is then isolated towards the receptor–ligand complex. Thus Manimala et al. isolated an aptamer that binds to the smallest organic ligand so far known. By using this chimeric approach, novel biosensors for very small ligands can be developed.

4.9 Conclusion

Aptamers may serve as powerful binders to small molecules for many possible applications, e.g. as “domains” for allosteric ribozymes, for biosensors, for artifi-

References

cal riboswitches or fluorescence tagging. In addition to their ability to efficiently interact with polypeptides, other bio-macromolecules, viruses, or even cells, aptamers are not only able to recognize small molecules with high specificity but also to differentiate between marginal differences within these small molecules. Aptamers are able, for example, to distinguish between caffeine and theophylline or the two highly similar amino acids citrulline and arginine.

Acknowledgments

We are grateful to Michael J. Knauer for reading the manuscript.

References Anderson, P. C., Mecozzi, S. (2005). Unusually short RNA sequences: design of a 13mer RNA that selectively binds and recognizes theophylline. J Am Chem Soc 127, 5290–5291. Araki, M., Okuno, Y., Hara, Y., Sugiura, Y. (1998). Allosteric regulation of a ribozyme activity through ligand-induced conformational change. Nucleic Acids Res 26, 3379– 3384. Babendure, J. R., Adams, S. R., Tsien, R. Y. (2003). Aptamers switch on fluorescence of triphenylmethane dyes. J Am Chem Soc 125, 14716–14717. Baugh, C., Grate, D., and Wilson, C. (2000). 2.8 A crystal structure of the malachite green aptamer. J Mol Biol 301, 117–128. Burgstaller, P., Famulok, M. (1994). Isolation of RNA aptamers for biological cofactors by in vitro selection. Angew Chem, Int Ed Engl 33, 1084–1087. Burgstaller, P., Kochoyan, M., Famulok, M. (1995). Structural probing and damage selection of citrulline- and arginine-specific RNA aptamers identify base positions required for binding. Nucleic Acids Res 23, 4769–4776. Burke, D. H., Gold, L. (1997). RNA aptamers to the adenosine moiety of S-adenosyl methionine: structural inferences from variations on a theme and the reproducibility of SELEX. Nucleic Acids Res 25, 2020–2024. Burke, D. H., Hoffman, D. C. (1998). A novel acidophilic RNA motif that recognizes coenzyme A. Biochemistry 37, 4653–4663.

Connell, G. J., Illangesekare, M., Yarus, M. (1993). Three small ribooligonucleotides with specific arginine sites. Biochemistry 32, 5497–5502. Desai, S. K., Gallivan, J. P. (2004). Genetic screens and selections for small molecules based on a synthetic riboswitch that activates protein translation. J Am Chem Soc 126, 13247–13254. Ellington, A. D., Szostak, J. W. (1990). In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822. Ellington, A. D., Szostak, J. W. (1992). Selection in vitro of single-stranded DNA molecules that fold into specific ligand-binding structures. Nature 355, 850–852. Famulok, M. (1994). Molecular recognition of amino acids by RNA-aptamers: An L-citrulline binding RNA motif and its evolution into an L-arginine binder. J Am Chem Soc 116, 1698–1706. Flinders, J., DeFina, S.C., Brackett, D.M., Baugh, C., Wilson, C., and Dieckmann, T. (2004). Recognition of planar and nonplanar ligands in the malachite green-RNA aptamer complex. Chembiochem 5, 62–72. Frauendorf, C., Jaschke, A. (2001). Detection of small organic analytes by fluorescing molecular switches. Bioorg Med Chem 9, 2521–2524. Gebhardt, K., Shokraei, A., Babaie, E., Lindqvist, B. H. (2000). RNA aptamers to Sadenosylhomocysteine: kinetic properties, divalent cation dependency, and comparison with anti-S-adenosylhomocysteine antibody. Biochemistry 39, 7255–7265.

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4 Aptamers to Small Molecules Geiger, A., Burgstaller, P., von der Eltz, H., Roeder, A., Famulok, M. (1996). RNA aptamers that bind L-arginine with sub-micromolar dissociation constants and high enantioselectivity. Nucleic Acids Res 24, 1029– 1036. Gold, L., Polisky, B., Uhlenbeck, O., Yarus, M. (1995). Diversity of oligonucleotide functions. Annu Rev Biochem 64, 763–797. Grate, D., Wilson, C. (1999). Laser-mediated, site-specific inactivation of RNA transcripts. Proc Natl Acad Sci USA 96, 6131–6136. Grate, D., Wilson, C. (2001). Inducible regulation of the S. cerevisiae cell cycle mediated by an RNA aptamer-ligand complex. Bioorg Med Chem 9, 2565–2570. Harada, K., Frankel, A. D. (1995). Identification of two novel arginine binding DNAs. EMBO J 14, 5798–5811. Harvey, I., Garneau, P., Pelletier, J. (2002). Inhibition of translation by RNA-small molecule interactions. RNA 8, 452–463. Holeman, L. A., Robinson, S. L., Szostak, J. W., Wilson, C. (1998). Isolation and characterization of fluorophore-binding RNA aptamers. Fold Des 3, 423–431. Huizenga, D. E., Szostak, J. W. (1995). A DNA aptamer that binds adenosine and ATP. Biochemistry 34, 656–665. Jenison, R. D., Gill, S. C., Pardi, A., Polisky, B. (1994). High-resolution molecular discrimination by RNA. Science 263, 1425–1429. Jeong, S., Eom, T., Kim, S., Lee, S., Yu, J. (2001). In vitro selection of the RNA aptamer against the Sialyl Lewis X and its inhibition of the cell adhesion. Biochem Biophys Res Commun 281, 237–243. Kato, T., Takemura, T., Yano, K., Ikebukuro, K., Karube, I. (2000a). In vitro selection of DNA aptamers which bind to cholic acid. Biochim Biophys Acta 1493, 12–18. Kato, T., Yano, K., Ikebukuro, K., Karube, I. (2000b). Interaction of three-way DNA junctions with steroids. Nucleic Acids Res 28, 1963–1968. Kiga, D., Futamura, Y., Sakamoto, K., Yokoyama, S. (1998). An RNA aptamer to the xanthine/guanine base with a distinctive mode of purine recognition. Nucleic Acids Res 26, 1755–1760. Knight, R. D., Landweber, L. F. (1998). Rhyme or reason: RNA-arginine interactions and the genetic code. Chem Biol 5, R215–220.

Knight, R. D., Freeland, S. J., Landweber, L. F. (1999). Selection, history and chemistry: the three faces of the genetic code. Trends Biochem Sci 24, 241–247. Koizumi, M., Breaker, R. R. (2000). Molecular recognition of cAMP by an RNA aptamer. Biochemistry 39, 8983–8992. Lauhon, C. T., Szostak, J. W. (1995). RNA aptamers that bind flavin and nicotinamide redox cofactors. J Am Chem Soc 117, 1246– 1257. Lin, C. H., Patel, D. J. (1996). Encapsulating an amino acid in a DNA fold. Nat Struct Biol 3, 1046–1050. Lin, C. H., Patel, D. J. (1997). Structural basis of DNA folding and recognition in an AMPDNA aptamer complex: distinct architectures but common recognition motifs for DNA and RNA aptamers complexed to AMP. Chem Biol 4, 817–832. Lin, C. H., Wang, W., Jones, R. A., Patel, D. J. (1998). Formation of an amino-acid-binding pocket through adaptive zippering-up of a large DNA hairpin loop. Chem Biol 5, 555– 572. Lorsch, J. R., Szostak, J. W. (1994). In vitro selection of RNA aptamers specific for cyanocobalamin. Biochemistry 33, 973–982. Lozupone, C., Changayil, S., Majerfeld, I., Yarus, M. (2003). Selection of the simplest RNA that binds isoleucine. RNA 9, 1315– 1322. Majerfeld, I., Yarus, M. (1994). An RNA pocket for an aliphatic hydrophobe. Nat Struct Biol 1, 287–292. Majerfeld, I., Yarus, M. (1998). Isoleucine: RNA sites with associated coding sequences. RNA 4, 471–478. Manimala, J. C., Wiskur, S. L., Ellington, A. D., Anslyn, E. V. (2004). Tuning the specificity of a synthetic receptor using a selected nucleic acid receptor. J Am Chem Soc 126, 16515–16519. Mannironi, C., Di Nardo, A., Fruscoloni, P., Tocchini-Valentini, G. P. (1997). In vitro selection of dopamine RNA ligands. Biochemistry 36, 9726–9734. Mannironi, C., Scerch, C., Fruscoloni, P., Tocchini-Valentini, G. P. (2000). Molecular recognition of amino acids by RNA aptamers: the evolution into an L-tyrosine binder of a dopamine-binding RNA motif. RNA 6, 520–527.

References Masud, M. M., Kuwahara, M., Ozaki, H., Sawai, H. (2004). Sialyllactose-binding modified DNA aptamer bearing additional functionality by SELEX. Bioorg Med Chem 12, 1111–1120. Meli, M., Vergne, J., Decout, J. L., Maurel, M. C. (2002). Adenine–aptamer complexes: a bipartite RNA site that binds the adenine nucleic base. J Biol Chem 277, 2104–2111. Nix, J., Sussman, D., Wilson, C. (2000). The 1.3 A crystal structure of a biotin-binding pseudoknot and the basis for RNA molecular recognition. J Mol Biol 296, 1235–1244. Robertson, M. P., Ellington, A. D. (2000). Design and optimization of effector-activated ribozyme ligases. Nucleic Acids Res 28, 1751–1759. Robertson, S. A., Harada, K., Frankel, A. D., Wemmer, D. E. (2000). Structure determination and binding kinetics of a DNA aptamer-argininamide complex. Biochemistry 39, 946–954. Roychowdhury-Saha, M., Lato, S. M., Shank, E. D., Burke, D. H. (2002). Flavin recognition by an RNA aptamer targeted toward FAD. Biochemistry 41, 2492–2499. Saran, D., Frank, J., Burke, D. H. (2003). The tyranny of adenosine recognition among RNA aptamers to coenzyme A. BMC Evol Biol 3, 26. Sassanfar, M., Szostak, J. W. (1993). An RNA motif that binds ATP. Nature 364, 550–553. Sazani, P.L., Larralde, R., and Szostak, J.W. (2004). A small aptamer with strong and specific recognition of the triphosphate of ATP. J Am Chem Soc 126, 8370–8371. Soukup, G. A., Emilsson, G. A., Breaker, R. R. (2000). Altering molecular recognition of RNA aptamers by allosteric selection. J Mol Biol 298, 623–632. Srisawat, C., Goldstein, I. J., Engelke, D. R. (2001). Sephadex-binding RNA ligands: rapid affinity purification of RNA from complex RNA mixtures. Nucleic Acids Res 29, E4. Stojanovic, M. N., Kolpashchikov, D. M. (2004). Modular aptameric sensors. J Am Chem Soc 126, 9266–9270. Stojanovic, M. N., Landry, D. W. (2002). Aptamer-based colorimetric probe for cocaine. J Am Chem Soc 124, 9678–9679. Stojanovic, M. N., de Prada, P., Landry, D. W. (2000). Fluorescent sensors based on apta-

mer self-assembly. J Am Chem Soc 122, 11547–11549. Stojanovic, M. N., de Prada, P., Landry, D. W. (2001). Aptamer-based folding fluorescent sensor for cocaine. J Am Chem Soc 123, 4928–4931. Stojanovic, M. N., Green, E. G., Semova, S., Nikic, D. B., Landry, D. W. (2003). Crossreactive arrays based on three-way junctions. J Am Chem Soc 125, 6085–6089. Suess, B., Fink, B., Berens, C., Stentz, R., Hillen, W. (2004). A theophylline responsive riboswitch based on helix slipping controls gene expression in vivo. Nucleic Acids Res 32, 1610–1614. Tao, J., Frankel, A. D. (1996). Arginine-binding RNAs resembling TAR identified by in vitro selection. Biochemistry 35, 2229–2238. Vianini, E., Palumbo, M., Gatto, B. (2001). In vitro selection of DNA aptamers that bind L-tyrosinamide. Bioorg Med Chem 9, 2543– 2548. Werstuck, G., Green, M. R. (1998). Controlling gene expression in living cells through small molecule-RNA interactions. Science 282, 296–298. Wilson, C., Szostak, J. W. (1995). In vitro evolution of a self-alkylating ribozyme. Nature 374, 777–782. Wilson, C., Szostak, J. W. (1998). Isolation of a fluorophore-specific DNA aptamer with weak redox activity. Chem Biol 5, 609–617. Yang, Q., Goldstein, I. J., Mei, H. Y., Engelke, D. R. (1998). DNA ligands that bind tightly and selectively to cellobiose. Proc Natl Acad Sci USA 95, 5462–5467. Yang, Y., Kochoyan, M., Burgstaller, P., Westhof, E., Famulok, M. (1996). Structural basis of ligand discrimination by two related RNA aptamers resolved by NMR spectroscopy. Science 272, 1343–1347. Yarus, M. (1998). Amino acids as RNA ligands: a direct-RNA-template theory for the code’s origin. J Mol Evol 47, 109–117. Yarus, M. (2000). RNA-ligand chemistry: a testable source for the genetic code. RNA 6, 475–484. Zimmermann, G. R., Jenison, R. D., Wick, C. L., Simorre, J. P., Pardi, A. (1997). Interlocking structural motifs mediate molecular discrimination by a theophylline-binding RNA. Nat Struct Biol 4, 644–649.

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5 Aptamers to Antibiotics

5 Aptamers to Antibiotics Christina Lorenz and Rene Schroeder

5.1 Introduction

The idea that RNA molecules are able to fold and form binding pockets to accommodate small molecules is now 20 years old. The story began with the discovery of self-splicing group I introns, which require a guanosine as cofactor to initiate the splicing reaction (Bass and Cech, 1984; Cech, 1990). The binding pocket of the guanosine cofactor is located in the core of the ribozyme and upon binding guanosine forms hydrogen bonds with a G-C pair (Michel et al., 1989). The observation that the amino acid arginine, which has similar hydrogen bonding capabilities as guanosine, was a competitive inhibitor of group I intron splicing was very exciting. This was the first evidence that RNA molecules can specifically bind amino acids. Even more unexpected was that the guanosine-binding pocket, and thus also the arginine-binding pocket, resides within arginine codons (Yarus, 1988). In the 1960s, when the genetic code was being studied, efforts were undertaken to fold codons in the RNA that would be able to recognize the side chains of amino acids (Watson, 1993). This, of course, failed and led to the postulation of adapter molecules for decoding, the tRNAs. Also in the 1980s, evidence for the pivotal role that the RNA components of the ribosome play during translation, accumulated due to the work of Harry Noller and co-workers, who showed that antibiotics, which inhibit translation, interacted with the ribosomal RNA (Moazed and Noller, 1987). In 1990, we reported that the antibiotic streptomycin, which has a guanidinium group like the group I intron cofactor guanosine (Table 5.1), was an inhibitor of group I intron splicing via competition with the guanosine cofactor (von Ahsen and Schroeder, 1990, 1991). The following year we observed that a large number of aminoglycoside antibiotics were strong non-competitive splicing inhibitors (von Ahsen et al., 1991), which had the ability to displace metal ions otherwise essential for the catalytic activity of the ribozyme (Hoch et al., 1998). The observation that many of the antibiotics that inhibit translation were also inhibitors of group I introns and other ribozymes clearly established antibiotics as a class of RNA-binding small molecules The Aptamer Handbook. Edited by S. Klussmann Copyright c 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-31059-2

5.1 Introduction

(reviewed in Wallis and Schroeder, 1997). Today, the number and diversity of small ligands that bind RNA is very complex and requires systematic analysis (Hermann, 2003). It was recently discovered that a number of small metabolites, such as cobalamin, amino acids, and nucleosides, have naturally occurring RNAbinding sites in the 5l-UTRs of their corresponding genes. Binding of the metabolite induces structural changes within these RNA stretches, resulting in the functional modulation of transcription and translation (Mandal and Breaker, 2004). These phenomena have been termed riboswitches and the RNAs that fold to form specific binding pockets for the small metabolites are naturally occurring RNA aptamers. As soon as antibiotics were recognized as RNA-binding compounds, the most interesting question was how small molecules bind to RNA and how they affect Table 5.1 Chemical structures of the antibiotics for which aptamers have been selected and the secondary structures of the RNA aptamers Antibiotic

Aptamer

Streptomycin

Streptomycin aptamer -

Neomycin

-

G

NH2

-

NH

N H

OH

-

HO

OH

U 20 G U C U A GA CU GCCC G CUGG CGGGG A C U A C 30 40 C -

NH

O O

A UU

-

O HO HOC

C G 5’ - G G A U C 3’ - C C U A G

NH

-

H2N

O OH

Bachler, 1999

10

H N

HO HO

Reference

Neomycin aptamer

Wallis, 1995

NH2 O H2N

NH2

20

OH

O

H2N

OH

-

O

HO O

HO HO

H2N

O O

G 10 5’ - N N N N G G G C A A 3’ - N N N N U U U G A A

-

HO HO

NH2

Tobramycin

Tobramycin minimal aptamer J6f1 Hamasaki, 1997 OH

-

HO

NH2

H2N

Viomycin

Viomycin aptamer vio112 OH H N

N H

NH2 O

NH

N H

NH

NH2 O

20

-

HO

O NH

10 G A A U 5’ - N N N N G C A NN N N C GCU A GG

--

O

OH O H N

-

H N

O HN

-

O

H N

H2N

-

H2N

-

HO O

-

O

-

HO

10 20 U A A U 5’ - G G C U U A G U G C G A G G U UA 3’ - C C G A G U C G U G C U C C A U CG A 40 30

O

HO H2N O

-

NH2

G A U C C -3’

Wallis, 1997

117

118

5 Aptamers to Antibiotics Table 5.1 Chemical structures of the antibiotics for which aptamers have been selected and the secondary structures of the RNA aptamers (continue) Antibiotic

Aptamer

Chloramphenicol

Chloramphenicol aptamer A

AA

AAA

A

G GA C G U U CU GU AA G

-

A

-

-

AAA A AA

50

O

CA GU G GU C A C

-

HO

N

5’ - G G G A U C 3’ - C C C U A G

-

O

-

Cl O

Burke, 1997 20

-

10

Cl

-

OH

Reference

30

40

Tetracycline

Tetracycline minimal aptamer cb28 Berens, 2001 minimer L2

N

HO 6

5

4

10

11

12

1

OH

O

OH OH O

OH CONH2

10

C G A U C U G GA

30 G

P3

A AGGUG

A G A

-

7

U U 20 U A P2 G A C AC U A C A A A A U C P1 C G G 5’

UCCA C C

A G G C C

50

G G U

A G C

40 A

U U

L3

60

3’

its structure. In the early 1990s, when this question arose, it seemed like an impossible mission to study RNA–antibiotic interactions at high resolution, because the most interesting targets, the ribosomal RNAs and the ribozymes, are very large and crystal structure determination of RNA molecules was still in its infancy. Strategies to circumvent the RNA size problem were either to design or to select smaller RNAs with high antibiotic binding affinity. The basis for the design of small RNAs was the modular nature of RNAs. Large RNAs are composed of independent domains, which can fold autonomously and still retain their binding properties (Murphy and Cech, 1993). For example, Seth Stern and colleagues showed that it was possible to dissect the aminoglycoside-binding site of the 16S ribosomal RNA, the so called A-site or decoding site (Purohit and Stern, 1994). This domain was extensively used to determine the structure of ribosomal Asite RNA from the small subunit bound to aminoglycosides (Vicens and Westhof, 2003; Yoshizawa et al., 1999). The second strategy used to obtain small RNAs that recognize antibiotics, was to de novo isolate small RNA aptamers out of a random pool via SELEX (systematic evolution of ligands by exponential enrichment). This review aims to describe the aptamers binding to antibiotics and to show what these aptamers have been used for.

5.3 Aptamers to Tetracyclines

5.2 RNA-binding Antibiotics

The chemical diversity of RNA-binding antibiotics is astonishing, showing what great effort nature has undertaken to produce these molecules (Table 5.1). Like most secondary metabolites they are complex compounds, whose synthesis requires sophisticated biosynthetic pathways. All RNA-binding antibiotics target the ribosome and most often they interact directly with the ribosomal RNAs. Once the structure of the ribosome was known, it became possible to study cocrystals of antibiotics with the ribosome and thus to analyze the structural basis for their recognition and their mode of action. Aptamers have been selected for a subclass of these translation-inhibiting antibiotics. We will discuss aptamers that bind to the aminoglycosides streptomycin and neomycin, the peptide antibiotic viomycin, chloramphenicol, and tetracycline. Streptomycin and aminoglycosides such as neomycin and paromomycin interact with the 16S ribosomal A-site RNA and interfere with decoding (Carter et al., 2000). Tetracycline was shown to have several binding sites on the ribosome (Oehler et al., 1997; Carter et al., 2000; Anokhina et al., 2004). Chloramphenicol interacts with the large subunit ribosomal RNA and interferes with peptide bond formation. Viomycin’s mode of action is still elusive, but it probably interferes with the dynamics of both subunits (Wank and Wallis, 2001). For a recent review that discusses the mode of action of these antibiotics on the bacterial ribosome, see Yonath and Bashan (2004).


Tetracycline is a structurally flat molecule composed of a four-ring system (Table 5.1). One side carries mainly hydrophilic functional groups that are able to form ionic interactions, while the other side has a hydrophobic face, thus allowing nonpolar and stacking interactions. Being an antibiotic of low toxicity and high cell permeability, tetracyclines are widely used as therapeutic agents against common pathogens. They act as inhibitors of prokaryotic translation by interfering with binding of the aminoacyl-tRNA to the ribosomal A-site (Epe et al., 1987; Spahn and Prescott, 1996). The affinity to the small ribosomal subunit, where, among other non-specific binding sites, they interact with the 16S rRNA, is in the low micromolar range. The crystal structure of the 30S subunit complexed with the antibiotic indicates that two molecules of tetracycline are present, one near the acceptor site for the aminoacyl-tRNA and the other in a region involved in the regulation of translation accuracy (Brodersen et al., 2000). Selection In order to study the mode of interaction with RNA, aptamers recognizing tetracycline were isolated by in vitro selection. A typical selection procedure is shown

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Fig. 5.1 In vitro selection scheme. A DNA pool containing a central region with random sequences flanked by defined sequences for primer binding and a T7 RNA polymerase promotor is transcribed into an RNA pool (1), which is applied to selection procedures, most often affinity chromatography (2). The ligand against which it is selected is covalently attached to a column resin of choice.

Non-binding RNA molecules are washed off the column (3) and specifically ligand-binding RNAs are eluted with the ligand (4). These RNAs are then reverse-transcribed (5) and PCR amplified (6), resulting in a selected DNA pool, which is submitted to consecutive rounds of selection until the desired affinity is obtained. Then, the selected DNA pool is cloned and sequenced.

in Fig. 5.1. Thereby, small RNAs were selected whose complexes with tetracycline had similar Kd-values to those of tetracycline with the small ribosomal subunit. The initial RNA pool consisted of random sequences of 74 nucleotides in length flanked by constant regions necessary for transcription, reverse transcription, and amplification. For the selection process itself, tetracycline was covalently attached to epoxy-activated sepharose on a column and specifically bound RNA was eluted stepwise with increasing concentrations of tetracycline. After 15 rounds of selection, several aptamers were isolated, and one of them, cb28, with a Kd of 1 mmol/L was further investigated. A secondary structure model of the RNA aptamer with and without tetracycline was established via lead cleavage and dimethylsulfate (DMS) modification experiments and a change of secondary structure upon tetracycline binding was ascertained (Berens et al., 2001). Upon tetracycline binding, the stem–loop P3–L3 with joining regions J1/2 and J2/3 change conformation. Since several regions of the aptamer showed no change in the lead cleavage pattern and therefore presumably are not involved in formation of the RNA-binding pocket, a minimal aptamer of 60 nucleotides was designed (Table 5.1). The minimal aptamer consists of three paired regions (P1–P3) and two loops (L2 and L3)


connecting the respective stems (P1, P2, and P3), as well as the joining regions J1/2, J2/3. G39 in loop 3 was identified as a nucleotide in close proximity to tetracycline upon binding via UV crosslinking, indicating that loop 3 is probably part of the tetracycline-binding site. Studies with tetracycline derivatives revealed that the hydroxyl group at position 6 of tetracycline is essential for recognition, while functional groups at positions 4, 5, and 7 do not interfere with RNA binding. A magnesium ion coordinated by the ketoenolate group at positions 11 and 12, which is involved in binding of tetracycline to its primary binding site on the 30S subunit, was found to be also necessary for tetracycline binding to the cb28 aptamer. Application With tetracycline binding strongly to a specific RNA sequence and thereby altering its secondary structure, a gene expression system in which translation is controlled by tetracycline binding to its aptamer was designed in yeast (Fig. 5.2). For this purpose the tetracycline aptamer was cloned into the 5l-UTR of an mRNA encoding green fluorescence protein (GFP). Thus, if the GFP mRNA is expressed without added tetracycline, GFP is expressed normally (though to a lesser extent, as the inserted aptamer influences expression). However, if tetracycline is added and binds specifically to its aptamer sequence in the nascent RNA, it changes the secondary structure of the RNA in a way that inhibits translation of GFP (Suess et al., 2003). Either binding of the ribosomal 40S preinitiation complex is inhibited or assembly of the 80S ribosome is prevented by hindering successful scanning of the 40S subunit (Hanson et al., 2003) depending on whether the aptamer sequence is located near the cap or in proximity to the start codon (Fig. 5.2). A similar approach was used by Werstuck and Green (1998), who inserted an aptamer sequence against Hoechst dye into the 5l-UTR of b-galactosidase. With such a system in hand, it is possible to modulate the structure of an RNA, resulting in a riboswitch which can turn translation on or off, providing a useful tool for the study of translational regulation.

Fig. 5.2 Tetracycline aptamer-mediated regulation of translation. The tetracycline-binding aptamer has been used as a target module to regulate translation in yeast via small molecules. The aptamer was introduced near the

cap proximal 5l end of the mRNA (a), or downstream, proximal to the start codon (b). The presence of the ligand, in this case tetracycline, stabilizes the aptamer, making it more difficult for the ribosome to start translation.

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5.4 Aptamers to Streptomycin

Streptomycin is a member of the aminocyclitol glycoside antibiotics (Table 5.1) and it interferes with translation by disturbing the decoding of genetic information. It induces misreading of the genetic code in vitro and suppresses missense and nonsense codons (Davies et al., 1965; Gorini, 1974). Streptomycin is also a competitive inhibitor of group I intron self-splicing (von Ahsen and Schroeder, 1990, 1991). Selection Streptomycin-binding aptamers were selected with a pool containing a 74-nucleotide-long randomized region, flanked upstream and downstream by constant regions (Klug and Famulok, 1994). Dihydrostreptomycin, which is irreversibly hydrolyzed at high pH, was coupled to Sepharose 6B rather than streptomycin. The selection conditions were 50 mmol/L Tris–HCl pH 7.6, 250 mmol/L NaCl, and 5 mmol/L MgCl2. Specifically binding RNAs were eluted from the column with 5 mmol/L streptomycin. Two different selection conditions were performed, one without counterselection and one using bluensomycin as counterselector, in order to obtain aptamers that can discriminate between these two antibiotics and recognize a specific group, namely guanidine. Bluensomycin differs from streptomycin in containing a carbamido group rather than a guanidino group in the para-position to the glycosidic bond of the streptose (Table 5.1) and it does not inhibit group I intron splicing. From 99 sequenced clones, 43 were unique, indicating that there are probably many ways how RNA can bind to the antibiotic streptomycin. Two motives, termed motif 1 and motif 2, occurred several times and were further analyzed. Motif 1 RNA was able to bind streptomycin four orders of magnitude better than bluensomycin, and the motif 2 RNA was able to bind both antibiotics. Both motives have similar secondary structures with large asymmetric internal loops disrupting a stem followed by a second loop, an internal loop for motif 1 and a terminal loop for motif 2. The asymmetric loops contain one nucleotide on one side, a G in motif 1 and an A in motif 2 and 7 nucleotides on the opposite side of the loop. Both motives require magnesium for binding of streptomycin. The X-ray crystal structure of the motif 1 aptamer complexed with streptomycin was solved at high resolution and shows that the antibiotic is clamped between the two loops (Tereshko et al., 2003). The aptamer adopts an L-shape, where the two loops of each arm of the L sandwich the ligand by forming a pocket and making contacts mainly by base-edges. Application The motif 1 streptomycin-binding aptamer was used to develop an affinity purification procedure for RNA-binding proteins. The aptamer functions as an affinity tag and is fused to an RNA domain of interest, which is recognized and bound by the corresponding RNA-binding protein (Fig. 5.3). The hybrid RNA, composed of

5.4 Aptamers to Streptomycin

Fig. 5.3 StreptoTag, an affinity purification method using the streptomycin-binding aptamer. The streptomycin-binding aptamer (Wallace and Schroeder, 1998) was used to develop an affinity purification method to isolate RNAbinding proteins. The aptamer is tagged to the RNA, which is thought to bind proteins and the

hybrid RNA is incubated with cell extracts, which contain the desired protein. The complexes are applied to a streptomycin-derivatized sepharose, the non-binding proteins are washed off the column and the desired RNA– protein complexes are affinity eluted with streptomycin.

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the streptomycin-binding tag and the RNA domain, is incubated with protein extracts and loaded onto a streptomycin-derivatized sepharose. Then, all proteins and other molecules that do not bind to the RNA are washed thoroughly away with buffer before the desired RNA–protein complex is eluted with streptomycin (Bachler et al., 1999).

5.5 Aptamers to Aminoglycosides

Aminoglycosides are a large family of antibiotics, which target the ribosomal decoding site. The basic building block is 2-deoxystreptamine, which is mono- or disubstituted with amino sugars attached by glycosidic bonds. Hydroxyl, methyl, and methylhydroxyl groups linked to the sugar rings contribute to the diversity of the aminoglycosides. The aminoglycosides are multiple positively charged compounds of high flexibility, which facilitates their accommodation into the mostly negatively charged binding pockets of the RNA. The natural binding site of aminoglycosides is in the internal loop of helix 44 of the 16S rRNA of prokaryotic ribosomes, the so-called A-site (Moazed and Noller, 1987). Binding of aminoglycosides into this loop shifts the position of the two adenines (A1492 and A1493), which sense the codon–anticodon interaction (Ramakrishnan, 2002). With the X-ray crystal structure of the ribosome in complex with antibiotics solved, our perception of the binding and action modes of aminoglycosides to the ribosomal RNA has reached a high degree of accuracy. Some of these principles had been foreseen through the analysis of antibiotic–RNA aptamer complexes. Selections Several laboratories have isolated aptamers with high affinity to aminoglycosides like tobramycin (Wang and Rando, 1995), lividomycin, kanamycin A (Lato et al., 1995), kanamycin B (Kwon et al., 2001), and neomycin B (Wallis et al., 1995). The selections were done with libraries containing from 30 to 74 random nucleotides (Klug and Famulok, 1994). All these selections resulted in the isolation of more or less specific aptamers, depending whether counterselection procedures were applied. For example, the K8 aptamer, which was isolated to bind kanamycin B, recognized tobramycin more than 10-fold better, but neomycin B 5-fold worse (Kwon et al., 2001). Most aptamers that bind lividomycin also recognize neomycin B and paromomycin (Lato et al., 1995). The W13 RNA that was isolated to bind tobramycin, binds neomycin B as well (Wang and Rando, 1995). These results make clear that although these aptamers differ in their binding patterns, they clearly have many things in common that can be considered as basic recognition elements in the RNA–aminoglycoside interactions. All structurally analyzed aminoglycoside-binding aptamers form stem–loop structures where the stems are often disturbed by single nucleotide bulges or small internal loops, which most probably widen the major grooves. The loops, which are the essential binding sites are quite variable in sequence, but seem to show a recurring motif. The so-

5.6 Aptamers to Chloramphenicol

lution structures for tobramycin as well as for neomycin B have been solved and revealed striking similarities (Jiang et al., 1997, 1999; Jiang and Patel, 1998). In all three cases, the loops close with non-Watson–Crick base pairs and accommodate the antibiotic in the floor of the widened major groove. For example, in the neomycin B aptamer, rings I and II of neomycin are encapsulated by an adenosine, which folds back, anchoring the antibiotic. Application The J6f1 tobramycin-binding aptamer has been used as tag for the purification of RNP complexes. The tobramycin aptamer was added cotranscriptionally to the 3l end of a pre-mRNA, which was immobilized onto a chromatography column via the tobramycin tag. With a solid-phase assembly scheme, spliceosomal particles were loaded onto the mRNA and the particles were released from the column under native conditions via competition with tobramycin (Hartmuth et al., 2004).

5.6 Aptamers to Chloramphenicol

Chloramphenicol is derived from the biosynthesis pathway of aromatic amino acids like phenylalanine and targets the peptidyltransferase center in the 23S ribosomal RNA. It inhibits peptide bond formation by binding to the peptidyltransferase loop of the 23S rRNA in the large ribosomal subunit. It is believed that chloramphenicol acts as a conformational analog of the 3l-O(aminoacyl)adenosine part of the incoming aminoacyl-tRNA (Bhuta et al., 1980; Drainas et al., 1993; Zemlicka et al., 1993) or of the transition state for peptide bond formation or a newly formed peptide bond (Bhuta et al., 1980). This implies that the interaction of chloramphenicol with the ribosome is somehow similar to the interaction of the ribosome with a component of the translational machinery. As the peptidyltransferase loop participates in peptide bond formation and chloramphenicol recognizes a part of the rRNA involved in this process, aptamers against chloramphenicol were selected in order to learn about how rRNA contributes to the formation of peptide bonds (Burke, 1997). Selection In order to select aptamers against chloramphenicol, two individual RNA pools were used. They consisted of 70 and 80 randomized nucleotides respectively, containing 1014 –1015 sequences. For the selection procedure an agarose resin was derivatized with chloramphenicol and antibiotic-binding RNAs were affinity eluted. Elution required long incubation times in order to bias the selection towards high-affinity aptamers with long koff rates. While the recovery of specific RNAs was low in the first seven cycles, it increased up to 50% in the 12th cycle, with Kd-values of 25 mmol/L (70 nt pool) and 65 mmol/L (80 nt pool). RNAs from both pools contained the sequence motif shown in Table 5.1, consisting of three helices interrupted by two asymmetric bulges with 4–6 adenosines across from a single

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adenosine. This motif was identified by determining the 3l and 5l boundaries of the sequence required for chloramphenicol binding, RNase S1 analysis and investigation of the activity of truncated RNAs. For the latter assay a 50-nt minimal RNA (Cm1) containing only the identified motif, as well as a 33-nt RNA (Cm2) containing only one bulge, that is one half of the otherwise symmetrical motif (Table 5.1), were synthesized. While Cm2 showed little activity, Cm1 displayed activities similar to those of the full-length RNAs. Thus, Cm1 was resynthesized with a 15% mutagenesis rate per position and subjected to reselection in order to further refine the sequence requirements for chloramphenicol binding. Moreover, the stoichiometry of the bound complex was analyzed by NMR spectroscopy and found to be 1:1. Therefore, two models for binding of chloramphenicol to its aptamer are possible. Either the two symmetric halves of the selected structure come together through tertiary interactions and form a single binding site, or the aptamer contains two identical binding sites which can be occupied only one at a time. As Cm2, corresponding to such a single binding site, shows only little affinity to chloramphenicol, the first possibility seems to be more likely. By comparing the sequence of the peptidyltransferase loop of the 23S rRNA with the sequence of the selected aptamer, certain parallels become obvious. By juxtaposing three arcs of the peptidyltransferase loop, it is possible to model a binding site similar to the selected one, containing nucleotides actually involved in chloramphenicol binding. However, this model is based on the independence of the two halves of Cm1, and therefore made assailable by the low activity of Cm2.

5.7 Aptamers to the Peptide Antibiotic Viomycin

A very interesting family of RNA-binding antibiotics are the tuberactinomycins, whose most prominent member, viomycin, is shown in Table 5.1. Viomycin is a basic peptide composed of six amino acids – two serines and four unusual amino acids, derivatives of alanine, arginine, lysine, and propionic acid. Its core structure forms a 16-membered ring with an intramolecular hydrogen bond between the a-amide proton of the guanidine moiety and the carboxyl oxygen of the serine residue 1. These peptides are not synthesized by the translational machinery, but by non-ribosomal peptide synthetases, a large family of complex and fascinating enzymes existing in microorganisms (Walsh, 2004). Viomycin is a member of the tuberactinomycin family of antibiotics, which were mainly used to fight tuberculosis. Viomycin has a single binding site at the interface of the ribosomal subunits and interferes with several stages of protein synthesis. It inhibits translation initiation, tRNA binding and translocation (Liou and Tanaka, 1976; Modolell and Vazquez, 1977; Wurmbach and Nierhaus, 1983). It also prevents ribosomal subunit dissociation. In addition to inhibiting the ribosome, viomycin is also a competitive inhibitor of group I intron splicing (Wank et al., 1994) and the human hepatitis delta virus ribozyme in vitro (Rogers et al., 1996). The

5.7 Aptamers to the Peptide Antibiotic Viomycin

diversity of the RNA target sites recognized by this antibiotic requested a more detailed analysis of how viomycin binds to RNA. Therefore, aptamers against viomycin were selected. Selection The in vitro selection of viomycin-binding RNAs, performed with the same pool as the selection for streptomycin and neomycin, resulted in a number of RNAs, of which 90% had a highly conserved region of 14 nucleotides (Wallis et al., 1997). Determination of the minimal binding motif and secondary structure analysis revealed a hairpin structure, whose loop is involved in a longrange pairing interaction forming a pseudoknot (Table 5.1). The most interesting aspect of this selection is that all known viomycin-binding sites are pseudo-knots, although all these pseudo-knots have different topologies. No high-resolution structure has been determined for this aptamer and therefore it is unknown how this drug interacts with RNA in detail. 5.7.1 The Peptide Antibiotic Viomycin as a Primordial Lead Molecule

Antibiotics are generally viewed as inhibitors but rarely as modulators of cellular functions. Julian Davies, however, has a different view about the origins of antibiotics and suggested that antibiotics, like other secondary metabolites, were not purely invented for the purpose of killing bacteria but rather to act as effector molecules in the regulation of many biological activities (Davies, 1990). RNA, due to its structural flexibility, is a perfect target for small molecules such as antibiotics to bind and to induce conformational changes leading to modulation of activity. A very exciting example of a stimulatory activity of an antibiotic in the sense of Julian Davies’ theory is viomycin. It was shown to induce oligomerization of group I introns, switching the intron from a cis-acting to a trans-acting ribozyme. In the presence of viomycin, the excised intron forms intron concatamers instead of circular molecules (Wank and Schroeder, 1996). Viomycin also enhances the cleavage activity of the Neurospora crassa-derived VS ribozyme and reduces its requirement for magnesium (Olive et al., 1995). The trans-activation potential of viomycin means that this molecule enhances RNA:RNA interactions, a characteristic activity that might have played an essential role during the evolution of the RNA world. In order to test this hypothesis, group I intron RNA was incubated with a pool of viomycin-binding aptamers in the presence of viomycin and as a consequence, viomycin did induce the intron RNA to become covalently linked to viomycin aptamers. Viomycin can thus be regarded as a small effector molecule that increases the activity profile of catalytic RNAs (Wank et al., 1999).

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5.8 What Have We Learned From the Antibiotic-binding Aptamers?

The aptamers to antibiotics were initially isolated to answer a very basic scientific question: How can RNA molecules fold to form pockets to accommodate antibiotics? Their natural binding sites, the ribosome, seemed too large to address this question structurally. The analyses of aptamers revealed basic principles of RNA–antibiotic recognition, which were then also seen when the high-resolution structures of the ribosomal subunits complexed with the antibiotics were solved or were further analyzed. These aptamers not merely served to study RNA–antibiotic interactions, they have also been used as research tools for molecular biology. The tobramycin and streptomycin aptamers were used as tags for affinity purification of RNA-binding proteins and RNA–protein particles. The tetracycline aptamer was used as an allosteric regulatory element to achieve tetracycline-dependent mRNA translation in yeast. A take-home message from these studies is that these aptamers were great fun to study, they are very handy molecules, and what we learned from them will be useful for the future isolation of aptamers against other targets.

Acknowledgments

We thank Paul Watson for the help with the preparation of the manuscript and all the members from the Schroeder lab for constant discussions. The studies on RNA-binding aptamers in our laboratory were funded by the European Community Biotechnology Programme (no. 930345) and ERBFMRXCT97-0154 and by the Austrian Research Fund FWF grants nos Z-72 and P16026.

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5 Aptamers to Antibiotics Rogers, J., Chang, A. H., von Ahsen, U., Schroeder, R., Davies, J. (1996). Inhibition of the self-cleavage reaction of the human hepatitis delta virus ribozyme by antibiotics. J Mol Biol 259, 916–925. Spahn, C. M., Prescott, C. D. (1996). Throwing a spanner in the works: antibiotics and the translation apparatus. J Mol Med 74, 423– 439. Suess, B., Hanson, S., Berens, C., Fink, B., Schroeder, R., Hillen, W. (2003). Conditional gene expression by controlling translation with tetracycline-binding aptamers. Nucleic Acids Res 31, 1853–1858. Tereshko, V., Skripkin, E., Patel, D. J. (2003). Encapsulating streptomycin within a small 40-mer RNA. Chem Biol 10, 175–185. Vicens, Q., Westhof, E. (2003). Molecular recognition of aminoglycoside antibiotics by ribosomal RNA and resistance enzymes: an analysis of x-ray crystal structures. Biopolymers 70, 42–57. von Ahsen, U., Schroeder, R. (1990). Streptomycin and self-splicing. Nature 346, 801. von Ahsen, U., Schroeder, R. (1991). Streptomycin inhibits splicing of group I introns by competition with the guanosine substrate. Nucleic Acids Res 19, 2261–2265. von Ahsen, U., Davies, J., Schroeder, R. (1991). Antibiotic inhibition of group I ribozyme function. Nature 353, 368–370. Wallace, S. T., Schroeder, R. (1998). In vitro selection and characterization of streptomycin-binding RNAs: recognition discrimination between antibiotics. RNA 4, 112– 123. Wallis, M. G., Schroeder, R. (1997). The binding of antibiotics to RNA. Prog Biophys Mol Biol 67, 141–154. Wallis, M. G., von Ahsen, U., Schroeder, R., Famulok, M. (1995). A novel RNA motif for neomycin recognition. Chem Biol 2, 543– 552. Wallis, M. G., Streicher, B., Wank, H., von Ahsen, U., Clodi, E., Wallace, S. T., Famulok, M., Schroeder, R. (1997). In vitro selection of a viomycin-binding RNA pseudoknot. Chem Biol 4, 357–366. Walsh, C. T. (2004). Polyketide and nonribosomal peptide antibiotics: modularity and versatility. Science 303, 1805–1810. Wang, Y., Rando, R. R. (1995). Specific binding of aminoglycoside antibiotics to RNA. Chem Biol 2, 281–290.

Wank, H., Schroeder, R. (1996). Antibioticinduced oligomerisation of group I intron RNA. J Mol Biol 258, 53–61. Wank, H., Wallis, M. G. (2001). The peptide antibiotic viomycin: some (mis)behavior. In: RNA-binding Antibiotics, M. G. W. Rene Schroeder, ed. Georgetown, Texas: Landes Bioscience, pp. 89–99. Wank, H., Rogers, J., Davies, J., Schroeder, R. (1994). Peptide antibiotics of the tuberactinomycin family as inhibitors of group I intron RNA splicing. J Mol Biol 236, 1001– 1010. Wank, H., Clodi, E., Wallis, M., Schroeder, R. (1999). The antibiotic viomycin as a model peptide for the origin of the co-evolution of RNA and proteins. Orig Life Evol Biosph 29, 391–404. Watson, J. D. (1993). Early speculation and facts about RNA templates. In: The RNA World, J. F. A. R.F. Gesteland, ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, pp. XV–XXIII. Werstuck, G., Green, M. R. (1998). Controlling gene expression in living cells through small molecule-RNA interactions. Science 282, 296–298. Wurmbach, P., Nierhaus, K. H. (1983). The inhibition pattern of antibiotics on the extent and accuracy of tRNA binding to the ribosome, and their effect on the subsequent steps in chain elongation. Eur J Biochem 130, 9–12. Yarus, M. (1988). A specific amino acid binding site composed of RNA. Science 240, 1751–1758. Yonath, A., Bashan, A. (2004). Ribosomal crystallography: initiation, peptide bond formation, and amino acid polymerization are hampered by antibiotics. Annu Rev Microbiol 58, 233–251. Yoshizawa, S., Fourmy, D., Puglisi, J. D. (1999). Recognition of the codon-anticodon helix by ribosomal RNA. Science 285, 1722– 1725. Zemlicka, J., Fernandez-Moyano, M. C., Ariatti, M., Zurenko, G. E., Grady, J. E., Ballesta, J. P. (1993). Hybrids of antibiotics inhibiting protein synthesis. Synthesis and biological activity. J Med Chem 36, 1239– 1244.

6.1 Introduction

6 Aptamers to Proteins Shahid M. Nimjee, Christopher P. Rusconi, and Bruce A. Sullenger

6.1 Introduction

The concept that nucleic acid ligands could modulate the activity of target proteins emerged from basic science studies of viruses. In the 1980s, research on human immunodeficiency virus (HIV) and adenovirus discovered that these viruses encode small structured RNAs that bind tightly and specifically to viral or cellular proteins. Consistent with their binding affinities, functional analyses of these viral RNA ligands showed that the viruses had evolved these aptamers either to control the activity of proteins essential for their replication (Cullen and Greene, 1989) or to impede the activity of proteins involved in cellular antiviral responses (Marciniak et al., 1990). An example of this phenomenon is HIV evolving short, structured RNA ligand trans-activation response (TAR) that binds to Tat, a viral protein along with the cellular protein cyclin T1 to modulate viral gene expression and replication (Fig. 6.1a) (Cullen and Greene, 1989). Adenovirus has evolved a short structured RNA aptamer called virus-associated (VA) RNA to inhibit protein kinase (PKR) activity induced by interferon and in so doing, circumvent the cell’s antiviral defense system (O’Malley et al., 1986; Burgert et al., 2002). The observation that viruses utilize RNA ligands for their ends suggested to translational researchers in the late 1980s that RNA ligands may have potential as therapeutic agents. The first study performed to evaluate aptamers as inhibitors of pathologic protein targets was published in 1990. This groundbreaking work demonstrated that a TAR RNA, evolved by HIV to recruit viral and cellular proteins to viral transcripts, could be turned against the virus to inhibit its replication (Fig. 6.1a) (Sullenger et al., 1990). The TAR aptamer sequence was expressed from a tRNA promoter to act as a decoy for the viral Tat and cellular cyclin T1 proteins in CD4+ T cells. Cells expressing high levels of the TAR aptamer were shown to be highly resistant to viral replication and cytotoxicity (Sullenger et al., 1990, 1991). These results described a novel means to inhibit HIV replication and demonstrated for the first time that RNA aptamers could be utilized as therapeutic agents to directly bind and inhibit the activity of clinically relevant proteins. The Aptamer Handbook. Edited by S. Klussmann Copyright c 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-31059-2

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Fig. 6.1 Aptamer–protein interaction. (a) TAR decoy RNA-mediated inhibition of Tat. The TAR decoy RNA is similar to the endogenous viral TAR RNA and assumes an analogous tertiary structure and in so doing, competes with viral TAR for Tat binding. (b) Anti-VEGF aptamer

inhibits VEGF receptor activation by VEGF by the same mechanism as (c) an antibody targeted to the same molecule. TAR. trans-activation response element; Tat, trans-activating regulatory protein; VEGF, Vascular endothelial growth factor.

A second groundbreaking discovery in the aptamer field was also published in 1990 by Tuerk and Gold (1990), who demonstrated that large libraries of RNAs could be screened in the test tube for RNA ligands that bind T4 DNA polymerase. This in vitro selection process was termed SELEX (systematic evolution of ligands by exponential enrichment) by Tuerk and Gold (1990) and RNA ligands were given the name aptamers by Ellington and Szostak (1990). The basic approach to SELEX is outlined in Fig. 6.2. The process begins by generating a large library of RNA sequences with fixed 5l and 3l ends and a random region, usually between 40 and 60 nucleotides. A library with these parameters contains between 1014 –1015 different RNA species that fold into numerous sequence-dependent structures. The library is incubated with the target protein and RNA molecules that bind the protein are separated from those that do not. The retained RNAs are subsequently amplified by reverse transcriptase polymerase chain reaction (RT-PCR) and transcribed in vitro to generate a pool of RNA with increased affi-

6.1 Introduction

Fig. 6.2 Systematic evolution of ligands by exponential enrichment (SELEX). The process involves incubating a library of DNA or RNA molecules (Z1 q 1014) with a given target. Oligonucleotides that bind to the protein are partitioned from those that do not. Bound nucleic acids are then reverse transcribed

(in the case of RNA) and amplified using polymerase chain reaction (PCR) resulting in a nucleic acid pool that is enriched for the protein. Typically 8–12 rounds are performed to isolate aptamers that bind with high affinity and specificity.

nity for the protein target. This process is repeated (usually 8–12 rounds) until a maximum amount of enrichment is seen in the RNA pool by binding analysis. These aptamers are then cloned and sequenced for further analysis. Thus the work by Tuerk and Gold (1990) on in vitro evolution of RNA aptamers to protein targets suggested that the concept introduced by Sullenger and colleagues (1990) that aptamers can inhibit pathogenic proteins may become broadly useful since SELEX could in principle be utilized to rapidly generate aptamers to many therapeutically relevant targets. As outlined in this chapter, it is becoming abundantly clear that aptamers have the potential to be a new and effective class of therapeutic molecules. Macugen or pegaptanib sodium has just received approval by the US Food and Drug Administration (FDA) to treat age-related macular degeneration (AMD), numerous aptamers are in preclinical development and a few of these are scheduled to begin clinical evaluation in the near future.

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6.2 Properties of Aptamers as Protein Inhibitors

The properties of aptamers make them an attractive class of molecules that meet and exceed the properties of antibodies, a predominant class of therapeutic compounds (Table 6.1). Like antibodies, they fold into a three-dimensional structure based on their nucleic acid sequence to bind to its target (Fig. 6.1b,c). Both DNA and RNA ligands bind their targets with dissociation constants (Kd) in the low picomolar (1 q 10 –12 mol/L) to low nanomolar (1 q 10 –9 mol/L) range. Aptamer binding to its target is a highly specific interaction, discriminating between related proteins that share common sets of structural domains (Doudna et al., 1995; Lee and Sullenger, 1997; Rusconi et al., 2000, 2002).

Table 6.1 Properties of aptamers versus antibodies Aptamers

Antibodies

Binding affinity in low nanomolar to picomolar range

Binding affinity in low nanomolar to picomolar range

Entire selection is a chemical process carried out in vitro and can therefore target any protein

Selection requires a biological system, therefore difficult to raise antibodies to toxins (not tolerated by animal) or non-immunogenic targets

Can select for ligands under a variety of conditions for in vitro diagnostics

Limited to physiologic conditions for optimizing antibodies for diagnostics

Iterative rounds against known target limits screening processes

Screening monoclonal antibodies time consuming and expensive

Uniform activity regardless of batch synthesis Activity of antibodies vary from batch to batch Pharmacokinetic parameters can be changed on demand

Difficult to modify pharmacokinetic parameters

Investigator determines target site of protein

Immune system determines target site of protein

Wide variety of chemical modifications to molecule for diverse functions

Limited modifications of molecule

Return to original conformation after temperature insult

Temperature sensitive and undergo irreversible denaturation

Unlimited shelf-life

Limited shelf-life

No evidence of immunogenicity

Significant immunogenicity

Cross-reactive compounds can be isolated utilizing toggle strategy to facilitate preclinical studies

No method for isolating cross-reactive compound

Aptamer-specific antidote can be developed to reverse the inhibitory activity of the drug

No rational method to reverse molecules

6.2 Properties of Aptamers as Protein Inhibitors

In preclinical studies doses 1000-fold in excess of doses used in animal and human therapeutic applications aptamers do not elicit immunologic reactions (White et al., 2000; Eyetech Study 2002, 2003). Nucleic acid bioavailability and pharmacokinetics can also be modified to tailor the drug for a particular application. Nuclease degradation is classically a problem with oligonucleotides. In order to protect against this enzymatic activity, selections are routinely performed with amino- or fluoro-modifications at the 2l position of pyrimidines (Jellinek et al., 1995; Willis et al., 1998) as well as postselection substitutions of O-methyl for OH residues at the 2l position of purines in a given aptamer (Aurup et al., 1992; Beigelman et al., 1995). Moreover, to further increase exonuclease degradation aptamers are often capped at their 3l end with a deoxythymidine (Beigelman et al., 1995). Truncated aptamers have a molecular weight of between 8 and 14 kDa, which corresponds to a length of 25–40 nucleotides. This small size results in renal clearance of the drug on the order of minutes. Aptamers are modified by site-specific addition of polyethylene glycol (PEG) and other moieties or by attaching them to a liposome surface to reduce such renal clearance (Willis et al., 1998; Tucker et al., 1999). As mentioned, aptamers have extreme specificity for their target. This can pose a significant challenge in preclinical evaluation of such compounds even though

Fig. 6.3 Toggle SELEX: Round 1 consists of incubating an oligonucleotide library with both the human and animal ortholog of a given protein target. Nucleic acid ligands that bind to both orthologs undergo reverse transcrip-

tion and PCR. The enriched pool is then “toggled” between the human and animal proteins to isolate oligonucleotides that bind to a conserved epitope on both orthologs.

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Fig. 6.4 Oligonucleotide control of aptamers. An aptamer folds into a specific three-dimensional conformation based on its sequence. An antidote is designed based on its sequence homology to the aptamer. The aptamer–antidote interaction occurs by Watson– Crick base pairing, preventing the aptamer assuming its requisite tertiary structure and rendering it inactive.

their activity is specific. To subvert this, a method called “toggle SELEX” was developed whereby an RNA library is exposed to the human form of a given protein and in the subsequent round, bound to the animal ortholog from the species in which preclinical studies will be conducted (Fig. 6.3) (White et al., 2001). Although the pharmacokinetic activity of aptamers can be modified, they still rely on the efficiency of the clearance mechanism. This creates an obstacle in the clinic where patients have multiple organ dysfunction, including hepatoand renal insufficiency. In order to afford aptamer activity independent of biological clearance, Rusconi and colleagues developed the concept of rationally designed antidote control of an aptamer (Rusconi et al., 2002). Utilizing Watson– Crick base pairing between the aptamer and the oligonucleotide antidote to alter the shape of the aptamer, the antidote binds to the aptamer and thereby prevents it from binding to its target (Fig. 6.4). This concept has created a unique niche for aptamer therapeutics by designing antidote-controlled drugs for any given clinical application. The properties of aptamers make them an attractive class of direct protein inhibitors. Their strong and specific binding for a given protein target leads to the potential to isolate ligands to virtually any target. The ability to adjust their bioavailability increases the diversity of their clinical application. By utilizing toggle SELEX, aptamers can be isolated that retain activity in multiple organisms, facilitating preclinical development, saving time and resources over raising antibodies in animal model systems. Finally, the development of antidotes that control aptamer activity is perhaps the most noteworthy recent development in nucleic acid therapeutics, creating safe, tightly controlled drug compounds. Here we present a summary of aptamers isolated to protein targets and their binding characteristics, inhibitory properties, and their preclinical and clinical development (Table 6.2).

RNA DNA

Basic fibroblastic growth factor

Platelet-derived growth factor

In nature In vitro (14)

RNA DNA DNA RNA

HIV RT

Transcription factor E2F

Nuclear factor kB

In nature

In vitro (9)

In nature

RNA

In nature

RNA

In vitro (11)

RNA

HIV-1 Rev

In nature

RNA

In vitro (12)

In vitro (11)

HIV-2 Tat

HIV-1 Tat

Nucleic acid-binding proteins

RNA

In vitro (11)

In vitro (17)

RNA

Interferon-g

Angiopoietin-2

Selection (rounds)

In vitro (12)

Nucleic acid type

RNA

Vascular endothelial growth factor

Cytokines/growth factors

Target

Table 6.2 Summary of aptamers to therapeutic targets

0.120

0.1

0.35

2.2

2.7

0.05–0.150

Kd (nmol/L)

Lebruska and Maher, 1999

Morishita et al., 1997; Sawa et al., 1997; Tomita et al., 2000; Ueno et al., 2001

Morishita et al., 1995; Mann et al., 1999; Mann et al., 1999; Ehsan et al., 2001

Tuerk et al., 1992; Joshi and Prasad, 2002

Lee et al., 1992; Bahner et al., 1996; Kohn et al., 1999

Browning et al., 1999

Yamamoto et al., 2000

Sullenger et al., 1990, 1991

Green et al., 1996; Floege et al., 1999; Pietras et al., 2001

Jellinek et al., 1995

White et al., 2003

Kubik et al., 1997

Ruckman et al., 1998; Tucker et al. 1999; Huang et al., 2001; Eyetech Study 2002, 2003; Kim et al., 2002

References

6.2 Properties of Aptamers as Protein Inhibitors 137

RNA

In vitro (15)

DNA

RNA DNA

P-Selectin

L-Selectin

Cell surface receptor/cell adhesion molecules

RNA

RNA

Immunoglobulin E

Cytotoxic T cell antigen 4

In vitro (9)

RNA

Monoclonal antibody to acetylcholine receptor

In vitro (17)

In vitro (12)

In vitro (9)

In vitro (12 +12 more)

RNA

In vitro (12)

In vitro (8)

In vitro (13)

Anti-insulin receptor antibody MA20

Antibodies/immunoglobulins

RNA

Factor IXa

In vitro (13)

RNA

Factor VIIa

In vitro (5)

DNA

a-Thrombin

In vitro (18)

In vitro (9)

RNA RNA-hybrid

In vitro (6)

Selection (rounds)

RNA

Nucleic acid type

Human neutrophil elastase

DNS3

Hepatitis C NS3

Serine proteases

Target

Table 6.2 Summary of aptamers to therapeutic targets (continued)

1.8

0.019–0.039

10

9

30

6–60

30

0.58

11

2.8

25–200

10

Kd (nmol/L)

Hicke et al., 1996; O’Connell et al., 1996


Santulli-Marotto et al., 2003

Wiegand et al., 1996


Lee and Sullenger, 1997; Seo and Lee, 2000; Hwang and Lee, 2002

Doudna et al., 1995; Lee and Sullenger, 1996

Rusconi et al., 2002


White et al., 2001

Bock et al., 1992; Griffin et al., 1993; DeAnda et al., 1994

Bless et al., 1997; Charlton et al., 1997

Fukuda et al., 2000; Hwang et al., 2000

Kumar et al., 1997

References

138


RNA

Trypanosoma cruzi

RNA RNA DNA RNA

Neuropeptide calcitonin gene-related peptide 1

Gonadotropin-releasing hormone

Neuropeptide nociceptin/orphanin FQ

RNA

DNA

RNA

Ghrelin

Peptides

Prion protein PrPSc

Prion proteins

Human non-pancreatic secretory phospholipase A2

Lipoproteins

Tenascin-C

Extracellular membrane proteins

Complement C5

RNA

RNA

Prostate-specific membrane antigen

Complement proteins

Nucleic acid type

Target

In vitro (13)

In vitro (12)

In vitro (15)

In vitro (17)

In vitro (12)

In vitro (17)

In vitro (13)

In vitro (12)

In vitro (7)

In vitro (6)

Selection (rounds)

Table 6.2 Summary of aptamers to therapeutic targets (continued)

300

20

2.5

35

1.8

4

0.02–0.04

172

Kd (nmol/L)

Faulhammer et al., 2004

Wlotzka et al., 2002

Vater et al., 2003

Helmling et al., 2004

Proske et al., 2002

Hicke et al., 1996; O’Connell et al., 1996

Ishizaki et al., 1996

Biesecker et al., 1999

Ulrich et al., 2002

Lupold et al., 2002

References

6.2 Properties of Aptamers as Protein Inhibitors 139

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6.3 Cytokines/Growth Factors 6.3.1 Vascular Endothelial Growth Factor (VEGF)

Angiogenesis plays a critical role in numerous physiological and pathological states. Vascular endothelial growth factor (VEGF) is the best characterized factor involved in benign and neoplastic angiogenesis and represents a promising target in anticancer and other anti-angiogenic therapies (Rosen, 2001, 2002). Elevated VEGF levels are associated with ocular neovascularizations including diabetic retinopathy (Adamis et al., 1994), retinopathy of prematurity (Pierce et al., 1995), and macular degeneration (Kvanta et al., 1996). Several selections have been performed against a 165-amino acid form of VEGF (VEGF165) (Jellinek et al., 1994; Green et al., 1995) with the latest of this series being a 2l-fluoropyrimidine RNA selection (Ruckman et al., 1998). Ruckman and co-workers performed 12 rounds of selection and isolated three aptamers with Kd-values ranging from 5 to 50 pmol/L (Ruckman et al., 1998). All but two of the 2l-OH purine positions were modified with 2l-O-methyl substitutions to further increase structure stability and nuclease resistance without significant loss of binding affinity (Kd-values between 49 and 130 pmol/L). These aptamers exhibited specificity for VEGF165 and showed no detectable binding with related proteins VEGF121 or placenta growth factor (PlGF129). The VEGF165 aptamers were then evaluated for their ability to inhibit VEGF binding to two of its receptors, fms-like tyrosine kinase (Flt-1) and kinase inset domain-containing receptor (KDR) (Fig. 6.1b). Using 125I-labeled VEGF, the IC50 for Flt-1 and KDR inhibition ranged from 50 to 300 pmol/L and 2 to 60 pmol/L, respectively. A Miles assay, which monitors VEGF inhibition in vivo by measuring vascular permeability or leakage in an animal model was used to assess the therapeutic potential of the VEGF aptamers. The test was conducted in adult guinea-pigs and the most effective aptamer was able to slow vascular leakage by 58% at a 1 mmol/L dose (Ruckman et al., 1998). Research into carrier molecules for VEGF aptamers showed that they improved the inhibitory activity of the aptamer in vitro and in vivo by increasing the circulating plasma concentration (Willis et al., 1998). Pharmacokinetic studies of the 2l-fluoropyrimidine and 2l-O-methylpurine aptamer to VEGF, renamed NX-1838, were conducted in Rhesus monkeys. This aptamer was conjugated to a 40 kDa PEG moiety and following intravenous administration showed a terminal half-life (t⁄2) of 9.3 h and a clearance rate of 6.2 mL/h. Subcutaneous administration resulted in almost 80% absorption into the plasma compartment with a time to a peak concentration of 4.9 mg/mL in 8–12 h (Tucker et al., 1999). Preclinical and clinical studies of NX-1838, renamed once more as Macugen, have been conducted by Eyetech Pharmaceuticals Inc., for the treatment of exudative age-related macular degeneration and diabetic macular edema (Eyetech Study 2002, 2003). 1

6.3 Cytokines/Growth Factors

The anti-angiogenic activity of the VEGF aptamer has been evaluated in rat corneal angiogenesis and mouse retinopathy of prematurity (ROP) models. These studies revealed significant inhibition of VEGF-mediated neovascularization with the aptamer reducing 80% of retinal neovascularization in the ROP model (Eyetech Study, 2002). Based upon these exciting preclinical results, a phase IA safety study was performed and showed no significant risks with a single intravitreal injection of the drug. Moreover, 80% of treated patients exhibited stable or improved vision 3 months after treatment and 27% demonstrated a three-line or greater improvement on the Early Treatment for Diabetic Retinopathy Study (ETDRS) (Eyetech Study, 2002). A phase II study reported that with multiple intravitreal injections of Macugen with or without photodynamic therapy (PDT), no drug-related serious side effects were observed and 87.5% of the treated patients exhibited stabilized or improved vision 3 months after treatment and 25% demonstrated three lines or greater improvement in vision on ETDRS. Furthermore, a 60% three-line gain after 3 months was seen in patients who received combination therapy of Macugen and PDT (Eyetech Study, 2003). A phase III clinical study set the stage for launching Macugen as the first aptamer-based therapeutic. In two prospective, randomized, double-blind, multicenter, dose-ranging study, Macugen was administered by intravitreal injection at a dose of 0.3, 1.0, and 3.0 mg every 6 weeks for 48 weeks (Gragoudas et al., 2004). The primary endpoint was prevention of loss of 15 letters of acuity (defined as three lines on the study eye chart) and in all three aptamer-treated groups, there was statistically significant improvement (0.3 mg, P I 0.001; 1.0 mg, P I 0.001; 3.0 mg, P = 0.03). In the 0.3 mg Macugen-treated group, 70% of patients lost less than 15 letters of visual acuity compared with 55% of controls (P I 0.001). Severe loss of visual acuity, defined as loss of 30 letters or more, was decreased from 22% in vehicle-injected group to 10% in patients receiving 0.3 mg of Macugen (P I 0.001). Moreover, in the aptamer-treated patient group, 33% maintained or gained visual acuity compared with 23% (P = 0.003). Analysis of lesion size in aptamer-treated patients revealed that there was a significant reduction in lesion size by week 54 of treatment in 0.3 mg and 1.0 mg Macugentreated group compared to control (P I 0.01) as well as a reduction in choroidal neovascularization and severity of leakage in patients that received 1.0 mg of Macugen compared with control (P I 0.01) (Gragoudas et al., 2004). No antibodies against Macugen were detected nor any reports of drug-inducted local or systemic hypersensitivity. Eyetech, in conjunction with Pfizer, has received approval from the FDA to use Macugen to treat AMD. These results are the first to show that in vitro-selected aptamers can be clinically efficacious drugs in humans and Macugen is the first aptamer-based compound to be approved for therapy. In addition to preventing ocular neovascularization, a logical potential therapeutic use for aptamers to VEGF is in cancer. Wilm’s tumor is a pediatric renal cancer, also called nephroblastoma. The aptamer isolated and optimized by Ruckman et al. (1998) was tested in a mouse model of this neoplasm (Huang et al., 2001). The kidneys of mice were inoculated with tumor cells and maintained

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for one week. Mice received daily injection of 200 mg of VEGF aptamer NX-1838 or phosphate-buffered saline (PBS) for 5 weeks. Renal histopathology revealed that there was an 84% reduction in tumor weight of the aptamer-treated kidneys compared with the control. Furthermore, lung metastases were seen in 20% of the aptamer-treated mice compared with 60% of control animals (Huang et al., 2001). The aptamer was also tested in a murine model of neuroblastoma, and showed 53% reduction in tumor growth compared with control (Kim et al., 2002). 6.3.2 Human Interferon g

The function of major histocompatibility complex (MHC) class I and II genes as well as intercellular adhesion molecule 1 (ICAM-1) are upregulated by interferong (IFN-g) in numerous cell types (Van Seventer et al., 1990). Secretion of IFN-g can result in inflammatory and autoimmune diseases. RNA selections using 2lfluoropyrimidine-, 2l-aminopyrimidine-modified RNA and a mixture of the two modifications (2l-F/2l-NH2) were screened for aptamers that bound to and inhibited IFN-g (Kubik et al., 1997). One particular aptamer 2l-NH2 -30 had a Kd of 2.7 nmol/L and inhibited IFN-g binding to its receptor on A549 human lung carcinoma cells with a Ki of 10 nmol/L. This aptamer also inhibited IFN-g-mediated induction of MHC class I antigen and ICAM-1 expression, exhibiting IC50 values of 700 nmol/L and 200 nmol/L respectively (Kubik et al., 1997). 6.3.3 Angiopoietin-2

Endothelial receptor tyrosine kinase Tie2 plays an important role in vascular stability. Angiopoietin-2 (Ang-2) is a naturally occurring Tie-2 antagonist that appears to be expressed only during active angiogenesis such as tumors (Holash et al., 1999). To investigate the inhibition of Ang-2 on vascular development with aptamers, 11 rounds of RNA selection were performed to isolate RNA molecules that specifically bound Ang-2 (White et al., 2003). The highest affinity aptamers bound Ang-2 with a Kd of 3.1 nmol/L and did not detectably bind Ang-1 (Kd i 1 mmol/L). This aptamer was truncated to a 41-mer with slightly improved binding affinity (Kd = 2.2 nmol/L). This truncated aptamer inhibited Ang-2 function in cell culture and in a rat corneal angiogenesis model, where the aptamer significantly inhibited neovascularization by over 40% (White et al., 2003). 6.3.4 Basic Fibroblastic Growth Factor

Pathological vascular neogenesis is a malfunction of tightly controlled processes that results in a number of diseases such as diabetic retinopathy, rheumatoid arthritis, and cancer. Basic fibroblastic growth factor (bFGF) is an angiogenic growth factor whose increased levels have been correlated with disease states

6.3 Cytokines/Growth Factors

including leukemia and lymphoma (Molica et al., 2002; Rimsza et al., 2003). Jellinek and co-workers used a 2l-aminopyrimidine-modified RNA library and performed 11 rounds of selection against recombinant human bFGF (Jellinek et al., 1995). Kinetic analysis identified an aptamer named m21A that bound to bFGF with a Kd of 0.35 nmol/L with a dissociation rate constant (koff ) of 1.96 q 10–3 s–1 and a calculated association rate constant (kon) of 5.6 q 106 L/ mol.s. Competitive binding studies revealed that the aptamer competed for bFGF binding with both unfractionated heparin and low molecular weight heparin. Inhibition activity of m21A was conducted in Chinese hamster ovary (CHO) cells where the aptamer prevented [125I]bFGF binding to its receptor with an ED50 of 1–3 nmol/L. Finally, the effect of m21A on endothelial cell motility was studied by analyzing the migration of endothelium cells to a denuded area in a bovine aortic cells (BAE) where endogenous bFGF is essential for activity. Aptamer m12A inhibited cell migration in a dose-dependent manner at concentrations i 50 nmol/L compared with controls (Jellinek et al., 1995). 6.3.5 Platelet-derived Growth Factor

Platelet-derived growth factor (PDGF) is a ubiquitous mitogen and chemotactic growth factor in the form of three disulfide-linked dimers made of two homologous chains, A and B (Heldin, 1992). PDGF is involved in wound healing and is linked to the progression of numerous diseases, including atherosclerosis and glomerulonephritis (Iida et al., 1991; Lindner et al., 1995; Lindner and Reidy, 1995). Moreover, one of the hallmarks of malignant transformation is the loss of dependence on exogenous mitogenic stimulation, and many tumor cell lines are thought to produce and secrete PDGF for this reason (Heldin, 1992). An in vitro DNA selection was performed against recombinant human PDGF-AB and after 12 rounds, DNA aptamers were isolated that bind with a Kd of 50 pmol/L (Green et al., 1996). Three aptamers efficiently inhibited binding of PDGF-BB to PDGF a and b receptors with Ki-values of 1 nmol/L. The PDGF aptamers also inhibited the mitogenic effects of PDGF-BB on cells expressing PDGF b receptors, with a Ki of approximately 2.5 nmol/L (Green et al., 1996). One PDGF aptamer, termed 36t, was further truncated and 2l-O-methyl-, 2lfluoro-modified, capped at the 3l end to increase nuclease resistance and conjugated to a 40 kDa PEG residue to increase its circulating half-life (Floege et al., 1999). The modified aptamer bound to the human protein with high affinity (Kd = 100 pmol/L) and was subsequently tested in a rat model of mesangioproliferative glomerulonephritis (Floege et al., 1999). In this model, administration of two intravenous doses per day of 2.2 mg/kg of the aptamer decreased mitoses by 64% on day 6 and by 78% on day 9. There was a 95% reduction of proliferating mesangial cells by day 9 and markedly reduced glomerular expression of endogenous PDGF B-chain. Finally, aptamer-treated animals displayed a reduced monocyte/macrophage influx and glomerular extracellular matrix overproduction on day 6 compared with scrambled-sequence-treated or PEG-alone-treated ani-

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mals (Floege et al., 1999). Further studies showed inhibition of other disease parameters by the aptamer in experimental glomerulonephritis (Ostendorf et al., 2001, 2002). Expression of PDGF b receptors in tumors is associated with increased interstitial fluid pressure (IFP) in dermis. This diminishes the gradient between capillaries and interstitium and impedes solute exchange over the capillary membrane, most importantly in cancer, of anticancer agents (Jain, 1987). Increasing this gradient may facilitate transport of anticancer drugs to tumors (Jain, 1996). To reduce IFP, the PDGF-B aptamer (Floege et al., 1999) was tested in a rat tumor model. Animals treated with PDGF-B aptamer had an IFP of 9.7 mmHg compared with 14.6 mmHg in scrambled-RNA-treated animals (Pietras et al., 2001).

6.4 Nucleic Acid Binding Proteins 6.4.1 HIV-1 Tat

The therapeutic utility of aptamers was first studied in HIV replication. The Tat protein is a potent activator of viral replication. The trans-activation response (TAR) element is required for Tat function. A 60-nucleotide RNA decoy was developed that contained HIV viral TAR transcripts (Fig. 6.1a) and overexpressed in CEM SS cells. This RNA decoy inhibited i 99% Tat-mediated HIV replication in vitro (Sullenger et al., 1990). Inhibition was achieved by expressing high levels of the TAR aptamer with a tRNA promoter in CD4+ T cells. Perturbation of the stem or loop of the TAR aptamer abolished the decoy’s ability to bind Tat and cyclin T1 and also abolished HIV inhibition (Sullenger et al., 1991). A selection has also been performed against HIV Tat protein in order to isolate a Tat-binding aptamer via SELEX. Using a 120-nucleotide random region and performing 11 rounds of selection, a truncated RNA ligand named RNATat bound Tat with a Kd of 120 pmol/L. This 37-mer inhibited HIV-1 in vitro and showed a 70% reduction in HIV-1 replication in cell culture (Yamamoto et al., 2000). Other groups have also developed similar RNA decoys that bind to Tat and inhibit HIV-1 (Lisziewicz et al., 1993; Bohjanen et al., 1996). After the development of TAR decoys that inhibited HIV-1, research showed that there was incompatibility between the TAR and Tat interactions of HIV-1 and HIV-2. While Tat-1 could transactivate HIV-2 through TAR-2, Tat-2 could not interact with TAR-1 to transactivate HIV-1 (Browning et al., 1999). This led to the development of a TAR-2 decoy aptamer that inhibited Tat-2 transactivation and inhibited both HIV-1 and HIV-2 replication (Browning et al., 1999).

6.4 Nucleic Acid Binding Proteins

6.4.2 HIV-1 Rev

Following this principle of sequence correspondence, a tRNA promoter was utilized to express the major Rev-binding site in the Rev response element (RRE) of HIV-1 (Lee et al., 1992). In cells expressing the chimeric tRNA–RRE aptamer, HIV-1 replication was inhibited by more than 90% (Lee et al., 1992). Other groups reported similar inhibition, (Bahner et al., 1996) and the results led to phase I clinical trial where the decoy was transduced in vitro in CD34+ cells from bone marrow of patients with HIV-1 and subsequently the cells were reinfused into the subject (Kohn et al., 1999). There were no adverse effects reported from aptamer administration, however, only low levels of the RRE aptamer gene remained in leukocytes 1 year after treatment because of low levels of gene transfer (Kohn et al., 1999). 6.4.3 HIV Reverse Transcriptase

An obvious target for SELEX in HIV therapy is reverse transcriptase (RT). Tuerk and co-workers, having recently published their landmark paper on SELEX (Tuerk and Gold, 1990), used RT as a target to isolate RNA ligands that inhibited HIV replication. Using a library randomized at 32 positions, they isolated an RNA ligand after nine rounds of selection that specifically bound to HIV RT and inhibited its activity (Tuerk et al., 1992). The structure of the RNA ligand interaction with RT was subsequently characterized (Jaeger et al., 1998) and cellular experiments showed a 90–99% reduction in HIV-1 replication (Joshi and Prasad 2002). Furthermore, Jurkat T cells expressing the aptamer completely blocked the spread of HIV in culture (Joshi and Prasad, 2002). 6.4.4 Transcription Factor E2F

Proliferation of cardiac and vascular cells is central in the development of cardiovascular diseases such as cardiac intimal hyperplasia, cardiac hypertrophy, and atherosclerosis, as well as the development of malignancies (Nevins, 1992; Hunter, 1993). Transcription factor E2F is essential in regulating cellular proliferation (Nevins, 1992). It binds with high specificity to double-stranded DNA containing an eight-base pair consensus sequence TTTCGCGC (Hiebert et al., 1989). A 14nucleotide double-stranded DNA decoy aptamer that contains the consensus sequence for E2F binding was constructed and tested first for its ability to inhibit E2F activity (Morishita et al., 1995). In vascular smooth muscle cells (VSMC), serum-stimulated to increase E2F binding activity, the 14-mer oligodeoxynucleotide (ODN) inhibited VSMC proliferation and expression of cell cycle control genes c-myc, cdc2, and proliferating-cell nuclear antigen (PCNA). In vivo transfection of the 14-mer ODN in a rat carotid balloon injury model showed marked sup-

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pression of neointima formation with a neointima/media ratio of 0.291 compared with untransfected segments with a ratio of 1.117 (P I 0.01). Moreover this inhibition was sustained for 8 weeks post transfection following single administration of the aptamer (Morishita et al., 1995). These exciting results led Dzau and colleagues to evaluate the E2F DNA decoy aptamer in human trials to determine if it could limit intimal hyperplasia in bypass vein grafts. The E2F aptamer was delivered into the infra-inguinal vein graft by ex vivo pressure-mediated transfection (Mann et al., 1999a,b). Mean cell transfection efficiency was 89% and c-myc and PCNA expression decreased by 73% and 70% respectively compared with the control-treated group. Impressively, at 12 months, fewer graft occlusions, revisions, or major stenosis were seen in the E2F–DNA aptamer group compared with the untreated group (hazards ratio 0.34, 95% confidence interval (CI) 0.12–0.99) (Mann et al., 1999b). The E2F– DNA aptamer is now being evaluated by Corgenetch Inc., in a phase III study to evaluate its efficacy at limiting coronary and peripheral vascular graft failure in a large double-blind clinical trial. Thus this naturally derived DNA aptamer is one of the aptamers closest to mainstream clinical use. The E2F–DNA decoy was also transfected into rabbit vein grafts to test longterm protection from neointimal hyperplasia and atherosclerosis (Ehsan et al., 2001). Hypercholesterolemic rabbits underwent a jugular vein to carotid artery interposition grafting and were stratified into E2F aptamer-treated, scrambled-oligonucleotide-treated, and vehicle-treated groups. After 6 months post transfection where the animals received cholesterol feeding, the E2F aptamer-treated grafts remained free of macroscopic plaque whereas the scramble-ODN-treated and vehicle-treated vessels had extensive plaque formation (Ehsan et al., 2001). Finally, SELEX has been performed on E2F1 protein to find in vitro selected RNA aptamers that bind to and inhibit E2F activity. After 13 rounds of selection, clone E1 RNA bound to E2F1 with a Kd of 4 nmol/L and blocked E2F1 binding to its DNA-binding site (Ishizaki et al., 1996). To test the ability of E1 RNA to arrest cell proliferation, human diploid fibroblast cells were treated with E1 RNA aptamer and induced to proliferate. The E2F RNA aptamer inhibited S-phase induction by 90% compared with controls by impeding E2F activity (Ishizaki et al., 1996). Thus, both natural and in vitro selected aptamers appear to be able to limit cell proliferation. 6.4.5 Nuclear Factor Kappa B

Transcription factor nuclear factor kappa B (NF-kB) activates genes involved in inflammation and the synthesis of chemokines, interferons, MHC proteins, growth factors, and cell adhesion molecules that play a role in ischemia-reperfusion injuries seen post myocardial infarction (Verma et al., 1995). It is also necessary in HIV-1 gene expression and downregulation of NF-kB potentates tumor cell death (Verma et al., 1995; Beg and Baltimore, 1996). A double-stranded DNA aptamer was found in nature that binds to NF-kB with high affinity. This NF-kB

6.5 Serine Proteases

DNA aptamer has been evaluated in several animal models. The aptamer has been studied in an in vivo model of cardiac ischemia-reperfusion and showed that the decoy-treated but not control-treated animals had a significant effect in inhibiting injury (Morishita et al., 1997). In a rat cardioplegic arrest model, animals transfected with the NF-kB DNA aptamer showed improved recovery of left ventricular function compared with scrambled DNA controls as well as coronary flow (97% versus 61%) 3 days post transfection (Sawa et al., 1997). The aptamer-treated group expressed a lower percentage of neutrophil adhesion to endothelial cells (38% versus 81%) and a lower level of interleukin 8 (109 versus 210 ng/mg) (Sawa et al., 1997). The NF-kB DNA aptamer was also studied in a murine model of nephritis, where the aptamer but not the scrambled-oligonucleotide control abolished glomerular inflammation and gene expression of inflammatory markers IL-1a, IL-1b, IL-6, ICAM-2, and VCAM-1 (Tomita et al., 2000). To study the potential of the aptamer in brain ischemia, a rat global brain ischemia model was employed whereby NF-kB aptamer or mutant control aptamer was administered through the carotid artery during 20 min of global brain ischemia (Ueno et al., 2001). One hour after ischemia, expression of tumor necrosis factor a (TNFa), IL-1b, and ICAM-1 was inhibited in the NF-kB aptamer-treated group compared with the control-treated group. Moreover, 7 days after ischemia, neuronal damage was also significantly attenuated in the NF-kB-aptamer-treated group compared with control (Ueno et al., 2001). An in vitro selected RNA aptamer has also been generated against p50, one of the two subunits of NF-kB (the other being p65) (Lebruska and Maher, 1999). Fourteen rounds of selection yielded RNA aptamers that bind to p50 with high affinity and are able to inhibit NF-kB binding to DNA by interrupting protein dimerization in vitro (Lebruska and Maher, 1999).

6.5 Serine Proteases 6.5.1 Hepatitis C Virus–NS3 (HSV–NS3)

Hepatitis C virus (HCV) is the main cause of both sporadic and post-transfusion non-A, non-B hepatitis (Kumar et al., 1997). The viral-encoded non-structural protein 3 (NS3) is a serine protease that has protease, nucleoside triphosphate, and helicase activities and therefore represents a good target for inhibition of HCV. A selection was performed with a library bearing a 120-nucleotide random region and after six rounds of selection, two aptamers were identified that bound to NS3 and inhibited its helicase and protease activities in vitro (Kumar et al., 1997). In order to identify an aptamer with specificity for the NS3 active site, Fukuda et al. (2000) performed a selection to the truncated polypeptide DNS3. Using a RNA library with a 30-nucleotide random region, they performed nine

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rounds of selection and identified 45 clones that bound to DNS3 (Fukuda et al., 2000). While the aptamers were divided into three families based on their sequence similarity, they all retained a GA(A/U)UGGGAC conserved region. These aptamers bound to DNS3 with a Kd of 10 nmol/L and inhibited 90% of protease activity of DNS3 and of full-length NS3 fused with maltose-binding protein (MBP– NS3). In vivo, HCV non-structural proteins are processed by NS3 and cofactor NS4A and in order to recapitulate physiologic condition, the effect of the aptamers of NS3 protease activity was tested in the presence of P41 peptide, which enhances MBP–NS3 activity seven-fold. In this simulated physiologic state, the aptamers inhibited 70% of MBP–NS3 protease activity. The same group subsequently characterized the RNA aptamer-binding site of NS3 (Hwang et al., 2000). 6.5.2 Human Neutrophil Elastase

Inflammatory disease such as acute respiratory distress syndrome (ARDS), septic shock, emphysema, arthritis, and ischemia-reperfusion all have neutrophil elastase (hNE) implicated in their pathogenesis (Jochum et al., 1993; Doring, 1994). A covalent inhibitor of hNE, a diphenyl phosphate derivative of valine (Oleksyszyn and Powers, 1989, 1991) was coupled to an RNA library to enhance the binding of the inhibitor with hNE (Smith et al., 1995). Ten rounds of in vitro selection were completed and an RNA aptamer conjugated to DNA:valP (RNA10.11:DNA:valP) was isolated that binds hNE with high affinity (Kd = 71 nmol/L). Human NE inhibition studies demonstrated that hNE aptamer was a potent inhibitor with a Ki of 5 nmol/ L. The second-order rate constant for irreversible inactivation of hNE was two orders of magnitude greater than the unconjugated hNE inhibitor (104 –106 L/mol. min). The blended molecule also inhibited lung inflammation in an ex vivo rat model for acute respiratory distress syndrome (ARDS), in contrast to the aptamer RNA 10.11 or DNA:valP alone (Smith et al., 1995). The same group also performed a valyl phosphonate:DNA library selection to find more potent inhibitors of hNE (Charlton et al., 1997). A single enantiomer form of the valyl phosphonate was used in this experiment compared with a racemic mixture used in the previous one (Smith et al., 1995) and inhibitors were selected against both purified elastase and secreted elastase in the presence of neutrophils (Charlton et al., 1997). After 18 rounds of selection, aptamer–inhibitor ED45 inhibited hNE formation with a second-order rate constant (kinact/Ki) of 1 q 108 L/mol.min, which is two orders of magnitude greater than the previous RNA.10.11:DNA:valP (Smith et al., 1995) and the most potent peptide phosphonate inhibitors (Oleksyszyn and Powers, 1991). The aptamer was truncated to a 42-mer, named NX21909, and tested in a rat model of lung inflammatory injury. The truncated aptamer–inhibitor exhibited a second-order rate constant for inactivation of 1 q 107 L/mol.min for rat neutrophil elastase and in an in vivo rat ARDS model, a 40 nmol dose of NX21909 inhibited neutrophil infiltration by 53% in the lung (Bless et al., 1997).


6.5.3 Thrombin

Thrombin is a key regulatory enzyme in the coagulation cascade. It is a serine protease, produced from prothrombin by the action of factor Xa. Thrombin, in turn, converts fibrinogen into fibrin, which is the building block of the fibrin matrix of blood clots (Coughlin, 2000). Thrombin also activates platelets, initiating a change in their shape, upregulating a number of substrates and activating molecules as well as mobilizing P-selectin, CD40 ligand, and activating integrin a2b3 (Henn et al., 1998; Coughlin, 2000). One of the first therapeutic aptamers isolated by SELEX was against human thrombin. A single-stranded DNA aptamer was isolated from a pool of Z1013 oligodeoxyribonucleotides containing a 60-nucleotide random region (Bock et al., 1992). After five rounds of selection, aptamers were identified that exhibited moderate binding affinities for thrombin (Kd = 25– 200 nmol/L). The majority of aptamers identified shared a common conserved hexamer GGTTGG. A 15-mer GGTTGGTGTGGTTGG prolonged a clotting time from 25 to 169 s in purified fibrinogen and from 25 to 43 s in human plasma. Studies have elucidated the structure of the aptamer as well as its binding to thrombin’s anion-binding exosite I (Wu et al., 1992; Macaya et al., 1993; Paborsky et al., 1993; Wang et al., 1993). The anticoagulant activity of the aptamer was tested in cynomolgus monkeys. The prothrombin time (PT), a clotting assay sensitive to thrombin inhibition, increased by 1.7-fold in 10 min and returned to baseline 10 min after termination of the infusion of the aptamer (Griffin et al., 1993). The aptamer also inhibited platelet aggregation and prolonged thrombininduced platelet activation (Griffin et al., 1993). The aptamer was also evaluated in a regional anticoagulation model via sheep hemofiltration and achieved PT values of 40–45 s (baseline 21.7 s) while the systemic PT remained close to baseline (Griffin et al., 1993). In a separate study, the aptamer was tested in a canine cardiopulmonary bypass (CPB) model to analyze its anticoagulant efficiency, changes in anticoagulant variables, and the aptamer clearance mechanisms and pharmacokinetics (DeAnda et al., 1994). Animals were stratified into two groups, the first received a bolus injection of heparin (300 IU/kg) and protamine was utilized to reverse heparin’s activity post CPB. The second set of animals received 0.3 and 0.5 mg/ kg · min infusion of the aptamer. In these animals there was an increase in PT, activated partial thromboplastin time (aPTT), and activated clotting time (ACT) that subsequently returned to baseline after the infusion of the aptamer ceased (Table 6.3). Pharmacokinetic studies using a canine CPB model revealed that the elimination half-life of the drug was approximately 1.9 min pre- and post-CPB, however during 60 min CPB the half-life increased to 7.7 min. These results indicated that the aptamer could function in animal models and that unmodified DNA aptamers have very rapid clearance. Currently this DNA aptamer is being evaluated in preclinical studies by Archemix Corp., in preparation for human clinical trials. RNA selections have also been carried out against thrombin (Kubik et al., 1994; Drolet et al., 1999). Using a library with a 40-nucleotide random region, seven

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6 Aptamers to Proteins Table 6.3 Representative thrombin aptamer anticoagulation profiles during phase I Rate (mg/kg · min)

Time

PT (s)

aPTT (s)

ACT (s)

0.3 (n = 1)

Baseline

10.3

10.5

113.0

Bypass

40.4

36.0

276.0

Post-CPB

13.5

18.2

162.0

Baseline

7.5

12.0

117.0

Bypass

27.7

57.3

494.0

Post-CPB

10.9

22.5

162.0

0.5 (n = 1)

Reproduced from DeAnda et al. (1994). PT, protrombin time; aPTT, activated partial thromboplastin time; ACT, activated clotting time.

rounds of selection were completed against human thrombin immobilized on microtiter plates. Clones from round seven revealed aptamers with a predominant conservation of a single sequence, UAAGAGCCCCUCCUGAUCGAAAACAAAG CUGGAGUACUUA within the variable region. The Kd of this aptamer for thrombin was 250 pmol/L (Drolet et al., 1999), however no functional experiments were conducted. Aptamers bind to their target with extremely high specificity, however such discrimination can be problematic in preclinical testing of these drugs as they often are so specific that they do not bind to non-human orthologs of the target protein. To overcome this problem, a “toggle” approach to selection was developed whereby the 2l-fluoro-modified RNA molecules were exposed to a mixture of human and porcine a-thrombin in round 1 and then exposed to human and porcine thrombin in alternate rounds of the selection (Fig. 6.3) (White et al., 2001). Thirteen rounds of selection revealed clones with a conserved region ACAAAGCUG RAGWACUUA, where R represents A or G and W represents A or U. The final molecule selected for inhibitory studies, Toggle-25 (Tog-25) contained this conserved region within its sequence GGGAACAAAGCUGAAGUACUUACCC that cross-reacted well with human and porcine thrombin. It bound to human thrombin with a Kd of 2.8 e 0.7 nmol/L and porcine thrombin with a Kd of 83 e 3 pmol/L. The aptamer increased the time for thrombin (10 nmol/L) to clot human plasma from 11.6 e 0.2 s to 22.6 e 1.4 s. In porcine plasma, Tog-25 increased the time for porcine thrombin to clot porcine plasma from 15.7 e 0.7 s to 61.9 e 1.2 s. The amelioration of thrombin-dependent platelet activation by the aptamer occurred in a dose-dependent manner, having a greater effect on porcine thrombin in activating platelets – a 10-fold excess of Tog-25 inhibited nearly 90% of porcine thrombin activity (White et al., 2001).


6.5.4 Factor VIIa

Factor VIIa (FVIIa) is a trypsin-like serine protease that, in conjunction with tissue factor (TF), is critical in the initiation of the coagulation cascade. An RNA selection to FVIIa was performed to isolate aptamers that inhibited TFdependent activation of factor X by FVIIa (Rusconi et al., 2000). After 16 rounds of selection using a 2l-amino-modified RNA library, aptamers were identified that bound FVIIa with a Kd of 11.3 e 1.3 nmol/L. The specificity of this aptamer was also studied and binding reactions were performed on factors XIa (FIXa) and Xa (FXa), revealing dissociations constants j1 mmol/L. The FVIIa aptamer was able to reduce the initial rate of FX activation by approximately 95% and experiments involving dilutions of the aptamer revealed that it inhibits FX activation in a dosedependent manner. The aptamer prolonged the clotting time by 175% in a prothrombin time (PT) assay, which corresponded to a PT international normalized ratio (PT INR) of approximately 3.8. 6.5.5 Factor IXa

Factor IX (FIX) is a serine protease that plays an important role in generating the critical quantity of thrombin necessary in coagulation. The TF/FVIIa complex cleaves FIX to generate FIXa. FIXa combines with FVIIIa on the platelet surface and activates FX to FXa, which finally converts prothrombin into thrombin (Schmidt and Bajaj, 2003). An RNA selection to FIXa was conducted by Rusconi and co-workers, and after eight rounds of selection, an aptamer was identified that bound to FIXa with a Kd of 0.65 e 0.2 nmol/L and exhibited greater than 5000-fold specificity for FIXa over structurally similar coagulation factors VIIa, Xa, XIa, and activated protein C (Rusconi et al., 2002). A truncated version of this FIXa aptamer (9.3t) retained high affinity for FIXa (Kd = 0.58 e 0.1 nmol/L) and completely blocked FX cleavage by the enzyme complex. The anticoagulant activity of 9.3t was tested in an activated partial thromboplastin time (aPTT) test that is sensitive to FIXa inhibition. The aptamer increased the clot time in a dose-dependent manner and was able to prolong clotting times to that of plasma that was FIX deficient ( Hemorrhage is a major cause of morbidity associated with anticoagulation therapy (Ginsberg et al., 2001; Moll and Roberts, 2002). Effective control of anticoagulation is paramount in establishing truly safe therapies for all patient populations. To achieve this, Rusconi and co-workers developed an RNA antidote that reversed 9.3t activity and ultimately produced the first rationally designed drug–antidote pair for anticoagulant therapy. By exploiting the sequence-specific structure of an aptamer, they constructed a second RNA oligonucleotide that is complimentary to a region of the 9.3t aptamer (Fig. 6.4). This antidote oligonucleotide reversed the anticoagulant activity of the FIXa aptamer within 10 min in a sustainable fashion for over 5 h (Rusconi et al., 2002). Almost 5% of the 12 million

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people who receive heparin annually develop heparin-induced thrombocytopenia (HIT) (Blakeman, 1999) and are therefore unable to receive the drug (Eikelboom and Hankey, 2002). Patients who require repeated anticoagulation, such has those requiring renal dialysis, require viable alternatives. In order to evaluate the activity of the factor IXa aptamer in vivo, Rusconi and colleagues developed a cholesterol-modified aptamer (Ch-9.3t) which retained binding affinity (Kd = 5.29 e 1.1 nmol/L) and potent anticoagulation activity (Rusconi et al., 2004). The aptamer was then tested in porcine and murine plasma and found to cross-react in both species with similar efficiency to that seen in human plasma. They showed using a porcine systemic anticoagulation model that the aptamer increased ACT and aPTT compared with negative controls without affecting PT. The difference in duration of effect between the two compounds was striking, with a 9.3t blood half-life of Z5–10 min and Z60–90 min for Ch-9.3t. Antidote 5-2C reversed i 95% of aptamer function within 10 min in these animals (Rusconi et al., 2004). In order to evaluate the antithrombotic effect of the aptamer, the drug was tested in a murine arterial injury model whereby the carotid artery was injured with ferric chloride in animals that received Ch-9.3t or a functionally inactive form of the aptamer. All the mice in the negative control group developed an occlusive thrombi in 8.1 e 0.1 min in contrast to the aptamer-treated group where i 80% remained patent after 30 min after injury (time to occlusive thrombus i 24.4 min, P I 0.0001) (Rusconi et al., 2004). To measure the effect of antidote 5-2C to ameliorate bleeding due to anticoagulation with Ch-9.3t, a murine tail-transection bleeding model was employed where the aptamer or negative control was administered and after 1 h the tail was clipped and blood loss was measured over 15 min. Animals treated with Ch-9.3t exhibited significantly more blood loss (176 e 23.7 mL) than controls (48 e 17.8 mL, P = 0.007). Administration of 5-2C immediately after tail transaction however, prevented the hemorrhage seen in aptamer-treated animals (blood loss 54.5 e 13.6 mL, P = 0.0001) (Rusconi et al., 2004). Regado Biosciences has developed an optimized version of this anti-FIXa aptamer–antidote pair, termed REG1, and is progressing rapidly towards phase I clinical testing.

6.6 Antibodies/Immunoglobulins 6.6.1 Anti-insulin Receptor Antibody MA20

Autoimmunity usually entails aberrant recognition of self-antigens by antibodies, which leads to a variety of pathologies. Autoantibodies generated against human insulin receptor share an a subunit epitope with murine antibody MA20 (Zhang and Roth, 1991). Eleven rounds of RNA selection were completed against the

6.6 Antibodies/Immunoglobulins

murine antibody, and an aptamer named RNA-9 was found to bind to the MA20 antibody with a Kd of 2 nmol/L and to completely inhibit the monoclonal antibody from binding insulin receptor (Doudna et al., 1995). In addition, Doudna and coworkers demonstrated that the RNA aptamer cross-reacts with autoantibodies in serum from patients with extreme insulin resistance (Doudna et al., 1995). These results suggested that RNA aptamers can structurally mimic protein epitopes. In order to develop an RNA aptamer against MA20 that was nuclease resistant, a 2l-aminopyrimidine-modified RNA selection was conducted on MA20 (Lee and Sullenger, 1996). After 12 rounds of SELEX, a modified RNA aptamer was identified that bound to MA20 with a Kd of 30 nmol/L. To measure its capacity to resist nuclease degradation, unmodified RNA and modified RNA was incubated in 10% human serum and while the unmodified RNA degraded in 15 s, most of the amino-modified aptamer was still present as a full-length ligand after 24 h. The aptamer also blocked MA20 binding to the insulin receptor by 95% on human lymphocytes. Lee and Sullenger then showed that the modified MA20 aptamer could protect cells from MA20-dependent and autoantibody-dependent downregulation of the insulin receptor expression by up to 90% and 80% respectively (Lee and Sullenger, 1996). 6.6.2 Monoclonal Antibody (MAb) to Acetylcholine Receptor

Myasthenia gravis is a neuromuscular disorder characterized by muscular weakness and fatigue resulting from antibody-mediated autoimmune response to acetylcholine receptors (AChR) (Willcox, 1993; Drachman 1994). Lee and Sullenger isolated 2l-amino-modified aptamers that bound to MAb198, a monoclonal antibody that recognizes the major immunogenic epitope on human AChR (Saedi et al., 1990; Lee and Sullenger, 1997). After 12 rounds of selection, a modified RNA aptamer was isolated that binds MAb128 with a Kd of 60 nmol/L. The MAb128 aptamer was able to protect TE671 cells from antigenic downmodulation in a dose-dependent manner, with an ED50 of 5 mmol/L and a maximum inhibition of 70% (Lee and Sullenger, 1997). Moreover, the aptamer could also protect AChR from autoantibodies found in patients with myasthenia gravis. A subsequent 2l-fluoropyrimidine-modified RNA selection was performed to generate an improved MAb128 aptamer that is even more effective at protecting AChR from the monoclonal and patient autoantibodies (Seo and Lee, 2000; Hwang and Lee, 2002). 6.6.3 Immunoglobulin E

Immunoglobulins are critical in host defense. Immunoglobulin E (IgE) plays an important role in protecting mammals from parasites (Gounni et al., 1994). Overproduction of IgE by exposure to environmental antigens, however, can result in IgE-mediated diseases such as allergies, atopic dermatitis, and allergic asthma

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(Sutton and Gould, 1993). A 2l-aminopyrimidine-modified RNA selection and a DNA selection were performed against human IgE which yielded aptamers that bind human IgE with high affinities (RNA, Kd = 30 nmol/L; DNA, Kd = 9 nmol/L) (Wiegand et al., 1996). Unfortunately, both of these aptamers had 300- to 1000fold greater binding human IgE than rat or mouse orthologs, which limited preclinical testing of the aptamers. Experiments to determine the ability of the aptamers to inhibit IgE binding to its receptor FceRI yielded a Ki = 21 nmol/L for the RNA aptamer and a Ki = 6 nmol/L for the DNA aptamer. The aptamers were also tested for their ability to prevent IgE-mediated cellular degranulation in serum of patients with a grass allergy. When triggered by grass extract, the IC50 for both molecules was 2–6 mmol/L and 200–300 nmol/L when triggered by anti-IgE antibodies. Competition studies revealed that both aptamers compete for the same binding epitope on IgE (Wiegand et al., 1996). 6.6.4 Cytotoxic T Cell Antigen 4

T cell activation is regulated by the cofactors CD28 and cytotoxic T cell antigen 4 (CTLA-4). While CD28 activates T cells, CTLA-4 inhibits this activation, and therefore impedes T cells in mounting an immune response against tumors (Chambers et al., 2001). In order to test the effect of aptamers in tumor immunotherapy, an RNA selection was performed against a murine CTLA-4/Fc fusion protein (Santulli-Marotto et al., 2003). After nine rounds of SELEX, an aptamer was isolated that binds CTLA-4 with a Kd of 10 nmol/L. In vitro functional studies revealed that the CTLA-4 aptamer was able to enhance T cell proliferation even more effectively than CTLA-4 inhibition by an a-CTLA-4 antibody (Santulli-Marotto et al., 2003). In order to increase the avidity of the CTLA-4 aptamer, a tetrameric derivative of the aptamer was constructed off a double-stranded DNA scaffold. This tetrameric aptamer exhibited a 10- to 20-fold greater inhibitory capacity for CTLA-4 compared with the monomer in cell culture studies and greatly enhanced tumor activity in mice. Finally, the CTLA-4 tetrameric aptamer was evaluated in conjunction with dendritic cell-based vaccination strategies. The polypeptide component of telomerase (TERT) elicits modest protective tumor immunity against several tumors (Nair et al., 2000). In melanoma tumor-bearing mice, 50 pmol/injection of the CTLA-4 tetrameric aptamer enhanced the efficacy of immunotherapy engendered by TERT mRNA-transfected dendritic cells. Therefore, an aptamer also can act as an adjuvant to enhance the potency of vaccines (Santulli-Marotto et al., 2003).

6.7 Cell Surface Receptor/Cell Adhesion Molecules

6.7 Cell Surface Receptor/Cell Adhesion Molecules 6.7.1 P-Selectin

The selectins, which include L-, E-, and P-selectin are a family of cell adhesion molecules that are expressed on leukocytes, endothelial cells, and platelets (Bevilacqua and Nelson, 1993; Rosen and Bertozzi, 1994). They have been implicated in a number of inflammatory diseases as well as tissue injury and infection (Bevilacqua and Nelson, 1993). A 2l-fluoropyrimidine-modified RNA library was screened by SELEX against human P-selectin/IgG fusion protein (PS-Rg) to isolate anti-inflammatory aptamers (Jenison et al., 1998). Twelve rounds of selection were performed, and yielded an RNA aptamer with a Kd of 18 pmol/L for PS-Rg. This P-selectin aptamer showed no detectable binding related to proteins CD22, E-selectin, and L-selectin. Truncated versions of the P-selectin aptamer were generated that retain low picomolar dissociation constants for P-selectin (Kd = 19 and 39 pmol/L respectively) and inhibited P-selectin binding to its ligand sialyl Lewis X (sLeX) with an IC50 of 2 nmol/L. Moreover, the P-selectin aptamer binds to its target on activated human platelets with subnanomolar affinity and inhibits both cellular adhesion of PS-Rg to neutrophils and completely blocks neutrophil rolling on activated platelets in vitro (Jenison et al., 1998). 6.7.2 L-Selectin

An RNA selection has also been conducted against an L-selectin/IgG fusion protein (LS-Rg) (O’Connell et al., 1996). Fourteen rounds of selection yielded two RNA aptamers with high affinity for L-selectin (Kd = 3 nmol/L at 22 hC). Both aptamers inhibited L-selectin adhesion in vitro, with IC50 -values of 9 nmol/L and 37 nmol/L. The aptamers also showed inhibition of L-selectin adhesion in endothelial venules of mouse lymph nodes, with IC50 -values of 9 nmol/L and 17 nmol/L (O’Connell et al., 1996). A DNA selection was also performed against LS-Rg to find ligands that could be tested in vivo (Hicke et al., 1996) as the aptamers isolated in the previous study lost affinity when incubated at 37 hC (O’Connell et al., 1996). Clones were sequenced from rounds 15 and 17 and revealed three distinct families (Hicke et al., 1996). A representative aptamer from each family, LD201, LD174, and LD196 all bound with Kd-values of 1.8 nmol/L at 37 hC. Truncated versions of these aptamers inhibited SL-Rg binding to sLeX with an IC50 of 3 nmol/L. Aptamer LD201t1 blocked L-selectin-mediated adhesion, of human lymphocytes and neutrophils in a flow system and inhibited human cell trafficking to peripheral and mesenteric lymph nodes in SCID mice (Hicke et al., 1996). Finally, a 2lfluoro-modified RNA selection was carried out against recombinant L-selectin/

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IgG fusion protein (LS-Rg) that yielded inhibitory aptamers to Ls-Rg that also had in vitro and in vivo activity (Watson et al., 2000). 6.7.3 Prostate-specific Membrane Antigen

Prostate cancer cells overexpress a well-characterized surface antigen, prostatespecific membrane antigen (PSMA) (Tasch et al., 2001). An RNA selection was conducted against the extracellular component of PSMA called sPSM (Lupold et al., 2002). After six rounds of SELEX, two aptamers were identified. The two aptamers named xPSM-9 and xPSM-10 inhibited xPSM with a Ki of 2.1 and 11.9 nmol/L respectively. Aptamer xPSM-10 was truncated (xPSM-10-3) and fluorescently end-labeled to evaluate its ability to bind PSMA-expressing cancer cells. Using PSMA-positive LNCaP and a PSMA-negative PC-3 prostate cancer cell line, xPSM-10-3 bound to LNCaP cells but not PC-3, showing specificity for PSMA and its potential in therapeutic development for this target. 6.7.4 Trypanosoma cruzi

Trypanosoma cruzi is a protozoan responsible for causing Chagas’ disease, which primarily affects the nervous system and heart. Symptoms include dementia, megacolon, megaesophagitis, and myocardial damage. Chagas’ is often fatal if left untreated. An RNA selection was performed against parasite receptors for host cell matrix molecules laminin, fibronectin, thrombospondin, and heparin sulfate (Ulrich et al., 2002). Seven rounds of selection were performed and RNA molecules that bound to T. cruzi trypomastigotes were eluted with a mixture of all four host cell matrix molecules. This pool of aptamers had a Kd of 172 nmol/L. An eighth round of selection was performed and the aptamers were eluted with each cell matrix protein individually. The resulting aptamers were divided into four classes based on the eluting molecule. Table 6.4 illustrates how aptamers were categorized based on their elution strategy. The consensus motifs for each class of aptamers were distinct from those of the other classes of aptamers. In analyzing inhibition of T. cruzi by the aptamers, the

Table 6.4 Aptamer characterization and binding affinity Class

Eluting agent

Binding affinity (Kd) (nmol/L)

Class I

Fibronectin

124

Class II

Thrombospondin

400

Class III

Heparin sulfate

Class IV

Laminin

40 209

6.10 Lipoproteins – Human Non-pancreatic Secretory Phospholipase A

round 8 pool decreased the number of parasites per invaded cell in a dose-dependent manner. The pools from rounds 7 and 8 as well as the displaced aptamers in classes I–IV were added at a dose of 1 mmol/L to LLC-MK2 cells and revealed inhibition of invasion by 50–70%.

6.8 Complement Proteins – Human Complement C5

Complement C5 is a serum glycoprotein that is cleaved to vasoactive C5a and C5b during complement activation (Morgan, 1995), and as a central component of inflammation, subsequently stimulates neutrophil–endothelial adhesion, cytokine and lipid mediator release and oxidant formation, which are associated with numerous inflammatory states and injury (Mulligan et al., 1997). An RNA library containing 2l-fluoropyrimidines were screened by SELEX for those RNA ligands that bind to human C5. After 12 rounds of selection, aptamers were identified that bound to human C5 with a Kd of 20–40 nmol/L and inhibited C5 in a human serum hemolytic activity assay (Biesecker et al., 1999).

6.9 Extracellular Membrane Protein – Tenascin-C

Tenascin-C (TN-C) is a large, extracellular matrix glycoprotein that is overexpressed during tissue remodeling such as wound healing, atherosclerosis, psoriasis, and tumor growth (Erickson and Bourdon, 1989; Jahkola et al., 1998). Furthermore, TN-C levels predict local tumor recurrence and correlate with invasiveness and metastasis (Jahkola et al., 1998). In order to isolate oligonucleotide that can target anticancer drug delivery, a 2l-fluoropyrimidine-modified RNA selection isolated ligands that bound to recombinant TN-C (Hicke et al., 2001). Eight rounds of selection improved the affinity of the pool to Kd of 3 nmol/L, 1000-fold greater than the starting library. TTA1 is a truncated version of an aptamer with a Kd of 5 nmol/L, which was a modest 5-fold decrease in affinity compared with the fulllength ligand (Hicke et al., 2001). The TN-C aptamer is currently being developed for tumor imaging applications (Hicke and Stephens, 2000).

6.10 Lipoproteins – Human Non-pancreatic Secretory Phospholipase A2

Human non-pancreatic secretory phospholipase A2 (hnps-PLA2) is a small, secreted, Ca2+-dependent enzyme, which when elevated is associated with diseases like acute pancreatitis, ARDS, bacterial peritonitis, and septic shock (Kramer et al., 1989; Rintala and Nevalainen, 1993). A 2l-fluoropyrimidine-modified RNA selection has been performed against hnps-PLA2 (Bridonneau et al., 1998). After 11

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rounds of selection, an RNA aptamer was isolated that binds to hnps-PLA2 with a Kd of 1.7 nmol/L and inhibits hnps-PLA2 activity with an IC50 of 4 nmol/L. In a tissue-based contraction assay of guinea-pig lung pleural strips, 0.3 mmol/L of the hnps-PLA2 aptamer inhibited 70–85% of hpns-PLA2 -mediated contraction at several concentrations, whereas the same dose of control oligonucleotide had no significant effect (Bridonneau et al., 1998).

6.11 Prion Proteins – Prion Protein PrPSc

Transmissible spongiform encephalopathies (TSEs) are a series of diseases that affect both humans and animals. In humans, these include Kuru and Creutzfeldt–Jakob disease and in animals, diseases like mad cow or mad deer. A critical initiating event in the pathophysiology of TSEs is the conversion of cellular prion protein (PrPc) to an aberrant isoform called PrPSc. The cellular prion protein was targeted for selection to prevent the conversion to PrPSc. Specifically, Famulok and co-workers performed 12 rounds of selection against residues 90–129 of the human prion protein (Proske et al., 2002). The RNA ligand identified was named DP7 and although selected against the human Prpc, the binding affinity of DP7 was 100 greater for mouse and hamster full-length PrPc than the human homolog. To analyze the functional activity of DP7, the aptamer was incubated with a persistently prion-infected murine neuroblastoma line and this treatment resulted in a 53% reduction in PrPSc formation after 16 h of incubation.

6.12 Peptides 6.12.1 Ghrelin

Ghrelin is a 28-amino-acid peptide that binds to growth hormone secretagogue receptor (GHS-R) and in so doing stimulates growth hormone release in a manner distinct from growth hormone regulation by the hypothalamic growth hormone-releasing hormone (GHRH) (Kojima et al., 1999). In addition to growth hormone release, it has also been linked to energy balance (Tschop et al., 2000) and parenteral administration of ghrelin led to increased food consumption in humans and caused obesity in rats (Wren et al., 2001a,b). Helmling et al. (2004) exploited a spiegelmer-based SELEX strategy to isolate a spiegelmerbased aptamer against ghrelin. Using biotinylated ghrelin bound to streptavidin or neutravidin beads, 17 rounds of selection were performed and binding and analysis identified a truncated 47-mer, L-NOX-B11 that bound to ghrelin with a Kd of 35 nmol/L. To measure inhibition of ghrelin binding to its receptor, CHO cells expressing human GHS-R1a were used and addition of L-NOX-B11 in ghre-

6.12 Peptides

lin-stimulated cells inhibited binding with an ED50 of 5 nmol/L. Ghrelin binding to its receptor GHS-R1a releases intracellular Ca2+. Aptamer L-NOX-B11 inhibited Ca2+ release with an IC50 of 5 nmol/L. To test the functional activity of L-NOX-B11, a 5l-PEG conjugated form of the spiegelmer was injected, and at doses of 15 and 30 nmol (in 5–10 excess of ghrelin challenge) no increase in growth hormone was seen in plasma when ghrelin was administered (Helmling et al., 2004). 6.12.2 Neuropeptide Calcitonin Gene-related Peptide 1

Cerebral vessel vasodilation is a widely accepted hypothesis for the cause of migraine headaches. Neuro calcitonin gene-related peptide 1 (a-CGRP) induces vasodilation, and inhibition of this target may play a role in treating such headaches (Renfrey et al., 2003). Using a tailored-SELEX approach with an RNAbased library, 15 rounds of selection against rat a-CGRP were performed and a spiegelmer named STAR-F12 bound to a-CGRP with a Kd of 2.5 nmol/L and inhibited a-CGRP-induced cAMP with an IC50 of 3 nmol/L (Vater et al., 2003). 6.12.3 Gonadotropin-releasing Hormone

Prostate cancer, breast cancer, and endometriosis are all conditions that are dependent on the presence of sex steroids (Kettel and Hummel, 1997; Schally, 1999). Gonadotropin-releasing hormone (GnRH) is a critical peptide involved in the signaling cascade for gonadotropin synthesis, and the production of leutenizing hormone (LH) and follicle-stimulating hormone (FSH) (Conn and Crowley, 1994) and therefore inhibition of GnRH may provide a therapeutic option for treating the above diseases. After 12 rounds of selection using a DNA library, a truncated 67-mer spiegelmer called NOX 1255 was identified with a Kd of 20 nmol/L (Wlotzka et al., 2002). Inhibition of GnRH by NOX 1255 and a 5lPEG-modified version, NOX 1257, was measured in a CHO cell line, resulting in an IC50 of Z20 nmol/L for both compounds. In vivo activity of the aptamers was evaluated in a rat model of GnRH. As previously mentioned, GnRH controls LH release, which subsequently regulates testosterone production. Rats that have been castrated lack testosterone, resulting in increased GnRH production and in turn, increased levels of LH. Inhibition of GnRH in castrated rats results in LH levels that are commensurate with those seen in non-castrated animals. Rats treated with NOX 1257 exhibited a significant downregulation in LH levels compared with negative controls and, furthermore, were equivalent to rats treated with Cetrorelix, a GnRH receptor antagonist (Wlotzka et al., 2002). Finally, the immunogenicity of the anti-GnRH spiegelmer was evaluated in rabbits. The antibody titer development was monitored over 14 weeks after receiving a standard immunization protocol of five doses of NOX 1255 over 6 weeks and two additional monthly boosts. No spiegelmer-specific antibody generation was detected, which validated other research about the low immunogenicity of aptamers.

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6.12.4 Neuropeptide Nociceptin/Orphanin FQ

G protein-coupled opioid receptor-like 1 (ORL1) receptor is found in both central and peripheral nervous systems (Meunier, 1997). Its agonist neuropeptide nociceptin/orphanin FQ (N/OFQ) is located in the central nervous system and is a critical element in biological responses such as pain and anxiety (Meunier, 1997). There is much controversy surrounding the biological action of N/OFQ as peptide inhibitors have shown to result in potentiating nociception centrally but ameliorating it peripherally (Tian and Han, 2000). Specific inhibitors of this peptide would prove useful in elucidating it as the mechanism of nociception and control of such pain. Using an RNA library, 13 rounds of selection were performed on immobilized N/OFQ and after truncation analysis, two spiegelmers, a 61-mer called NOX 2149 and a 65-mer called NOX 2137a/b, were identified, having dissociation constants of 300 nmol/L and 600 nmol/L respectively (Faulhammer et al., 2004). Both aptamers were tested for their ability to inhibit N/OFQ from binding to its receptor, resulting in IC50 of 110 nmol/L for NOX 2149 and 300 nmol/L for NOX 2137a/b. The aptamers were also evaluated in a functional GTPgS, where the aptamers were measured for their ability to block N/OFQmediated [35S]GTPgS into membranes containing human ORL1 receptor. Both aptamers prevented such incorporation, with NOX 2149 having the most significant inhibition. Finally, in ORL1-GIRK1-cRNA oocytes, addition of N/OFQ produced an inward current of –318 nA that was ameliorated by 50% when NOX 2149 was concurrently added (P I 0.0001) (Faulhammer et al., 2004).

6.13 Conclusion

In nature nucleic acid-based ligands or aptamers have been utilized by viruses and cells to bind to target proteins for millions of years. However, the concept of utilizing aptamers to bind and inhibit the activity of pathogenic proteins in the laboratory or the clinic has only been around for 15 years. Nevertheless remarkable progress has been made in turning this concept into clinical reality in a very short period of time. With the approval of the Macugen aptamer, and with many more aptamers that target proteins in the clinical pipeline (Table 6.2), it is apparent that the research and clinical utility of protein targeted aptamers will almost assuredly be tremendous. Aptamers can now be generated against most target proteins and moreover in most cases they are able to inhibit the activity of the target protein. How aptamers accomplish this is much less well understood, but future atomic resolution structural studies should result in a more thorough understanding of how aptamers can bind to and inhibit the activity of proteins, especially those that do not naturally interact with nucleic acids.

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7.1 Introduction

7 Aptamers to Nucleic Acid Structures Jean-Jacques Toulm, Fabien Darfeuille, Carmelo Di Primo, and Eric Dausse

7.1 Introduction

Nucleic acid–nucleic acid interactions constitute the basis for the transmission of genetic information. Watson–Crick base pairing mediates the formation of the DNA double helix as well as that of DNA–RNA heteroduplexes transiently formed during transcription and reverse transcription. The formation of A-U and G-C pairs is also responsible for long-range interactions in RNA that generate the secondary structure of the molecule. Regulatory RNA species (antisense, microRNA) make also use of Watson–Crick base pairing for the identification of their target site. This provided the basis for the rational design of inhibitory oligonucleotides targeting pre-mRNA, mRNA, or viral RNA (Crooke, 2004; Scanlon, 2004). This has been extended to the targeting of double-stranded DNA by triplex-forming oligonucleotides through the formation of Hoogsteen or reverse Hoogsteen pairs (Thuong and Hlne, 1993). The use of powerful structural methods (nuclear magnetic resonance, crystallography, molecular modeling) has revealed a number of additional interactions and motifs that provides RNA with tertiary and functional structures such as the adenosine platform or the tetraloop and its receptor (for reviews see Brion and Westhof, 1997; Westhof and Patel, 1997; Batey et al., 1999). Such interactions, which are difficult to predict and even more difficult to use rationally, suggest that, beyond Watson–Crick base pairing one could take advantage of the potential of nucleic acid chains for generating oligonucleotide ligands of DNA or RNA. In vitro selection (SELEX) has been widely used for the identification of specific ligands to small molecules (amino acids, nucleotides, antibiotics) or to proteins (Gold et al., 1995; Osborne and Ellington, 1997; Jayasena, 1999). Much less work has been devoted to the targeting of nucleic acids (Toulm et al., 2001). What is of no interest in the case of single-stranded DNA or RNA makes sense when the target is a folded chain for which reading the primary sequence by the complementary one is not favored or is even hampered by intramolecular interaction. Aptamers to nucleic acids might help us to understand RNA structure The Aptamer Handbook. Edited by S. Klussmann Copyright c 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-31059-2

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as exemplified by the GNRA tetraloop receptor (Costa and Michel, 1997) or the dimerization signal of the HIV-1 RNA (Lodmell et al., 2000). They are also of interest for designing oligonucleotides of therapeutic value if the target is a nucleic acid region of a pathogen (Toulm et al., 2003). In this chapter we will review a few examples of aptamers that bind with a strong affinity and a high selectivity to nucleic acid structures.

7.2 Targeting Double-stranded Nucleic Acids

The development of artificial ligands of double-stranded DNA is of great interest for gene therapy. One possible approach is oligonucleotide-directed triple helix formation. The association of the oligonucleotide to duplex DNA is mediated by the formation of base triplets (Le Doan et al., 1987; Moser and Dervan, 1987). Two differents motifs are known. In the purine motif A A.T and G G.C triplets are formed by reverse Hoogsteen association of purine-rich oligomers (“ ” denotes the interaction between the third strand and the double helix). The third strand is antiparallel to the purine strand of the duplex DNA and the resulting complex is relatively insensitive to pH conditions. In contrast the triple helical pyrimidine motif requires mildly acidic conditions to allow the formation of T A.T and C+ G.C base triplets. The binding of the third oligopyrimidine strand in the major groove of the double helix through Hoogsteen pairing results in a parallel orientation of this strand to the purine strand of the target duplex. A number of studies have shown that the formation of triple-stranded structure is restricted to the recognition of homopurine–homopyrimidine tracts (Thuong and Hlne, 1993). As triple-helical complexes have been reported to have a specific inhibitory effect on transcription it is of interest to extend the repertoire of target recognition (Giovannangeli and Hlne, 2000). To this end several groups have undertaken in vitro selection experiments in order to identify new triple-helical motifs. In a pioneering work Schultz and colleagues have screened an RNA library containing 1010 –1012 sequences with a 50-nucleotide randomized region (Pei et al., 1991). The sequences identified after five rounds of selection carried out at pH 5.5 against a DNA duplex containing a tract of 16 purines on one strand and, consequently, 16 pyrimidines on the second strand, were essentially oligoribopyrimidines matching the target DNA (formation of canonical U A.T and C+ G.C triplets). Most of the sequences contained internal loops or hairpin loops. A few non-standard triplets were observed. These “mismatches” did not prevent the binding of the selected sequence to their target as, following conjugation of aptamers to staphylococcal nuclease, they were able to drive the cleavage of DNA at the expected position (i.e. next to the “complementary” sequence) (Pei et al., 1991). A similar approach was developed for the selection of RNAs that bind to duplex DNA at neutral pH (Soukup et al., 1996). Two different libraries with 60 or 42 x

x

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random nucleotides were screened against a homopurine–homopyrimidine DNA duplex 22 nucleotides long. The selection buffer contained both magnesium ions and spermidine, reagents known to promote triple helix formation. The pH of the buffer was progressively increased from 5.0 to 7.4 from the 10th to the 21st round. The selected candidates were able to form stable complexes under neutral pH condition, likely using the pyrimidine recognition motif. However the binding constant was 10- to 100-fold lower at pH 7.4 than at pH 6.0. The selection was certainly oriented by the acidic conditions used for the initial rounds. DNA triplexes were identified by in vitro selection using an approach in which a DNA duplex randomized over 19 base pairs was selected at pH 8.4 against a 19mer containing exclusively G and T residues. The selected sequences were obtained by the use of restriction endonuclease cleavage protection (Hardenbol and van Dyke, 1996). All of them possessed a large homology to the sequence able to bind through the purine recognition motif (i.e. mostly G G.C, T A.T base triplets). A few G A.T triplets were also obtained in agreement with known rules for triplex formation. Therefore these independent selections on DNA DNA.DNA and RNA DNA.DNA triplexes did not allow the identification of new recognition motifs in addition to the canonical ones. Triple-stranded complexes can also be generated from a hairpin by binding a third strand to the double-stranded stem of the hairpin. SELEX was used to identify oligonucleotides able to bind a DNA stem–loop structure. The candidates displayed a 16-nucleotide-long random region next to a fixed sequence complementary to the single-stranded sequence adjacent to the 5l end of the stem. This fixed part anchored the candidate sequence next to the hairpin, allowing the random region to fold back on the stem structure (Mishra and Toulm, 1994). After four rounds of selection three sequences were identified that were able to bind to the target hairpin with increased affinity compared to the anchor, indicating that the 16 nt selected sequence did contribute to the stability of the aptamer–hairpin complex. Indeed, the cleavage of the hairpin stem by a restriction endonuclease was prevented by the binding of the aptamers indicating its interaction with the stem–loop structure (Mishra et al., 1996). The selected sequence did not match the rules of triplex-forming oligonucleotides even though it contained mostly G and T residues. Interestingly this complex involving some kind of triple-stranded structure was stable at neutral pH in contrast to a rationally designed oligopyrimidine giving rise to regular Y R.Y triplets. A similar observation was reported for aptamers selected against a DNA hairpin corresponding to the template for in vitro transcription of the trans-activating responsive (TAR) element of the human immunodeficiency virus type 1 (HIV-1) (Boiziau et al., 1997). These oligomers engage non-identified interaction in addition to Watson–Crick base pairing with the bottom of the double-stranded stem of the DNA hairpin. x

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7.3 Loop–Loop Interactions

Intramolecular RNA folding generates stem–loop structures that frequently constitute scaffolds for triggering a biological process or that are binding sites of proteins (Hentze and Kuhn, 1996; Altuvia and Wagner, 2000). These structures might therefore be targets of interest for the design of ligands that will interfere with these processes or compete with RNA-binding proteins. The different strategies developed over the last 15 years for targeting RNA sequences with nucleic acids are poorly adapted to the recognition of RNA structures (Toulm et al., 2001). Indeed antisense oligonucleotides, ribozymes, or small interfering RNAs (siRNAs) require a single-stranded complementary sequence for binding. Intramolecular interactions involved in RNA structures weaken the association between the regulatory species (antisense, ribozyme, siRNA) and the target, and consequently the effect induced by these oligomers (Mir and Southern, 1999). Different possibilities have been considered, including the design of high-affinity chemically modified oligonucleotides, which invade the structure and shift the equilibrium toward the unfolded form (Compagno et al., 1999). Oligonucleotides have also been designed that bind to the double-stranded stem of RNA hairpins (Brossalina and Toulm, 1993; Franois et al., 1994; Le Tinvez et al., 1998). In vitro selection offers the unique opportunity to identify oligonucleotides that take advantage of structural peculiarities (loops, bulges, etc.) to establish both canonical (Watson–Crick base pairing) and non-canonical interactions. 7.3.1 RNA–RNA Kissing Complexes

Kissing interactions correspond to the formation of a loop–loop helix between two hairpin loops of complementary sequences. This is a frequently observed intermolecular RNA–RNA interaction (see Brunel et al., 2002 for a review). It turns out that several SELEX experiments run against RNA hairpins identified candidates that organize themselves as hairpins able to generate loop–loop interactions. In vitro selection carried out with an RNA library of candidates showing a 60-nucleotide random region against the TAR element of the virus HIV-1 led to the identification of RNA hairpins with a conserved octameric loop 5lGUCCCAGA (Ducong and Toulm, 1999). Interestingly, the six central bases of this consensus are complementary to the apical hexaloop of the imperfect TAR hairpin. RNase footprinting studies performed with both TAR and the aptamer (R06) exhibiting the lower Kd demonstrated that the complex involves kissing interactions (Fig. 7.1). Under the selection conditions (140 mmol/L K+, 20 mmol/L Na+, 3 mmol/L Mg2+) the equilibrium dissociation constant is about 10 nmol/L. The formation of the TAR–R06 complex is highly dependent on the loop complementarity. The introduction of a point mutation in the TAR loop is detrimental to complex formation. The compensatory mutation in the aptamer loop restores the interaction. However, substituting three GU or three AU pairs for the three GC pairs


Fig. 7.1 Schematic representation of TAR kissing complex formed with either a DNA (D04) or an RNA (R06) aptamer (Collin et al., 2000) (Ducong and Toulm, 1999). Only the apical regions of the TAR RNA (orange; nt 20–42) and of aptamers (black) are shown.

originally present in the loop–loop helix strongly weakens the complex (Ducong and Toulm, 1999). Tertiary interactions account for the high affinity of the aptamer for its target hairpin: the hexamer that corresponds to the central part of the aptamer loop and that consequently retains the base-pairing properties of the aptamer is a weak binder characterized by an equilibrium association constant lowered by more than two orders of magnitude compared with the parent aptamer R06. The aptamer stem has been minimized: starting from the selected R06 (98 nucleotides long) the oligomer was shortened down to 14 nucleotides, leaving a 3 base pair stem, which was sufficient to provide a Kd equivalent to that of the parent aptamer. Even a truncated aptamer with a stem as short as two GC pairs was still able to bind although with a 5-fold reduced efficiency (Kd z 50 nmol/L) (Darfeuille et al., 2004). The two residues closing the aptamer loop are part of the consensus: 45 out of the 46 clones showed a 5lG and a 3lA at these positions. The only exception contained the reverse combination (i.e. 5lA and 3lG). Surface plasmon resonance measurements with a series of R06 variants showed that these positions were indeed crucial for binding to TAR. The Kd increased in the order 5lGA i 5lAG i 5lGG i 5lGU = 5lAA i 5lGC i 5lUA, from about 17 nmol/L to about 200 nmol/L (Ducong et al., 2000). The difference originated essentially in the dissociation rate constant that increased from 1.1 q 10–3 s–1 to 13 q 10 –3 s–1. Some combination such as 5lCU did not give rise to a detectable complex. Interestingly TAR*, a hairpin with a hexameric loop, complementary to the TAR loop, closed by a 5lUA combination was previously rationally designed for structural studies (Chang and Tinoco, 1994, 1997). Both TAR* and R06 behave similarly for binding to TAR at high (10 mmol/L) magnesium concentration. But the association of R06 is less magnesium dependent than that of TAR*. If all TAR loop residues are engaged in the loop–loop helix a direct link is established through the major groove from the 5l position of

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the last C residue in the TAR stem to the 3l position of the C residue at the “top” of the loop–loop duplex, six base pairs away. The structure of the TAR–R06 complex has not been solved yet. However a molecular dynamic study carried out from the structure of the TAR–TAR* complex, suggested that the GA combination was selected for closing the aptamer loop due to optimized stacking interactions at the stem–loop junction and the formation of interbackbone hydrogen bonds (Beaurain et al., 2003). In a similar SELEX experiment Scarabino and co-workers raised RNA aptamers against yeast phenylalanine tRNA (Scarabino et al., 1999). From a pool of about 4 q 1015 independent sequences containing an 80-nucleotide random sequence, aptamers were identified after six rounds of selection. Two unique sequences accounted for more than 50% of the candidates. Under the selection conditions (0.25 mol/L Na+, 50 mmol/L Tris–HCl pH 7.5, 10 mmol/L Mg2+, 2 mmol/L spermidine, 0.2 mmol/L EDTA) the aptamer tRNA complex was characterized by a Kd of about 20 nmol/L. A large proportion of the candidates showed a region complementary to the anticodon loop of tRNAPhe, 7 nt long. This sequence was displayed in the loop of a hairpin structure, similarly to the results obtained with R06, the anti-TAR aptamer, thus allowing kissing interaction to take place. As noted for the anti-TAR aptamer the linear 7-nt antisense sequence corresponding to the anticodon complement did not bind the tRNA efficiently in fair agreement with previous results (Grosjean et al., 1976). Another shared feature between the anti-tRNAPhe and the anti-TAR aptamers is the presence of G,A residues at positions closing the loop. The RNA genome of retroviruses is diploid. Two homologous RNA molecules interact through loop–loop interactions. The association of the HIV genome has been extensively characterized. The dimerization initiation site (DIS) is a hairpin structure. In HIV-1 the 9-nt loop involved in the interaction shows a six-base palindromic sequence (Skripkin et al., 1994). Interestingly only two of the potential 64 combinations are found, suggesting that not all palindromic sequences can promote the formation of stable dimers. In order to identify the DIS motifs that preferentially lead to kissing complexes the loop was partly randomized: two positions of the palindrome and the three other loop residues were randomized, generating a population of 1024 candidates with a random loop sequence 5l NNGUNNACN. After 5–7 selection rounds Lodmell and co-workers (Lodmell et al., 2000, 2001) were able to identify several categories of interacting hairpins. G and C residues were predominantly selected at the central positions. Interestingly, the selected flanking residues also showed a strong bias: A was over-represented at the 5l position and a non-complementary combination (A,A) was strongly preferred for closing the loop (i.e. the one existing in nature). The in vitro selection therefore mostly led to sequences that are naturally present. In other words the DIS elements evolved for generating the most stable dimers. Of note, the non-canonical purine, purine pair closing the DIS hairpin loop contributes to the overall stability of the complex as described above for the anti-TAR and anti-tRNAPhe aptamers. NMR (Mujeeb et al., 1998) and crystallographic data (Ennifar et al., 2001) obtained for the DIS complex underlined stacking interac-


tions involving the sheared A,A pair closing the loop, in fair agreement with the results of a molecular dynamic study carried out on the TAR–R06 complex. Magnesium plays a critical role in kissing complex formation. The stabilization brought by Mg2+ ions on loop–loop interactions is well documented for natural complexes (Gregorian and Crothers, 1995; Jossinet et al., 1999). Two magnesium ions bind to the RNAI–RNAII kissing complex (Lee and Crothers, 1998). The same value was obtained for the R06 aptamer–TAR complex and for its variants. Nevertheless the R06–TAR association is less dependent on magnesium ions than the one formed with the aptamer mutant in which the G,A residues closing the loop were substituted by U,A (Ducong et al., 2000). This might be related to the interbackbone distance: the G,A combination might allow a larger phosphate– phosphate distance, thus reducing the electrostatic repulsion as suggested by molecular dynamics (Beaurain et al., 2003). Kissing interactions constitute a recurrent solution to in vitro selection against RNA hairpins. Indeed several additional examples have been described against different targets. This includes the domain II of the internal ribosome entry site of the hepatitis C virus (HCV) mRNA (Kikuchi et al., 2003) and the 16S RNA A-site (Tok et al., 2000). In the former case a constrained library was used in which the 30-nt-long random region was framed by complementary sequences, thus generating a library of hairpins. The aptamers identified showed a heptanucleotide conserved sequence complementary to the apical loop of the domain II. The best ones gave rise to complexes with a Kd of about 10 nmol/L. 7.3.2 DNA–RNA Kissing Complexes

It is well known that the nucleic acid chemistry drives the conformation; whereas double-stranded DNA adopts a B-type helix structure, the RNA double helix is A-type. These helices are characterized by very different parameters such as the number of residues per turn, the sugar conformation, the groove width, etc. Therefore, not surprisingly the DNA oligomer corresponding to the aptamer R06 does not bind to TAR (Ducong and Toulm, 1999). It has been reported that RNA or DNA aptamers can be identified against the same target, which displays similar properties. For instance both DNA and RNA aptamers have been selected against the reverse transcriptase of HIV-1. They are characterized by identical binding constants, and are competitive inhibitors for binding to the retroviral enzyme, even though their primary and secondary structures are different (Tuerk et al., 1992; Schneider et al., 1995). In vitro selection was carried out against the TAR RNA hairpin using a DNA library with a 30-nt-long random window. Aptamers obtained after 15 rounds displayed an imperfect stem–loop structure, with a consensus octamer 5ld(ACTCCCAT) in the apical loop (Boiziau et al., 1999). The six central bases could potentially pair with the top part of TAR (Fig. 7.1) assuming the opening of the last base pair of the TAR stem, next to the loop. In contrast to what is observed for the TAR–R06 kissing complex, the best DNA aptamer, D04, showed an

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imperfect upper stem suggesting that several nucleotides might connect the aptamer stem and the loop–loop helix. The secondary structure of the TAR–D04 complex was solved by NMR (Collin et al., 2000). This study showed that only five base pairs actually constitute the loop–loop heteroduplex, the upper part of the TAR stem remaining doublestranded (Fig. 7.1). The complex displays a quasi-continuous helix formed by the two stems and the central RNA–DNA helix stacked on the 3l side of each stem. The gap between the 5l residue on the top of each stem and the next residue at the other extremity of the loop–loop helix is filled by one residue (C) on the TAR side and two residues (dC and dA) on the DNA aptamer side (Fig. 7.1). This is a major difference with RNA–RNA kissing complexes, in particular for the TAR–R06 one in which the connection is ensured by a single phosphodiester link. This undoubtedly reflects the different geometries of the RNA–RNA, DNA–DNA, and RNA–DNA helices involved in the TAR–R06 and TAR–D04 complexes. Interestingly, the dC and dA residues constituting the connector for the TAR– D04 kissing complex are part of the consensus; in other words these two nucleotides have been selected, suggesting that they contribute to the stability of the kissing complex. The investigation of the binding properties of point mutants at one connecting position (either dA16 or dC17; see Fig. 7.2a) has been undertaken. None of the D04 variant in which dC17 was substituted by either dA, dG, or dT resulted in a detectable complex by electrophoretic mobility shift assay (Fig. 7.2). A similar result was obtained for dA16. However, a faint band migrating much faster than the wild-type complex was observed when dG was substituted for dA, indicating the formation of a complex less stable than the wild-type one and a conformation

Fig. 7.2 Recognition elements of the anti-TAR DNA aptamer. (a) Analysis of the stem/loop– loop helix connector of the D04 aptamer. The bases at positions 16 and 17 (see Fig. 7.1) of D04 were systematically varied. The TAR-binding ability of the resulting variants was analyzed using an electrophoretic mobility shift assay. Complexes formed by TAR (1 mmol/L) and 32P 5l end-labeled D04 aptamer (1 nmol/L) were either wild-type (wt) or mutated at position 16 or 17 as indicated at the bottom of each lane. Experimental conditions were as described previously (Boiziau et al., 1999). Only the aptamers showing A and C at the 16th and 17th positions, respectively, are fully bound to TAR under these conditions. A16 and C17 were the nucleotides identified (wt) during the SELEX procedure. (b) Determination of the D04-binding motif. Chemical interference analysis has been carried out as described (Pileur et al., 2003). Briefly, 32P 5l end-labeled D04 aptamer has been modified (less than one modification per D04 molecule on average) by

potassium permanganate (T), with dimethyl- n sulfate (G) or by depurination treatment (A,G). Such modified aptamers are then challenged with TAR RNA in a gel-shift experiment, as described in Fig. 7.2a. Retarded bands containing modified D04 for which the modification does not interfere with TAR binding and non-retarded bands containing D04 for which the modified base prevents binding to TAR are eluted separately. After cleavage at the modified positions, the oligomers of each sample, bound (B) or free (F) are analyzed on a sequencing gel side by side (left panel). Bands present in lanes F and absent in lanes B show positions at which modifications strongly interfere with the interaction. The sequence of the DNA oligomer is given on the left side of the autoradiograph. The nucleic acid bases crucial for TAR binding are indicated by a black bar (side of left panel) and by underlined letters on the hairpin structure of the D04 39-mer used for this experiment (right panel).


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different from that of the TAR–D04 kissing complex (Fig. 7.2). These results are in excellent agreement with the outcome of the selection. Whether these connecting residues dA16 and dC17 directly engage some interaction with another part of the molecule or they provide an optimal conformation of the junctions is not known. As in the case of the R06 aptamer, the top part of the D04 stem is also adapted to the interaction with TAR. The upper part of the stem of every selected DNA aptamer was characterized by a predicted low stability (i.e. the presence of unpaired residues or non-canonical base pairs combined with a low G,C content) (Boiziau et al., 1999). The NMR study revealed the formation of a dT,dT pair adjacent to a single dA residue that remained stacked within the aptamer stem (Boiziau et al., 1999). The substitution of one of the paired dT by any residue did not weaken the complex whereas its deletion was detrimental to the association. Chemical interference was used for delineating the role played by the D04 residues in the kissing interactions. Neither the loop, the connector, nor the upper part of the stem tolerate modification, underlining the crucial role played by a large part of the aptamer in the kissing complex formation (Fig. 7.2b). 7.3.3 Double RNA–RNA Kissing Loops

Different natural processes make use of kissing interactions for initiating the association between RNA molecules (Altuvia and Wagner, 2000). In several instances more than one loop–loop interaction is involved. Binding of RNA-I to the 5l end of RNA-II, which controls the initiation of plasmid ColE1 replication, is mediated by the formation of at least two loop–loop interactions (Eguchi et al., 1991). Several small RNA molecules have been suggested to regulate gene expression in E. coli. For instance oxyS, which is induced in response to oxidative stress, represses the translation of two mRNAs coding for the stress signal factor of RNA polymerase rpoS and the transcriptional activator encoded by the fhla gene. In the latter case it was suggested that the two long hairpins which constitute oxyS bind to two non-adjacent stem–loop structures of fhla mRNA, thus sequestering the ribosome-binding site. Both kissing interactions are contributing to the binding as point mutations introduced in either loop weaken the complex (Argaman and Altuvia, 2000). Given these observations it seems that aptamers consisting of two hairpins able to bind simultaneously to two stem–loop structures of a target RNA would be stronger and more specific ligands than a single hairpin. We validated this idea using the aptamer R06 targeted to the TAR RNA motif of HIV-1. The selection described above was carried out against the TAR hairpin of the BRU variant whose loop sequence is 5lCUGGGA. The MAL variant differs from BRU at one position: 5lCCGGGA. Consequently the R06 aptamer selected against BRU is a poor binder of TAR MAL due to an A,C mismatch in the loop–loop helix (Fig. 7.3). A compensatory ApG mutation introduced on the aptamer side restores the association with TAR MAL (Ducong and Toulm, 1999). This rationally de-

7.3 Loop–Loop Interactions Fig. 7.3 Bifunctional kissing aptamer. A bifunctional structures have been chemically synthesized by covalent linking of truncated TAR and R06 variants through a polyethylene glycol (PEG) linker. The BRU variant of TAR has been used as a target for selecting the R06 (BRU) aptamer. The MAL variant of TAR shows a UpC mutation in the loop which is recognized by R06 MAL in which the compensatory ApG mutation has been introduced.

signed R06 MAL does form detectable complexes with TAR BRU thanks to the formation of a G,U pair. A bifunctional target (dTAR) has been synthesized by covalently connecting the 5l end of a TAR MAL hairpin to the 3l end of a TAR BRU stem–loop. Similarly a double dR06 was designed by covalent linking of R06 BRU and R06 MAL hairpins (Fig. 7.3). Both electrophoretic mobility shift assays and surface plasmon resonance showed that the bifunctional aptamer dR06 did bind to the double hairpin target dTAR much more strongly than to either single hairpin. The difference originates essentially in the off rate constant resulting in a Kd lowered by at least one order of magnitude (Boucard et al., in press). In order to confirm these results in vitro selection was carried out using the 3l terminal end of the HCV mRNA as a target. The last 80 nucleotides form two hairpins called SL1 and SL2. We screened a library of about 2 q 1014 candidates with a 128-nt random region. We expected that such long sequences would generate complex structures. If bimodal interactions provide a selective advantage, the population should be enriched in oligonucleotides able to simultaneously engage two (or more) interactions with the target. Indeed, 18 candidates out of 46 cloned sequences at the end of the nineth round displayed two consensus complementary to both SL1 and SL2 loops. Interestingly, secondary structure prediction indicated that these aptamers fold in a two-hairpin structure with the two consensus sequences located in the loops. This means that such aptamers can potentially give rise to a double kissing complex with the SL1–SL2 RNA, equivalent to our model dR06–dTAR (Ledan-Schuester et al., unpublished). The binding properties of the most represented candidate, A1, which was found in seven of the eighteen double hairpin candidates, were investigated by bandshift assay: whereas the aptamer binds to the SL1–SL2 RNA target with a Kd of about 20 nmol/L under the selection conditions, it shows a much reduced affinity

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with the individual hairpins SL1 or SL2 (Kd i500 nmol/L), suggesting that both loops of the aptamer actually bind simultaneously to SL1 and SL2 loops. These results account for the evolution of the population and emphasize the interest of bifunctional kissing aptamers. 7.3.4 Apical Loop–Internal Loop Interactions

The presence of purine–purine “pairs” closing the loop of RNA hairpins giving rise to kissing complexes both in naturally interacting RNA species (the DIS element of HIV-1) and in selected RNA motifs (aptamers to TAR or to tRNAPhe) suggested that this might be a rule for generating stable loop–loop interactions. Indeed the substitution of a GC pair by a G,A combination at the top of the stem of the hairpin RNA Il stabilized its interaction with RNA IIl, two stem–loops derived from the structure involved in plasmid Col E1 replication (Ducong et al., 2000). However, the rational design according to these rules of a kissing hairpin potentially able to interact with the SL1 structure of the HCV mRNA (i.e. a hairpin with a loop complementary to that of SL1 framed by G and A residues) led to a complex of weak stability. Therefore, not every loop is prone to kissing complex formation. This is related to both the size and the sequence of the loop. The most common loop size for kissing complexes is 6–7 nucleotides (Brunel et al., 2002). When a larger loop is involved only part of it contributes to loop–loop helix formation. This is actually driven by the distance that can be spanned by a single ribose phosphodiester unit for connecting the 3l end of a stem to the 5l end of the loop–loop helix, stacked on the 3l strand of the stem, through the major groove of the “kiss” (Haasnoot et al., 1986). RNA aptamers selected against the SL1 hairpin within a library of 1013 candidates with a 40-nt random window led, after 10 rounds of selection, to aptamers containing 6–10 contiguous nucleotides complementary to the apical part of SL1 (Aldaz-Carroll et al., 2002). This might allow the formation of a helix comprising the entire 6-nt loop and the top part of the stem, of the target hairpin assuming the opening of the double-stranded region. The consensus motif was invariably located in a loop of the aptamers. Interestingly the selected sequences could be ranked in two different classes. Hairpins in which the consensus motif is displayed in the apical loop led to mediocre ligands characterized by a Kd > 1 mmol/L, confirming the result obtained with the rationally designed kissing aptamer (see above). The largest family of anti-SL1 aptamers actually corresponds to imperfect hairpins with a prominent internal loop complementary to the apical loop of the target, thus generating apical loop–internal loop (ALIL) complexes (Fig. 7.4) as demonstrated by footprinting and mutational analysis for the strongest ligand, the aptamer 5–39 (Aldaz-Carroll et al., 2002). As previously discussed for the kissing RNA aptamer R06 targeted to TAR, tertiary interactions contribute to the stability of the ALIL complex in addition to the formation of the loop–loop helix.

7.3 Loop–Loop Interactions Fig. 7.4 Aptamer giving rise to an apical loop–internal loop (ALIL) complex. The consensus sequence (orange) of such an aptamer is invariably located in an internal loop complementary to the apex of the target hairpin (thin lines in the right scheme), giving rise to a loop–loop helix through base pairing (blue dots). Note that the loop–loop helix may involve only part of the internal loop.

First, the deletion of the top part of the aptamer 5–39 (above the internal loop) abolishes its binding to SL1 and secondly an antisense RNA 9-mer identical to the internal loop sequence displays a Kd about 20-fold higher than that of the intact aptamer. In both cases the molecules retained the base-pairing properties of the parent aptamer. In contrast, the deletion of the apical loop or its substitution by a non-nucleotide linker did not prevent the binding of the 5–39 aptamer to SL1 (Aldaz-Carroll et al., 2002). Interestingly the upper and lower stems flanking the internal loop of the aptamers selected against SL1 displayed a very high G,C content: the upper stem of the aptamer 5–39 is composed of four GC pairs whereas five out of the six pairs of the lower stem adjacent to the internal loop are also GC ones. Such ALIL-forming aptamers have been independently selected in our laboratory against three different targets: namely the hairpins corresponding to domain IV (Aldaz-Carroll et al., 2002) and to the top part of domain II of the internal ribosome entry site (IRES) of the hepatitis C virus mRNA (Da Rocha Gomes et al., 2004). These aptamers shared the following properties which consequently can be considered as characteristics: An internal loop containing a sequence complementary to the apical loop of the target hairpin. The loop–loop helix contained potentially 6–9 base pairs. A few nucleotides (two or three) connect the sequence, giving rise to the loop–loop helix on either the lower or the upper stem of the aptamer. On the target a single nucleotide constitutes a link between the stem and the loop–loop helix. This is at variance from the kissing complexes for which no nucleotide is required in the connector, even though the same number of base pairs (6–7) are formed in the loop–loop association. G,C-rich double-stranded stems on both 5l and 3l sides of the internal loop. x

x

x

Kikuchi et al. (2005) isolated aptamers targeted to domain IIId of the HCV IRES which fulfilled several of the above criteria. The strongest ligands showed five bases, complementary to part of the apical loop of the domain IIId, in a 6-ntlong internal loop. The authors demonstrated the formation of an ALIL complex using RNase footprinting. Recently a study aiming at identifying the determi-

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nants of the interaction between tRNA and the tyrST box antiterminator of Bacillus subtilis led to the unanticipated selection of aptamers that generate ALIL associations (Fauzi et al., 2005). In this complex a seven base pair loop–loop helix could be formed. The internal loop involved in the interaction is flanked by two GC-rich stems: seven out of the eight base pairs are GC pairs. The internal loop is connected to the stems by one or three residues depending on the formation of a GU pair at the bottom of the upper stem. Therefore this fulfills the criteria of ALIL complexes. Interestingly, natural RNA–RNA complexes are known that do engage ALIL interactions. A first example is constituted by the bicoid (bcd) mRNA that codes for a protein involved in the development pattern of Drosophila melanogaster (Ferrandon et al., 1997). cis-Acting sequences have been identified within the 3l-UTR of bcdmRNA that are responsible for the dimerization of the message. These regions contain two complementary sequences AAGCCC and GGGCUU located in an apical loop and an internal loop, respectively. Indeed, dimers can be formed by these elements that involve two ALIL interactions. The B. subtilis bacteriophage phi29 provides a second example of such interactions. The packaging of the phage DNA involves the virus-encoded pRNA that shows the propensity to generate an hexamer (Guo et al., 1998). The packaging unit results from an ALIL association giving rise to “hand-by-arm” interaction. Therefore ALIL complexes constitute a motif for RNA–RNA recognition and assembly.

7.4 Chemically Modified Aptamers Recognizing RNA Targets

For a number of reasons it might be desirable to introduce additional functions into an aptamer. For instance the selected oligonucleotides might be converted into beacons or used as inhibitors of an enzyme. The use of RNA (or DNA) aptamers in a biological context is of limited interest as natural nucleic acids are rapidly degraded by nucleases. This problem has been addressed for many years by a number of investigators in the field of antisense oligonucleotides. Numerous nuclease-resistant analogs have been synthesized over the last two decades (Crooke, 2004; Toulm, 2001). However their use in the frame of in vitro selection raises two kinds of problems. First, only a limited number of modified nucleoside triphosphates are incorporated by polymerases used in the different steps of SELEX. This includes 2l-fluoro- and 2l-aminopyrimidines (Eaton and Pieken, 1995), 2l-O-methyl-nucleosides (Chelliserrykattil and Ellington, 2004) and phosphorothioates (Androla et al., 2000) as well as modification on the C,5l position of pyrimidines (Latham et al., 1994). Alternatively, chemical modification can be introduced post selection, but care should be taken to avoid changing the binding properties of the selected sequence. In general, converting the full-length RNA or DNA aptamer into a chemically modified derivative weakens or abolishes the properties of the oligomers. Therefore one should carefully identify the positions that can be modified, as exemplified by the RNA pseudo-knot selected against the


reverse transcriptase of HIV-1. In this case interference analysis was used to identify the two positions that had to be retained as ribo residues. This allowed the design of a 2l-O-methyl derivative of the 26-nt-long aptamer, modified at every nucleoside except these particular two, which displayed binding properties similar to the originally selected molecule (Green et al., 1995). A few examples of chemically modified aptamers targeted to nucleic acids are available. The only attempt at generating nuclease-resistant derivatives through a standard SELEX procedure was carried out using DNA enantiomers, so-called “spiegelmers”. In this approach natural d-DNA or d-RNA sequences are selected against the mirror image of the target. The l-versions of the identified aptamers are then chemically synthesized as ligands of the natural target. This strategy has been successfully used against amino acids and peptides (Klussmann et al., 1996; Williams et al., 1997 ; Wlotzka et al., 2002). We attempted to identify l-DNA ligands of the TAR motif. A truncated l-TAR RNA hairpin 27 nt long was chemically synthesized. As a proof of concept we showed that the l version of D04, a DNA aptamer, previously selected against the natural TAR RNA (see above), was able to bind to l-TAR: the l-TAR/l-D04 complex was characterized by a Kd identical to that of d-TAR/d-D04 (Toulm et al., 2002). A SELEX experiment was carried out with a DNA library containing sequences with a 50-nt randomized region. We failed to identify aptamers able to recognize l-TAR with an affinity lower than 100 mmol/L. As l-oligomers form very poor complexes with d-nucleic acids (Garbesi et al., 1993) this suggests that there is no alternative to the recognition of natural nucleic acids by l-oligomers. In some studies, chemical modifications aiming at designing nuclease-resistant aptamers targeted to RNA hairpins have been introduced post selection into RNA aptamers. A number of possibilities have been investigated for designing derivatives of R06, the RNA aptamer targeted to the TAR hairpin, generating a loop– loop complex with the HIV-1 RNA motif. In this particular case it was tempting to focus on analogs known to generate duplexes that mimic RNA–RNA helices (i.e. which retain the A-type geometry) (Fig. 7.5). 2l-O-Methyl oligoribonucleotides (OMe) (Lamond and Sproat, 1993) and N3lpP5l phosphoramidate oligodeoxyribonucleotides (NP) (Ding et al., 1996) fulfill this requirement. Indeed fully OMeor NP-modified R06 aptamers bind to TAR with rate constants and equilibrium constants similar to those of the original R06 RNA aptamer (Table 7.1) (Darfeuille et al., 2001, 2002a,b). In contrast, the aptamer fully modified with “locked nucleic acid” (LNA) residues did not form a detectable complex using either UV absorption-monitored melting transition (Tm) or surface plasmon resonance (Darfeuille et al., 2004), whereas the analog containing exclusively “hexitol nucleic acid” (HNA) showed a reduced affinity for TAR, mostly due to an increased off rate (Kolb et al., 2005) (Table 7.1). LNA and HNA oligomers have residues locked in positions mimicking the furanose ring in the 2l exo, 3l endo conformation, thus leading to regular duplexes of increased stability with complementary RNA sequences (Orum and Wengel, 2001). Such a conformation might not be adapted to kissing interactions all over the aptamer sequence. In particular, even though the structure of the

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Fig. 7.5 Chemically modified aptamers. (a) Modifications that have been used in the synthesis of RNA (R06) anti-TAR aptamer analogs: OMe, 2l-O-methyl; NP, N3lpP5l phosphoramidate; HNA, hexitol nucleic acid;

LNA, locked nucleic acid. (b) LNA–DNA (right) and HNA–RNA (left) chimeric derivatives of R06 discussed in the text and listed in the Table 7.1. Modified residues are indicated in orange.

R06–TAR loop–loop complex is not yet solved, it is likely that the conformation of the loop closing nucleosides is critical, as indicated by the selection of G and A residues at these positions. Indeed, LNA–DNA and HNA–RNA chimeras known to adopt A-type geometries, for which the key G and A residues were kept unmodified, displayed improved binding properties compared with fully modified LNA and HNA derivatives (Darfeuille et al., 2004; Kolb et al., 2005). Among several combinations of modified and unmodified residues, some were able to bind to TAR with an affinity similar to that of the parent R06 aptamer (Table 7.1). The introduction of LNA or HNA residues in the stem of the aptamer allowed it to truncate down to four base pairs, leading to a folded structure more stable than the selected RNA oligomer R06.


183

Table 7.1 Properties of unmodified RNA aptamer targeted to the TAR element of HIV-1 (R06) and of chemically modified analogs (N3lpP5l phosphoramidate (NP), 2l-O-methyl (OMe), locked nucleic acid (LNA), hexitol nucleic acid (HNA) and of LNA–DNA and HNA–RNA chimeras (see Fig. 7.5) Tm (hC)

kon q 104 (L/mol.s)

koff q 10 –4 (s–1)

Kd (nmol/L)

Tat IC50 References comp. (nmol/L)

R06

30.3 e 1.4

3.9 e 0.9

2.6 e 0.5

6.9 e 2.9

–

i 4000

Ducong et al., 2000

R06 NP

28.6 e 0.4

10.2 e 0.1

3.5 e 0.3

3.4 e 0.3

+

400

Darfeuille et al., 2002b

R06 OMe

29.0 e 0.2

9.0 e 0.3

8.9 e 0.6

9.9 e 1.0

nd

400

Darfeuille et al., 2002a

R06 LNA

I 10

nd

nd

nd

–

nd

R06 LNA–DNA

31.8 e 0.4

3.5 e 0.2

8.2 e 1.3

21.1 e 2.1

+

R06 HNA

20.0 e 0.3

13.9 e 6.4

115 e 7.0

82.6 e 5.5

–

nd

Kolb et al., 2005

9.0 e 2.1

1.6 e 0.1

1.7 e 0.3

–

I 750

Kolb et al., 2005

R06 HNA–RNA 28.0 e 2.1

Darfeuille et al., 2004 Darfeuille et al., 2004

Tm-values have been deduced from equilibrium melting curves as described in the references listed in the last column to the right. Generally 1 mmol/L of each oligomer were mixed in a buffer containing 140 mmol/L potassium chloride, 20 mmol/L sodium chloride, and 0.3 mmol/L magnesium chloride. The kon, koff, and Kd values were calculated from surface plasmon resonance measurements carried out in 20 mmol/L HEPES buffer pH 7.3 containing 140 mmol/L potassium acetate, 20 mmol/L sodium acetate, and 3 mmol/L magnesium acetate at room temperature, assuming a pseudo-first order model. The competition assay (Tat comp.) between a 36-amino-acid long Tat peptide and the aptamer derivatives was analysed by electrophoresis. The “+” and “–“ indicate that the addition of the aptamer resulted (or not, respectively) in the concentration-dependent decrease of the band corresponding to the Tat36 –TAR complex and in the subsequent increase of that corresponding to the aptamer–TAR complex. IC50 is the aptamer concentration that induced 50% reduction in the transcription efficiency of a TAR-containing template in a cell-free assay (see text). nd, not determined.

In addition, these chimeras exhibit an improved nuclease resistance: whereas R06 was fully degraded after 1 h incubation in cell culture medium containing 10% fetal calf serum, no significant degradation of the LNA–DNA chimera was observed after 20 h (Darfeuille et al., 2004). These chemically modified aptamers retain a high specificity of interaction with the target TAR hairpin: (1) the introduction of point mutation in the loop of the aptamer abolishes complex formation, (2) the modified aptamers discriminate between the BRU and MAL variants which differ by a UpC mutation in the loop, (3) the G,A combination at closing positions remains crucial for kissing interactions; its substitution by other ones weakens or abolishes the association of the aptamer with TAR.

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7.5 Biological Properties of Aptamers Targeted to Nucleic Acids

The strong and highly selective ligands identified by SELEX are of potential interest for artificial regulation of gene expression. The nuclease-resistant oligomers targeted to the TAR element of HIV-1 are particularly suitable for such a purpose. This 57-nt-long imperfect hairpin is transcribed from the R region of the long terminal repeat of the HIV-1 genome (Karn, 1999). The TAR hairpin located at the very 5l end of the retroviral mRNA serves as the binding site of the HIV protein Tat and of two cellular proteins, namely cyclin T1 and CDK-9 (Karn, 1999; Rana and Jeang, 1999). This Tat-associated kinase phosphorylates the C-terminal domain of RNA polymerase and consequently trans-activates the transcription of the viral genome. Whereas the cellular proteins bind to the apex of the TAR hairpin, the viral protein Tat recognizes a tripyrimidine bulge on the 5l side of the hairpin, located four base pairs away from the apical loop. Therefore the aptamers forming a kissing complex with the TAR loop might compete with the proteins and consequently interfere with the trans-activation of transcription. Electrophoretic mobility shift assays have shown that both the NP analog (Darfeuille et al., 2001) and the LNA–DNA chimera of aptamer R06 displaced a 36-amino-acid Tat peptide (Tat36), which retains the binding properties of the full-length protein (Churcher et al., 1993) from TAR. This competition occurred even though the magnesium concentration was not optimal for aptamer binding: as the Tat36 peptide did not bind under the selection conditions (3 mmol/L) the competition was carried out at low magnesium concentration (20 mmol/L). The Tat36 peptide was specifically displaced by these aptamer derivatives; variants of the loop closing combination that do not bind to TAR did not show any effect on the Tat36 –TAR complex. As there is no overlap between the binding sites of the two ligands this suggests that the conformation of the bulge in these TAR–aptamer complexes is not appropriate for Tat binding. It is known that TAR is rearranged upon association with Tat (Aboul-Ela et al., 1995). This induced conformational change might be precluded in the TAR–aptamer kissing complex. However, this does not constitute a general rule for anti-TAR aptamer derivatives. Indeed no Tat36 displacement is induced by the HNA–RNA chimeric aptamer (Kolb et al., 2005). Instead a trimolecular complex was detected due to the simultaneous binding of the Tat peptide and HNA derivative to TAR. This indicates that different conformations are adopted by the top part of TAR in the presence of the aptamer derivatives even though the stability of the complexes is very similar. Interestingly enough, the LNA–DNA chimeric octamer that corresponds to the top loop of the anti-TAR aptamer does not compete with Tat for binding to TAR even though the Tm-values of the TAR–aptamer and TAR–octamer complexes are identical (Darfeuille, unpublished results). This underlines the finding that the conformation of the loop– loop helix is different from that of a sense–antisense duplex. As mentioned above the TAR element plays a key role in the trans-activation of transcription. The effect of anti-TAR aptamers was investigated in an in vitro tran-

7.6 Conclusion

scription assay using a template corresponding to the 5l portion of the HIV genome (strain NL4–3). This DNA fragment directed the synthesis of two RNAs in HeLa cell nuclear extract corresponding to a synthetic terminator and to run-off products. The synthesis of these two fragments is stimulated upon the addition of the Tat protein, as expected for TAR-dependent transcription. The addition of the RNA aptamer did not induce any effect. The unmodified aptamer is likely rapidly degraded in the cell extract. In contrast the 2l-O-methyl (Darfeuille et al., 2002a) and the phosphoramidate fully modified analogs of R06 (Darfeuille et al., 2001b) as well as the LNA–DNA and HNA–RNA chimeras (Kolb et al., 2005) induced a dose-dependent inhibition of the transcription with an IC50 of about 500 nmol/L (Table 7.1). This effect is specific and should be related to the aptamer–TAR kissing association, as mutated chemically modified aptamers for which the G,A closing residues have been substituted by bases that abolish the binding to TAR do not show any inhibitory property. These aptamer derivatives decrease TAR-dependent transcription but do not induce any effect on the transcription driven by the cytomegalovirus (CMV) promoter. Therefore these aptamers constitute specific inhibitors of transcription. RNA aptamers giving rise to ALIL complexes with the HCV mRNA were also evaluated from the standpoint of their biological interest. The aptamers targeted to the SL1 structure in the 3l-UTR and to the domains II and IV of the IRES showed a weak or no effect on in vitro translation of HCV IRES-dependent mRNA translation (Aldaz-Carroll et al., 2002; Tallet-Lopez et al., 2003). The anti-SL1 aptamer also had no effect on RNA replication with the viral RNA-dependent RNA polymerase in an in vitro assay (Staedel and Astier-Gin, unpublished results). In contrast, the aptamer selected against the IIId domain of the IRES induced a decrease of luciferase activity both in a cell-free assay and in HeLa cells transiently transfected with a construct in which the reporter gene was under the control of the HCV IRES (Kikuchi et al., 2005). The transfection of 0.5 pmol of the aptamer reduced the luciferase activity to 45%. This effect was moderately specific. Whereas no effect was observed when RNA from the starting pool was used, mutated aptamers in which the internal loop was destroyed showed some inhibition, suggesting that other interactions, in addition to loop–loop associations, might contribute to the effect (Kikuchi et al., 2005). However these aptamer variants were not inhibitory in a cell-free assay in rabbit reticulocyte lysate.

7.6 Conclusion

Aptamers have great potential in many applications as shown by multiple studies over the last 15 years, both in diagnostics and in therapeutics. A smaller number of investigations have been dedicated to the targeting of nucleic acids than to proteins or small molecules. Limited information has been obtained from in vitro selection carried out against double-stranded DNA. The solutions for recognizing regular double helix might be restricted to the previously characterized triple

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helix formation. It may be of interest to investigate the recognition of abnormal local structures resulting, for instance, from the presence of damage. In the light of investigations on RNA structures this would likely lead to the selection of aptamers of interest. Multiple natural RNA–RNA complexes are known that involve loop–loop interactions; most of them are responsible for the control of important biological processes (replication, packaging, translation, etc.). The in vitro selection performed against several RNA hairpins both in our laboratory and in others clearly identified two types of RNA–RNA loop–loop interactions. The association between the apical loops of the two partners, namely the target and the aptamer hairpins, results in the formation of kissing complexes. Alternatively, the RNA–RNA interaction might favor the involvement of an internal loop. The parameters that drive the formation of either kissing or ALIL complexes are presently unknown but undoubtedly reside in the loop characteristics and the loop–stem junctions. A fournucleotide motif, YUNG, has been recognized as stabilizing sense–antisense RNA kissing complexes in prokaryotes (Brunel et al., 2002). In vitro selection carried out recently on pools of hairpins with a randomized loop identified new motifs promoting the formation of stable kissing complexes (Dausse, unpublished results). As previously demonstrated for other classes of targets, aptamers selected in vitro retain their properties in a cell context. The ALIL-forming aptamers to the IIId domain of the HCV IRES reduce mRNA translation (Kikuchi et al., 2005). The aptamer R06 targeted to the TAR element expressed from an appropriate construct in a stably transfected HeLa cell line specifically inhibits the expression of the b-galactosidose gene under the control of the HIV-1 long terminal repeat (Reigadas and Kolb, unpublished results). The design of numerous oligonucleotide analogs in antisense studies together with the structural information available on complexes that such derivatives form with RNA reduces the risk of failure when introducing chemical modifications in the aptamers once selection is completed. Indeed, successful results were obtained with several RNA mimics of the anti-TAR aptamer. An LNA-2l-O-methyl chimeric derivative of R06, which is a strong and specific binder of the TAR element, displays anti-HIV activity in cultured cells (Reigadas and Di Primo, unpublished results). Recently a 2l-O-methyl analog of a bifunctional aptamer targeted to the 3l-UTR of the HCV mRNA was shown to display interesting binding properties. In addition it reduced the replication of HCV RNA in cultured cells (Ledan-Schuester and Staedel, unpublished results). The results discussed in this chapter demonstrate that in vitro selection carried out against RNA elements brings interesting information on tertiary interactions. This will contribute to the extension of our knowledge on the 3D structure of RNA. The effect of aptamers on gene expression either produced in situ or transfected in a recipient cell as chemically modified analogs also has potential interest both for deciphering the function of a target gene and for the design of therapeutic agents. Aptamers extend the repertoire of oligonucleotides that can be used for functional genomics.

References

Acknowledgments

The contribution of past and present collaborators from our laboratory on the work described here is gratefully acknowledged. Particular thanks to those who shared unpublished results: D. Boucard, G. Kolb, E. Ledan-Schuester, S. Reigadas, C. Staedel. This work has been supported by INSERM, ANRS, SIDACTION and the European Union (5th FWP).

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Boiziau, C., Dausse, E., Yurchenko, L., Toulm, J. J. (1999). DNA aptamers selected against the HIV-1 TAR RNA element form RNA/DNA kissing complexes. J Biol Chem 274, 12730–12737. Brion, P., Westhof, E. (1997). Hierarchy and dynamics of RNA folding. Annu Rev Biophys Biomol Struct 26, 113–137. Brossalina, E., Toulm, J. J. (1993). A DNA hairpin as a target for antisense oligonucleotides. J Am Chem Soc 115, 796–797. Brunel, C., Marquet, R., Romby, P., Ehresmann, C. (2002). RNA loop-loop interactions as dynamic functional motifs. Biochimie 84, 925–944. Chang, K. Y., Tinoco, I. (1994). Characterization of a “kissing” hairpin complex derived from the human immunodeficiency virus genome. Proc Natl Acad Sci USA 91, 8705– 8709. Chang, K. Y., Tinoco, I. (1997). The structure of an RNA “kissing” hairpin complex of the HIV TAR hairpin loop and its complement. J Mol Biol 269, 52–66. Chelliserrykattil, J., Ellington, A. D. (2004). Evolution of a T7 RNA polymerase variant that transcribes 2l-O-methyl RNA. Nat Biotechnol 22, 1155–1160. Churcher, M. J., Lamont, C., Hamy, F., Dingwall, C., Green, S. M., Lowe, A. D., Butler, J. G., Gait, M., Karn, J. (1993). High affinity binding of TAR RNA by the human immunodeficiency virus type-1 tat protein requires base-pairs in the RNA stem and amino acid residues flanking the basic region. J Mol Biol 230, 90–110. Collin, D., Heijenoort, C., Boiziau, C., Toulm, J. J., Guittet, E. (2000). NMR characterization of a kissing complex formed between the TAR RNA element of HIV-1 and a DNA aptamer. Nucleic Acids Res 28, 3386–3391.

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7 Aptamers to Nucleic Acid Structures Compagno, D., Lampe, J. N., Kutyavin, I. V., Yurchenko, L., Lukhtanov, E. A., Bourget, C., Gorn, V., Gamper, H. B., Toulm, J. J. (1999). Antisense oligonucleotides containing modified bases inhibit translation of Leishmania amazonensis mRNAs by invading the mini-exon hairpin. J Biol Chem 274, 8191–8198. Costa, M., Michel, F. (1997). Rules for RNA recognition. EMBO J 16, 3289–3302. Crooke, S. T. (2004). Progress in antisense technology. Annu Rev Med 55, 61–95. Da Rocha Gomes, S., Dausse, E., Toulm, J. J. (2004). Determinants of apical loop-internal loop RNA-RNA interactions involving the HCV IRES. Biochem Biophys Res Commun 322, 820–826. Darfeuille, F., Cazenave, C., Gryaznov, S., Ducong, F., Di Primo, C., Toulm, J. J. (2001). RNA and N3lpP5l kissing aptamers targeted to the trans-activation responsive (TAR) RNA of the human immunodeficiency virus-1. Nucleosides Nucleotides Nucleic Acids 20, 441–449. Darfeuille, F., Arzumanov, A., Gait, M. J., Di Primo, C., Toulm, J.-J. (2002a) 2l-Omethyl-RNA hairpins generate loop-loop complexes and selectively inhibit HIV-1 Tat-mediated transcription. Biochemistry 41, 12186–12192. Darfeuille, F., Arzumanov, A., Gryaznov, S., Gait, M. J., Di Primo, C., Toulm, J. J. (2002b) Loop-loop interaction of HIV-1 TAR RNA with N3lpP5l deoxyphosphoramidate aptamers inhibits in vitro Tat-mediated transcription. Proc Natl Acad Sci USA 99, 9709–9714. Darfeuille, F., Hansen, J. B., Orum, H., Di Primo, C., Toulm, J. J. (2004). LNA/DNA chimeric oligomers mimic RNA aptamers targeted to the TAR RNA element of HIV-1. Nucleic Acids Res 32, 3101–3107. Ding, D., Gryaznov, S. M., Lloyd, D. H., Chandrasekaran, S., Yao, S., Ratmeyer, L., Pan, Y., Wilson, W. D. (1996). An oligodeoxyribonucleotide N3lpP5l phosphoramidates form an A-type helix in solution. Nucleic Acids Res 24, 354–360. Ducong, F., Toulm, J. J. (1999). In vitro selection identifies key determinants for looploop interactions: RNA aptamers selective for the TAR RNA element of HIV-1. RNA 5, 1605–1614.

Ducong, F., Di Primo, C., Toulm, J. J. (2000). Is a closing “GA pair” a rule for stable loop-loop RNA complexes? J Biol Chem 275, 21287–21294. Eaton, B. E., Pieken, W. A. (1995). Ribonucleosides and RNA. Annu Rev Biochem 64, 837–863. Eguchi, Y., Itoh, T., Tomizawa, J. (1991). Antisense RNA. Annu Rev Biochem 60, 631–652. Ennifar, E., Walter, P., Ehresmann, B., Ehresmann, C., Dumas, P. (2001). Crystal structures of coaxially stacked kissing complexes of the HIV-1 RNA dimerization initiation site. Nat Struct Biol 8, 1064–1068. Fauzi, H., Jack, K. D., Hines, J. V. (2005). In vitro selection to identify determinants in tRNA for Bacillus subtilis tyrS T box antiterminator mRNA binding. Nucleic Acids Res 33, 2595–2602. Ferrandon, D., Koch, I., Westhof, E., NussleinVolhard, C. (1997). RNA-RNA interaction is required for the formation of specific bicoid mRNA 3l UTR-STAUFEN ribonucleoprotein particles. EMBO J 16, 1751–1758. Franois, J. C., Thuong, N., Hlne, C. (1994). Recognition and cleavage of hairpin structures in nucleic acids by oligodeoxynucleotides. Nucleic Acids Res 22, 3943–3950. Garbesi, A., Capobianco, M. L., Colonna, F. P., Tondelli, L., Arcamone, F., Manzini, G., Hilbers, C. W., Aelen, J. M. E., Blommers, M. J. J. (1993). L-DNAs as potential antimessenger oligonucleotides – a reassessment. Nucleic Acids Res 21, 4159–4165. Giovannangeli, C., Hlne, C. (2000). Triplexforming molecules for modulation of DNA information processing. Curr Opin Mol Ther 2, 288–296. Gold, L., Polisky, B., Uhlenbeck, O., Yarus, M. (1995). Diversity of oligonucleotide functions. Annu Rev Biochem 64, 763–797. Green, L., Waugh, S., Binkley, J. P., Hostomska, Z., Hostomsky, Z., Tuerk, C. (1995). Comprehensive chemical modification interference and nucleotide substitution analysis of an RNA pseudoknot inhibitor to HIV-1 reverse transcriptase. J Mol Biol 247, 60–68. Gregorian, R. S., Crothers, D. M. (1995). Determinants of RNA hairpin loop-loop complex stability. J Mol Biol 248, 968–984. Grosjean, H., Soll, D. G., Crothers, D. (1976). Studies of the complex formed between

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Kolb, G., Reigadas, S., Boiziau, C., Van Aerschot, A., Arzumanov, A., Gait, M., Herdewijn, P., Toulm, J. J. (2005). Hexitol nucleic acid-containing aptamers are efficient ligands of HIV-1 TAR RNA. Biochemistry 44, 2926–2933. Lamond, A. I., Sproat, B. S. (1993). Antisense oligonucleotides made of 2l-O-alkylRNA – their properties and applications in RNA biochemistry. FEBS Lett 325, 123–127. Latham, J. A., Johnson, R., Toole, J. J. (1994). The application of a modified nucleotide in aptamer selection: novel thrombin aptamers containing 5-(1-pentynyl)-2l-deoyuridine. Nucleic Acids Res 22, 2817–2822. Le Doan, T., Perrouault, L., Chassignol, M., Thuong, N. T., Hlne, C. (1987). Sequencetargeted chemical modifications of nucleic acids by complementary oligonucleotides covalently linked to porphyrins. Nucleic Acids Res 15, 8643–8659. Le Tinvez, R., Mishra, R. K., Toulm, J. J. (1998). Selective inhibition of cell-free translation by oligonucleotides targeted to a mRNA structure. Nucleic Acids Res 26, 2273–2278. Lee, A. J., Crothers, D. M. (1998). The solution structure of an RNA loop-loop complex: the ColE1 inverted loop sequence. Structure 6, 993–1005. Lodmell, J. S., Ehresmann, C., Ehresmann, B., Marquet, R. (2000). Convergence of natural and artificial evolution on an RNA loop-loop interaction: The HIV-1 dimerization initiation site. RNA 6, 1267–1276. Lodmell, J. S., Ehresmann, C., Ehresmann, B., Marquet, R. (2001). Structure and dimerization of HIV-1 kissing loop aptamers. J Mol Biol 311, 475–490. Mir, K. U., Southern, E. M. (1999). Determining the influence of structure on hybridization using oligonucleotide arrays. Nat Biotechnol 17, 788–792. Mishra, R. K., Toulm, J. J. (1994). In vitro selection of antisense oligonucleotides targeted to a hairpin structure. CR Acad Sci Paris 317, 977–982. Mishra, R. K., Le Tinvez, R., Toulm, J. J. (1996). Targeting nucleic acid secondary structures by antisense oligonucleotides designed through in vitro selection. Proc Natl Acad Sci USA 93, 10679–10684.

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7 Aptamers to Nucleic Acid Structures Moser, H. E., Dervan, P. B. (1987). Sequencespecific cleavage of double helical DNA by triple helix formation. Science 238, 645–650. Mujeeb, A., Clever, J. L., Billeci, T. M., James, T. L., Parslow, T. G. (1998). Structure of the dimer initiation complex of HIV-1 genomic RNA. Nat Struct Biol 5, 432–436. Orum, H., Wengel, J. (2001). Locked nucleic acids: a promising molecular family for gene-function analysis and antisense drug development. Curr Opin Mol Ther 3, 239– 243. Osborne, S. E., Ellington, A. E. (1997). Nucleic acid selection and the challenge of combinatorial chemistry. Chem Rev 97, 349–370. Pei, D., Ulrich, H. D., Schultz, P. G. (1991). A combinatorial approach toward DNA recognition. Science 253, 1408–1411. Pileur, F., Androla, M. L., Dausse, E., Michel, J., Moreau, S., Yamada, H., Gaidamakov, S. A., Crouch, R. J., Toulm, J. J., Cazenave, C. (2003). Selective inhibitory DNA aptamers of the human RNase H1. Nucleic Acids Res 31, 5776–5788. Rana, T. M., Jeang, K. T. (1999). Biochemical and functional interactions between HIV-1 Tat protein and TAR RNA. Arch Biochem Biophys 365, 175–185. Scanlon, K. J. (2004). Anti-genes: siRNA, ribozymes and antisense. Curr Pharm Biotechnol 5, 415–420. Scarabino, D., Crisari, A., Lorenzini, S., Williams, K., Tocchini-Valentini, G. P. (1999). tRNA prefers to kiss. EMBO J 18, 4571– 4578. Schneider, D. J., Feigon, J., Hostomsky, Z., Gold, L. (1995). High-affinity ssDNA inhibitors of the reverse transcriptase of type 1 human immunodeficiency virus. Biochemistry 34, 9599–9610. Skripkin, E., Paillart, J. C., Marquet, R., Ehresmann, B., Ehresmann, C. (1994). Identification of the primary site of the human immunodeficiency virus type 1 RNA dimerization in vitro. Proc Natl Acad Sci USA 91, 4945–4949. Soukup, G. A., Ellington, A. D., Maher, L. J. (1996). Selection of RNAs that bind to duplex DNA at neutral pH. J Mol Biol 259, 216–228.

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8.1 Introduction

8 Riboswitches: Natural Metabolite-binding RNAs Controlling Gene Expression Adam Roth, Rdiger Welz, and Ronald R. Breaker

8.1 Introduction

The realization that RNA molecules are not just conduits for the storage and transmission of genetic information, but can adopt complex tertiary structures in order to catalyze chemical transformations, profoundly altered our perspective on the function of this biopolymer (Cech, 2002). Not long after this discovery, the scope of RNA function was widened yet further with the demonstration that, in addition to their natural roles as catalysts, RNA molecules selected in vitro can serve as specific and avid receptors for small molecule ligands (Hermann and Patel, 2000). As more examples of these ligand-binding RNAs, or aptamers, were identified in the laboratory, it became quite clear that catalysis and molecular recognition, long considered the duties of proteins exclusively, were roles that could also be fulfilled by RNA. Moreover, the capacity of RNA for interdomain communication had been demonstrated by engineered chimeric RNAs, in which distinct aptamer and ribozyme modules were linked to generate allosteric ribozymes whose activities could be regulated by concentrations of the relevant target ligand (Soukup and Breaker, 2000). In light of this newly appreciated potential, the question was posed broadly as to whether nature had exploited this capacity of RNA (Gold et al., 1995). Indeed, several intriguing cases had been reported concerning the regulation of bacterial genes involved in the biosynthesis or transport of the coenzymes adenosylcobalamin (AdoCbl) (Wei et al., 1992), thiamine pyrophosphate (TPP) (Miranda-Rios et al., 2001), and flavin mononucleotide (FMN) (Kreneva and Perumov, 1990). In these instances, the corresponding 5l non-coding regions contained sequences that in some cases were known to be highly conserved and that were necessary for the feedback inhibition of gene expression. As no auxiliary protein factors had been identified that effected this regulation (Nudler and Mironov, 2004), it was speculated that coenzyme concentrations might be monitored through direct interactions with the RNA motifs embedded within the 5l untranslated regions (5l-UTRs) of the respective mRNAs (Gelfand et al., 1999; Nou and Kadner, The Aptamer Handbook. Edited by S. Klussmann Copyright c 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-31059-2

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2000; Stormo and Ji, 2001). Subsequent biochemical and genetic analyses of these conserved sequence elements demonstrated that, indeed, they encode RNA structures, or riboswitches, that control expression of downstream genes through direct binding interactions with their respective coenzyme ligands (Mironov et al., 2002; Nahvi et al., 2002; Winkler et al., 2002a, 2002b). Analogous reports in the literature of non-coding motifs implicated in gene regulation spurred the discovery of additional natural aptamers, including motifs specific for S-adenosylmethionine (SAM) (Epshtein et al., 2003; McDaniel et al., 2003; Winkler et al., 2003), guanine (Mandal et al., 2003), adenine (Mandal and Breaker, 2004) and lysine (Grundy et al., 2003; Sudarsan et al., 2003b). More recently, strategies employing bioinformatics to search for sequence conservation within non-coding regions of bacterial genomes have uncovered riboswitches responding to the metabolites glycine and glucosamine 6-phosphate (GlcN6P) (Barrick et al., 2004; Mandal et al., 2004; Winkler et al., 2004), as well as an additional, structurally distinct natural aptamer specific for SAM (Corbino et al., 2005).

8.2 Genetic Control by Riboswitches

The riboswitches that have been identified to date occur predominantly in Grampositive organisms, although certain examples are more widespread among the eubacteria. Typically, natural aptamers reside in the 5l-UTRs of mRNAs encoding proteins involved in the metabolism or transport of the metabolite ligands. Such an arrangement creates a negative feedback loop in which the embedded aptamer participates in gene control by allowing high concentrations of the cognate metabolite to downregulate those proteins that would result in its continued accumulation. Natural aptamers may exert their control of gene expression through a variety of mechanisms, the majority of which exploit the conformational equilibrium that exists between alternatively folded RNA structures. In this way, the occupation state of the aptamer domain influences the conformation adopted by a separate, but interdependent downstream element, termed the expression platform, which directly mediates genetic control. Among organisms of the Bacillus/Clostridium group, the mode of genetic regulation most frequently utilized by riboswitches is the control of transcription termination. In cases where this type of expression platform is used, formation of an aptamer–ligand complex influences the stability of an intrinsic terminator, which is an extended stem–loop structure typically trailed by at least six U residues. Upon encountering an intrinsic terminator, RNA polymerase is induced to dissociate from the elongation complex, resulting in termination of transcription (Gusarov and Nudler, 1999; Yarnell and Roberts, 1999). Most of the identified riboswitches using this strategy act to repress gene expression in response to ligand binding (Fig. 8.1a). However, in cases where riboswitch-controlled genes are involved in metabolite efflux or catabolism (Mandal and Breaker, 2004; Mandal et al., 2004), ligand-induced activation of gene expression is required, and this

8.2 Genetic Control by Riboswitches

Fig. 8.1 Riboswitch control of gene expression at the level of transcription termination. Metabolite binding to the aptamer domain of a riboswitch induces structural rearrangements, resulting in (a) repression of gene expression by stabilizing an intrinsic terminator stem or

(b) activation of gene expression by disrupting a terminator stem. Riboswitches can also act at the level of translation initiation through structural rearrangements that govern the accessibility of the ribosome binding site.

is typically achieved by a riboswitch organization in which the structures of the ligand-bound aptamer and intrinsic terminator are mutually exclusive (Fig. 8.1b). Riboswitch-mediated gene regulation can also be exercised at the level of translation initiation simply by mating the aptamer domain with a distinct expression platform. The metabolite-induced structural change would then modulate the accessibility of the Shine–Dalgarno sequence, a short, purine-rich motif proximal to the start codon that is important for ribosome binding. The variety of post-transcriptional gene control mechanisms with which natural aptamers may interface is not limited to those known to be employed by eubacterial organisms. Certain examples of the TPP-binding motif, which is the only natural metabolite-binding aptamer to have been identified in the archaeal and eukaryotic domains, underline the potential versatility of riboswitch-mediated genetic control. Eukaryotes rely on post-transcriptional genetic control mechanisms that differ to some extent from prokaryotic strategies. Nevertheless, a sequence encoding a functional TPP aptamer occurs in the 3l-UTRs of plant genes involved in thiamine biosynthesis (Sudarsan et al., 2003a), suggesting a regulatory role for this motif, perhaps in modulating mRNA stability. Aptamers of the TPP-specific class have also been identified in fungi, where their locations within 5l-UTR in-

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trons imply roles in splicing control (Kubodera et al., 2003; Sudarsan et al., 2003a). Natural aptamers, then, may not necessarily be restricted to operating in the context of eubacterial expression platforms, and potentially could be coupled with a variety of RNA-based control mechanisms that are appropriate to the organism in which they occur.

8.3 Aptamer Domains of Riboswitches

The lack of conservation observed among expression platforms reflects the degree to which they may be interchanged or adapted to control different processes. Ligand-binding elements, however, tend to be evolutionarily more constrained compared with the expression platforms with which they interface. Unlike RNA elements that interact with protein factors, natural aptamers are prevented from significantly changing their recognition strategies simply because their metabolites targets are immutable, unable to co-evolve in step with their receptors. As genetic control elements, riboswitches are challenged with operating in an environment of enormous chemical complexity. Therefore, in order to avoid inappropriate genetic responses, their aptamer domains must be capable of discriminating with precision against compounds with related structures. These high levels of specificity exhibited by natural aptamers toward their ligands are often accompanied by impressive binding affinities, which in several instances extend into the nanomolar range (Table 8.1). Presumably, the avidity of a particular RNA–ligand interaction is tuned in order to respond appropriately over the physiologically relevant Table 8.1 Known riboswitch classes Riboswitch class

Gene/operon controlled

Aptamer size (nt)

Kd (nmol/L)

Referencesa

Coenzyme B12

E. coli btuB

202

300

Nahvi et al., 2002

TPP

E. coli thiM

78

30

Winkler et al., 2002a

Glycine

V. cholera gcvT

220b

30 000

Mandal et al., 2004

FMN

B. subtilis ribD

121

5

Winkler et al., 2002b

SAM I

B. subtilis yitJ

118

4

Winkler et al., 2003

SAM II

A. tumefaciens metA

55

1000

Corbino et al., 2005

Guanine

B. subtilis xpt

66

5

Mandal et al., 2003

Adenine

B. subtilis ydhL

64

300

Mandal and Breaker, 2004

Lysine

B. subtilis lysC

169

1000

Sudarsan et al., 2003b

GlcN6P

B. subtilis glmS

150

200 000

Winkler et al., 2004

a

b

References pertain to the specific aptamer constructs used in determinations of Kd-values. Size of the tandem motif containing two aptamers.

8.3 Aptamer Domains of Riboswitches

concentration range for a given metabolite. It should be noted, however, that binding constants determined in vitro do not necessarily reflect the metabolite concentrations to which riboswitches respond in vivo. Analysis of an FMN-specific riboswitch in Bacillus subtilis has revealed that the decision whether to terminate transcription is made before thermodynamic equilibrium is attained, indicating that kinetic parameters are likely to have a greater impact (Wickiser et al., 2005). The impressive conservation of individual aptamer domains in no way implies that a particular motif represents the only solution by which RNA may recognize the respective ligand. Just as selections in vitro have generated multiple aptamers recognizing the same target molecule (Huang and Szostak, 2003; Sassanfar and Szostak, 1993; Sazani et al., 2004), nature also should have access to the numerous solutions in sequence space. An example of this has recently been provided with the identification of a second, distinct motif, occurring primarily in a-proteobacteria, that recognizes the coenzyme SAM (Corbino et al., 2005). This newly identified aptamer is significantly smaller and has a less complex secondary structure than the previously reported SAM-binding domain, which has been observed almost exclusively among Gram-positive organisms. Despite its unique architecture, the a-proteobacterial SAM aptamer binds its target with high affinity and discriminates quite effectively against structurally related analogs. The non-overlapping distribution of these two SAM-specific aptamer classes indicates that different bacterial subgroups can employ distinct recognition strategies for RNA– metabolite interactions. Despite the impressive phylogenetic conservation exhibited by aptamer domains of a given riboswitch class, these motifs are subject to subtle degrees of variation. Among individual TPP aptamers, for example, an extraordinarily wide range of sizes of the P3 stem–loop is tolerated, presumably because this element has little impact on the global architecture of this motif (Rodionov et al., 2002; Sudarsan et al., 2003a). Similarly, helical elements commonly occurring at defined positions within a riboswitch class need not be present in all examples, as is the case with the RNA motif recognizing glycine (Barrick et al., 2004). Although more drastic deviations from consensus structures are not commonly observed among known riboswitches, substantial change to an aptamer core is not unprecedented. For example, a variant of the AdoCbl-binding motif has been identified in which nearly one half of the consensus structure is absent (Nahvi et al., 2004). Despite the severity of the modification, the truncated motif nonetheless binds AdoCbl with high affinity. This abbreviated aptamer contains a short sequence element not found in larger versions and discriminates less efficiently against AdoCbl analogs varying at the adenosyl moiety, suggesting that different subdomains may interact with distinct ligand moieties in a modular fashion. These observations demonstrate that an aptamer can depart significantly from its consensus while maintaining specificity toward its cognate ligand. Conversely, aptamers also exist in which ligand specificities may be changed without substantial alterations to their global architectures. The first such demonstrations occurred in the laboratory, where artificial aptamers were subjected to further evolution in vitro in attempts to create new ligand specificities (Famulok, 1994; Souk-

195

196


up et al., 2000). For example, an arginine-selective aptamer, which was evolved from an RNA motif recognizing the related compound citrulline, differs from its parent at only three nucleotide positions (Famulok, 1994). Structural studies have revealed that the two aptamers share a similar overall architecture, but discriminate between the two ligands through subtle changes to the binding pocket effected by the nucleotide substitutions (Yang et al., 1996). Nature has relied on a similar approach in recognizing the related metabolites guanine and adenine with distinct but closely related motifs. These two classes of natural aptamers, despite exhibiting preferential binding to their respective purine targets, possess nearly identical architectures. The features of these motifs, including detailed structural views that have emerged from the first X-ray crystallographic studies of natural aptamers, are discussed in more detail below.

8.4 Natural Aptamers Specific for Guanine and Adenine

The pur and xpt-pbuX operons in B. subtilis, containing genes involved in purine biosynthesis and salvage, respectively, had been reported to be controlled in part by transcription termination mechanisms in response to levels of guanine and hypoxanthine (Ebbole and Zalkin, 1987; Christiansen et al., 1997). Although it was speculated that an unidentified protein might mediate this component of the genetic regulation (Ebbole and Zalkin, 1987; Christiansen et al., 1997), no such factor had been isolated. The 5l-UTR corresponding to the xpt-pbuX operon is 185 nucleotides in length, large enough plausibly to accommodate a riboswitch element. Since aptamer domains corresponding to an individual riboswitch class tend to be highly conserved in both sequence and structure, a database search was undertaken to determine whether elements of this 5l leader sequence were conserved in additional B. subtilis loci or in genomes of other bacteria (Mandal et al., 2003). This bioinformatic approach identified more than 30 motifs related to the xpt-pbuX 5l-UTR in sequence and predicted secondary structure in Gram-positive and Gram-negative organisms. These elements were associated most often with genes involved in purine nucleotide metabolism or transport, consistent with their presumed functions as genetic control modules. The consensus structure deduced from this phylogeny comprises a three stem junction (Fig. 8.2a), in which most of the sequence conservation exists in the unpaired joining regions. The potential for canonical base-pairing interactions between certain conserved nucleotides of the L2 and L3 regions, together with the conserved lengths of the corresponding helices, suggested the possibility of a pseudoknot interaction between these stem–loops. Taken together, the conserved features of this motif seemed to indicate a structured RNA, perhaps one that could function as an aptamer in the context of a genetic control mechanism. In order to assess this possibility, RNA corresponding to one such conserved element from B. subtilis was assayed for structural changes in the presence of various purine ligands using a technique termed in-line probing.

Fig. 8.2 Secondary and tertiary structure depictions of natural purine-binding RNA motifs. (a) Secondary structures of the guanine-specific xpt-pbuX motif from B. subtilis (top) and the adenine-specific add motif from V. vulnificus (bottom). The Watson–Crick interactions between ligands (circled) and their respective aptamers are depicted with dashed lines. (b) Three-dimensional structure of the guanine riboswitch aptamer in complex with hypoxanthine

(graphic kindly provided by R. Batey; see Batey et al. (2004)). (c) Exploded views of the ligand-binding sites corresponding to the aptamers in (a). Structures were rendered with coordinates from Serganov et al. (2004) using PyMOL (http://pymol.sourceforge.net/) and nuccyl (http://www.mssm.edu/students/ jovinl02/research/nuccyl.html).

8.4 Natural Aptamers Specific for Guanine and Adenine 197

198


This protocol relies on the fact that a typical RNA receptor, while relatively unstructured in the absence of its cognate ligand, becomes structurally more constrained when complexed with its target. Because the geometry of each internucleotide linkage has a large impact on its rate of spontaneous transesterification, RNA sequences whose conformations are restricted due to ligand binding tend to undergo strand scission at different rates compared with the more flexible forms that occur in the absence of ligand (Soukup and Breaker, 1999). When this assay was performed with RNA containing the conserved portion of the B. subtilis xptpbuX 5l-UTR, the pattern of cleavage products resulting from spontaneous transesterification in the presence of certain purine compounds was markedly different from the one generated in the absence of ligand (Mandal et al., 2003). Specifically, regions of the RNA corresponding largely to the unpaired strands of the three-stem junction experienced significantly reduced rates of cleavage when guanine, hypoxanthine, or xanthine were added to the reactions at concentrations of 1 mmol/L. These changes reflect structural modulations that are induced selectively, as incubations with concentrations of adenine as high as 1 mmol/L had no observed effect on the pattern of spontaneously generated cleavage products. Under these assay conditions, an upper limit on the Kd value for the RNA–guanine interaction was determined to be approximately 5 nmol/L, while the corresponding values for hypoxanthine and xanthine are approximately 10-fold higher. Taken together, these observations indicated that certain 5l-UTRs of genes related to purine metabolism contain aptamers that recognize guanine and closely related compounds with high affinity. For such an aptamer to function as a component of a regulatory apparatus, however, a highly specific binding pocket would be required in order to discriminate against the abundance of purine-related compounds within the cellular environment. Indeed, measurements of apparent Kd values for a panel of guanine nucleobase analogs demonstrated that the substitution or removal of nearly any of the functionalized positions on the guanine heterocyclic ring structure resulted in a significant loss of binding affinity. Interestingly, alterations at the exocyclic amine of guanine were tolerated with only moderately detrimental effects on binding affinity, indicating that hypoxanthine and xanthine, as natural metabolites lacking this functional group, might be relevant targets of this aptamer. The more severe effects of chemical substitutions at all of the other positions examined, however, suggested that this class of aptamer employs contacts with nearly every functional group in order specifically and avidly to bind guanine. The existence of a class of guanine-binding RNAs embedded in the 5l-UTRs of genes involved in purine metabolism strongly implied a role for this motif in genetic control. Such a role was confirmed by a series of riboswitch mutants in which the ligand-binding function of the aptamer was correlated with its ability to influence expression of a reporter gene in vivo (Mandal et al., 2003). As expected, aptamers containing mutations that violated the consensus or that disrupted individual stem elements were significantly impaired in their abilities to bind guanine. Moreover, riboswitches bearing these attenuated aptamer domains failed to modulate the expression of a transcriptionally fused b-galactosidase re-

8.4 Natural Aptamers Specific for Guanine and Adenine

porter gene in response to exogenous guanine. In contrast, aptamer mutants in which the integrity of individual stems was restored by compensatory mutations recovered their abilities to bind guanine and also to exert control over reporter gene expression. Typically, aptamer domains of riboswitches adhere closely to the consensus structures and sequences that define the particular class. Close inspection of the phylogeny containing the guanine-binding motifs, however, revealed three sequences in which there was a slight deviation from the consensus (Mandal and Breaker, 2004). In these cases, a C residue that occurred in the P3/P1 joining region, and that was otherwise absolutely conserved, was substituted with a U residue. Interestingly, two of these variant sequences were associated with genes encoding adenine deaminase, raising the possibility that mutations at this position might contribute to an alteration of ligand selectivity. Specifically, if this riboswitch class employed a Watson–Crick base-pairing interaction with its target ligand as a component of molecular recognition, then a C to U change at the pertinent position might be expected to result in greater selectivity for adenine instead of guanine. In order to test this possibility, a series of binding assays was performed with the aptamer domain associated with the B. subtilis ydhL gene (Mandal and Breaker, 2004). This RNA motif, which contains the C to U substitution at position 74 (Fig. 8.2a), displays an altered ligand specificity, binding adenine with an apparent Kd of Z300 nmol/L while failing to undergo structural modulation in guanine concentrations as high as 10 mmol/L. The degree of selectivity exhibited by the adenine aptamer was comparable to that of the guanine-binding motif. Also, the functional group modifications of purine ligands that disrupted binding of the ydhL aptamer were generally analogous to those that abrogated binding of the guanine-specific motif, indicating that the binding pockets of these two aptamers are of similar construction. The only apparent distinction between the ligand-binding strategies of these aptamers existed in the mode of recognition at the Watson–Crick base-pairing face. This predicted canonical base-pairing interaction appeared to be the primary determinant of molecular discrimination between guanine and adenine, as the specificities of these riboswitch classes could be interconverted via single base substitutions at this position in the respective aptamer domains. Expression of the ydhL gene, which is predicted to encode a purine efflux pump, is activated by its associated riboswitch in response to elevated adenine concentrations. This example of ligand-induced positive regulation stands in contrast to most guanine riboswitches, which serve commonly to repress the expression of genes involved in purine biosynthesis or import. Inspection of the expression platform corresponding to the ydhL riboswitch reveals that the sequence of an intrinsic terminator stem overlaps considerably with that of the aptamer itself, such that the formation of these structures is mutually exclusive. In this way, adenine-induced stabilization of the aptamer domain precludes terminator formation, resulting in upregulation of the full-length mRNA. This illustrates that aptamers of a given class can harness ligand-induced allosteric structural changes to elicit different genetic control responses.

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8.5 High-resolution Aptamer Structures

The first detailed views of natural aptamers complexed with their ligands have recently been provided by X-ray crystallographic (Batey et al., 2004; Serganov et al., 2004) and NMR spectroscopic (Noeske et al., 2005) analyses of the purine-binding RNA motifs. The ligand-binding domain of the xpt-pbuX riboswitch from B. subtilis has been crystallized separately with the biologically relevant targets hypoxanthine (Batey et al., 2004) and guanine (Serganov et al., 2004). Strikingly, the ligand in each case is almost completely enveloped by the aptamer (Fig. 8.2b), with solvent effectively excluded from the binding pocket. The aptamer describes a tuning fork arrangement, in which the close packing of the P2 and P3 stems in a parallel alignment is anchored by interactions between their terminal loops. The purine ligand is then ensconced by nucleotides of the three-way junction, which form a multi-layered array of base triples stacked upon the P1 stem. The juxtaposition of the P2 and P3 helices requires several bound cations to neutralize unfavorable electrostatic interactions, but relies primarily on the association of the respective capping loops via an intricate hydrogen bonding network. At the core of this tertiary interaction are two novel base quadruplexes, each consisting of a Watson–Crick nucleotide pair docked with a non-canonical base pair. Mutation of any one of these quadruplex nucleotides would be expected to disrupt the intimate association of the loops, explaining why the identities of these residues are phylogenetically conserved. Evidence from biochemical studies (Mandal et al., 2003) indicates that this highly structured loop interaction occurs even in the absence of the target ligand. This would lend a certain degree of pre-organization to the aptamer, thereby providing a structural context that allows the junction nucleotides to associate efficiently with the target to form the compact aptamer core. The core of the RNA–ligand complex comprises essentially five tiers of base triplets, whose component nucleobases are contributed by conserved residues of the three-way helical junction. The ligand itself occupies the central layer, where it exists as part of a base triplet with U51 and C74 (Fig. 8.2c). Additional hydrogen bonds are formed with two other nucleotides, resulting in recognition of all heteroatoms of guanine or hypoxanthine by peripheral residues. The ligand is positionally constrained also by pairs of base triples above and below its base plane, which generate extensive base stacking interactions among these tiers. A structural comparison of the guanine- and adenine-binding RNAs reveals the nearly identical organization of their overall structures and, indeed, of the binding pockets themselves. The only significant difference lies in the identity of the pyrimidine at position 74, which, because it forms a Watson–Crick base pair with the target ligand, is the primary determinant of molecular discrimination between guanine and adenine (Fig. 8.2c). The crystal structures of the purine-binding RNAs clearly demonstrate that the ligands themselves are integral to the bound conformations of the aptamers. The basis for interdomain communication is then apparent, as ligand binding can be

8.6 The Glycine Riboswitch

used to influence the thermodynamic balance between an aptamer domain and its associated expression platform. Such RNA-based interdomain communication provides an efficient means of genetic control and appears to be the mechanism most often employed by riboswitches. Recently, however, a variation of this strategy has been observed in a glycine-binding RNA in which interdomain communication exists not only between an aptamer and the expression platform, but also between two separate aptamer domains.


The glycine riboswitch was identified in many different bacterial genomes using a bioinformatic approach (Barrick et al., 2004). In most cases this RNA element is located in the 5l-UTRs of genes that encode proteins involved in glycine utilization as an energy source. Since elevated glycine concentrations trigger increased expression of these proteins, this motif, like the adenine riboswitch described previously, provides an example of RNA-mediated activation of gene expression in response to ligand binding. Individual glycine riboswitches have been observed that mediate these effects at either the transcriptional or translational levels. Interestingly, the glycine aptamer occurs predominantly in a tandem arrangement, where two highly similar motifs are joined by a short linker sequence and cooperatively bind two glycine molecules. Such an arrangement reveals an additional layer of complexity that is available to RNA-based regulatory systems. The aptamer sequence associated with the Vibrio cholerae gcvT operon, which encodes enzymes of the glycine cleavage system, is shown in Fig 8.3a. RNA constructs containing both aptamers (orange line) bind two glycine molecules, while truncated versions containing only the second aptamer (blue line) interact with a single glycine. In-line probing assays yield apparent dissociation constants of 30 mmol/L for the tandem aptamer and 20 mmol/L for the single aptamer, indicating that there is no significant difference in the affinities of these constructs for glycine. In contrast, the binding curves corresponding to the single and double aptamer constructs differ substantially (Fig. 8.3b). For the RNA containing both aptamers (VC I + II), the transition between the unbound and bound states is steeper compared with that of the single aptamer (VC II). Fitting the data to the Hill equation for cooperative systems results in a Hill coefficient of 1.64 for the RNA containing two aptamers. (The value of the Hill coefficient can range from a minimum of 1 [no cooperativity] to a maximum that is equal to the number of binding sites [perfect cooperativity].) The degree of cooperativity per binding site is comparable to that of hemoglobin, a homotetramer whose four oxygen binding sites exhibit a Hill coefficient of 2.8 (Edelstein, 1975). Consistent with this cooperative binding effect, it was determined that the occupation of one binding site by glycine increases the ligand affinity of the other binding site by as much as 1000-fold (Mandal et al., 2004). This cooperative behavior results in greater sensitivity of the tandemly arranged riboswitch to

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Fig. 8.3 Cooperative binding by a glycine riboswitch. (a) Secondary structure of the glycine aptamer domain associated with V. cholerae gcvT. The orange line defines the RNA construct containing the tandem aptamer arrangement (VC I + II), while the blue line defines the truncated version containing only one aptamer (VC II). Nucleotides in red are highly

conserved. (b) Binding curves for both RNA constructs derived from in-line probing data. The dynamic range (DR), defined as the change in glycine concentration needed to adjust the fraction of bound RNA from 10% to 90%, is decreased for VC I + II by about 10-fold relative to VC II.


changing glycine levels, effectively compressing the range of ligand concentrations through which the maximum regulatory effect is achieved. Despite the fact that the glycine riboswitch recognizes a ligand containing only 10 atoms, it discriminates against related metabolites such as L-alanine or L-serine, which do not induce RNA structural modulation even at concentrations of 10 mmol/L. The affinity of the V. cholerae aptamer domain is likely to be tuned to respond to glycine concentrations where catabolism of this amino acid would be favorable. Cooperative binding would provide an advantage by enabling cells to adapt nimbly to changing glycine concentrations. Thus, glycine cleavage system genes would be rapidly upregulated to catabolize glycine as an energy source when it is present in excess, while these same genes would be swiftly repressed as glycine levels decrease in order to maintain a concentration adequate for protein synthesis (Tucker and Breaker, 2005). The mechanism of the evolution of this cooperative riboswitch poses an interesting question. The phylogeny of the glycine riboswitch and the experimental data pertaining to the sequence found in V. cholerae suggest that this tandem arrangement could have emerged from simple duplication. Individual aptamer domains within a given glycine riboswitch possess similar, highly conserved core sequences, suggesting a common lineage. In addition, a single aptamer construct has been demonstrated to bind glycine with an affinity similar to that of a double aptamer construct, implying that a single motif might be sufficient for gene regulation. In fact, examples of putative glycine riboswitches bearing just one aptamer domain have been identified (Mandal et al., 2004), supporting the possibility that tandemly arranged aptamers evolved by duplication events from single glycine-binding domains. The example of cooperative binding by the glycine-specific tandem aptamer demonstrates that conventional riboswitch mechanisms can be elaborated, presumably in order to achieve genetic control responses that confer maximum benefit to the host organism. Another RNA element has recently been described whose genetic control strategy deviates somewhat from that of more typical riboswitch motifs. This conserved RNA structure resides in 5l-UTRs of mRNAs encoding glucosamine 6-phosphate synthase, and interacts selectively with a product of this protein enzyme, glucosamine 6-phosphate (GlcN6P) (Winkler et al., 2004). Rather than relying exclusively on ligand-induced structural changes to modulate gene expression, however, this RNA undergoes site-specific self-cleavage at greatly increased rates in the presence of GlcN6P. This particular riboswitch, then, exploits the action of a metabolite-responsive self-cleaving ribozyme to regulate gene expression. While technically not an aptamer, this RNA is nevertheless capable of impressive molecular discrimination, as evidenced by considerably reduced cleavage rates in the presence of compounds closely related to GlcN6P. The level of sophistication exhibited by natural metabolite-binding RNAs, together with the variety of gene control mechanisms they employ, invites comparisons with small-molecule-binding RNAs that have been isolated in the laboratory. In cases where direct comparisons can be made, aptamers selected in vitro tend to be smaller and to bind their targets less avidly than natural aptamers recognizing

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8 Riboswitches: Natural Metabolite-binding RNAs Controlling Gene Expression Table 8.2 Comparison of natural and engineered aptamers Engineered aptamers References Riboswitch aptamersa Ligand Size Kd Ligand Size Kd (nt) (nmol/L) (nt) (nmol/L) Coenzyme B12 202

300

Vitamin B12

35

90

FMN

121

5

FMN

30

500

Burgstaller and Famulok, 1994

guanine

66

5

guanine

32

1300

Kiga et al., 1998

SAM I

118

4

SAM

35

100

Burke and Gold, 1997

SAM II

55

1000

a

Lorsch and Szostak, 1994

For references see Table 8.1.

the same compounds (Table 8.2). The smaller sizes of engineered aptamers are largely a function of the lengths of the pool molecules from which they were selected, and these reduced aptamer sizes may correlate generally with structures of lower complexity (Carothers et al., 2004). Natural metabolite-binding RNAs, in contrast, have not been subjected to the same size constraints and, because they must function in cells efficiently and reliably, the demands on their performance are likely to have been quite severe. Given the vastly different selective pressures experienced by these two categories of metabolite-binding RNAs, the superior performance of natural aptamers is not surprising. Engineered aptamers often suffer the added disadvantage of having limited access to the surface of their target. In vitro selection procedures commonly require immobilization of the ligand on a solid support, thereby precluding envelopment of the compound by RNA. The accumulated examples in the literature of artificial aptamers indicate that RNA is capable of the specific recognition of small molecules that would have been required for an ancient, RNA-guided metabolism. The more recent identification of natural RNAs with the same capability has only served to reinforce this viewpoint. The targets of known riboswitches are fundamental to contemporary metabolism, and many of these are either nucleobases themselves or nucleotide-containing coenzymes (Table 8.1). Because of the central biochemical roles of these compounds, and especially because coenzymes with nucleotide character are proposed to be vestiges of an RNA world era (White, 1976, 1982), it is tempting to speculate that the highly conserved RNA motifs recognizing these compounds may also have originated during this era. The widespread distributions of many of these metabolite-binding RNAs may also argue in support of their ancient origins. The possibilities cannot be excluded, however, that these motifs arose more recently during evolution and that horizontal transfer has contributed to their pervasive occurrence. The number of riboswitches is certain to expand as research into RNAmediated genetic control intensifies. Approaches relying on bioinformatics have

References

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Nahvi, A., Sudarsan, N., Ebert, M. S., Zou, X., Brown, K. L., Breaker, R. R. (2002). Genetic control by a metabolite binding mRNA. Chem Biol 9, 1043. Nahvi, A., Barrick, J. E., Breaker, R. R. (2004). Coenzyme B12 riboswitches are widespread genetic control elements in prokaryotes. Nucleic Acids Res 32, 143–150. Noeske, J., Richter, C., Grundl, M. A., Nasiri, H. R., Schwalbe, H., Wohnert, J. (2005). An intermolecular base triple as the basis of ligand specificity and affinity in the guanine- and adenine-sensing riboswitch RNAs. Proc Natl Acad Sci USA 102, 1372– 1377. Nou, X., Kadner, R. J. (2000). Adenosylcobalamin inhibits ribosome binding to btuB RNA. Proc Natl Acad Sci USA 97, 7190– 7195. Nudler, E., Mironov, A. S. (2004). The riboswitch control of bacterial metabolism. Trends Biochem Sci 29, 11–17. Rodionov, D. A., Vitreschak, A. G., Mironov, A. A., Gelfand, M. S. (2002). Comparative genomics of thiamin biosynthesis in procaryotes. New genes and regulatory mechanisms. J Biol Chem 277, 48949–48959. Sassanfar, M., Szostak, J. W. (1993). An RNA motif that binds ATP. Nature 364, 550–553. Sazani, P. L., Larralde, R., Szostak, J. W. (2004). A small aptamer with strong and specific recognition of the triphosphate of ATP. J Am Chem Soc 126, 8370–8371. Serganov, A., Yuan, Y. R., Pikovskaya, O., Polonskaia, A., Malinina, L., Phan, A. T., Hobartner, C., Micura, R., Breaker, R. R., Patel, D. J. (2004). Structural basis for discriminative regulation of gene expression by adenine- and guanine-sensing mRNAs. Chem Biol 11, 1729–1741. Soukup, G. A., Breaker, R. R. (1999). Relationship between internucleotide linkage geometry and the stability of RNA. RNA 5, 1308–1325. Soukup, G. A., Breaker, R. R. (2000). Allosteric nucleic acid catalysts. Curr Opin Struct Biol 10, 318–325. Soukup, G. A., Emilsson, G. A., Breaker, R. R. (2000). Altering molecular recognition of RNA aptamers by allosteric selection. J Mol Biol 298, 623–632. Stormo, G. D., Ji, Y. (2001). Do mRNAs act as direct sensors of small molecules to control

References their expression? Proc Natl Acad Sci USA 98, 9465–9467. Sudarsan, N., Barrick, J. E., Breaker, R. R. (2003a). Metabolite-binding RNA domains are present in the genes of eukaryotes. RNA 9, 644–647. Sudarsan, N., Wickiser, J. K., Nakamura, S., Ebert, M. S., Breaker, R. R. (2003b). An mRNA structure in bacteria that controls gene expression by binding lysine. Genes Dev 17, 2688–2697. Tucker, B. J., Breaker, R. R. (2005). Riboswitches as versatile gene control elements. Curr Opin Struct Biol 15, 342–348. Wei, B. Y., Bradbeer, C., Kadner, R. J. (1992). Conserved structural and regulatory regions in the Salmonella typhimurium btuB gene for the outer membrane vitamin B12 transport protein. Res Microbiol 143, 459– 466. White, H. B., III (1976). Coenzymes as fossils of an earlier metabolic state. J Mol Evol 7, 101–104. White, H. B., III (1982). Evolution of coenzymes and the origin of pyridine nucleotides. In: The Pyridine Nucleotide Coenzymes, J. Everse, B. Anderson, K. You, eds. New York: Academic Press, pp. 1–17.

Wickiser, J. K., Winkler, W. C., Breaker, R. R., Crothers, D. M. (2005). The speed of RNA transcription and metabolite binding kinetics operate an FMN riboswitch. Mol Cell 18, 49–60. Winkler, W., Nahvi, A., Breaker, R. R. (2002a). Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 419, 952–956. Winkler, W. C., Cohen-Chalamish, S., Breaker, R. R. (2002b). An mRNA structure that controls gene expression by binding FMN. Proc Natl Acad Sci USA 99, 15908–15913. Winkler, W. C., Nahvi, A., Sudarsan, N., Barrick, J. E., Breaker, R. R. (2003). An mRNA structure that controls gene expression by binding S-adenosylmethionine. Nat Struct Biol 10, 701–707. Winkler, W. C., Nahvi, A., Roth, A., Collins, J. A., Breaker, R. R. (2004). Control of gene expression by a natural metabolite-responsive ribozyme. Nature 428, 281–286. Yang, Y., Kochoyan, M., Burgstaller, P., Westhof, E., Famulok, M. (1996). Structural basis of ligand discrimination by two related RNA aptamers resolved by NMR spectroscopy. Science 272, 1343–1347. Yarnell, W. S., Roberts, J. W. (1999). Mechanism of intrinsic transcription termination and antitermination. Science 284, 611–615.

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Part 3 In Vitro Selection of Short, Catalytically Active Oligonucleotides


9.1 Introduction

9 Catalytically Active RNA Molecules: Tools in Organic Chemistry Barbara-Sylvia Weigand, Andreas Zerressen, Jrg C. Schlatterer, Mark Helm, and Andres Jschke

9.1 Introduction

Until the early 1980s, the notion of biocatalysis and enzymatic function was commonly associated with proteins. Beginning in 1982, this point of view started to change after the discovery, in the laboratories of Sid Altman and Tom Cech, of RNAs with catalytic function (Kruger et al., 1982; Guerrier-Takada et al., 1983). While the ability of RNA molecules to catalyze hydrolysis and ligation of phosphodiester bonds has become textbook knowledge, the full range of chemical reactions that RNA can accelerate remains to be elucidated. Like proteins, nucleic acids can form tertiary structures including binding sites and, consequently, catalytic centers. RNA molecules containing the first artificial binding sites against an artificially selected target were presented in 1990, when Tuerk and Gold published the first RNA aptamer, directed against bacteriophage T4 DNA polymerase, which they had obtained by a new combinatorial technique termed SELEX (systematic evolution of ligands by exponential enrichment). Only in hindsight, with the knowledge on catalytic antibodies in mind, does it seem inevitable that biomolecules with binding properties similar to antibodies would eventually prove to be capable of catalysis. Starting from synthetic combinatorial libraries, the labs of Gold, Szostak, and Joyce isolated RNA molecules, socalled aptamers, featuring binding properties similar to antibodies (Beaudry and Joyce, 1990; Ellington and Szostak, 1990; Tuerk and Gold, 1990). This technique eventually even produced RNA with catalytic activities. Neither of these newly found properties were in any way related to the hitherto presumed biological functions of nucleic acids. SELEX is a very delicate technique, with the success of the experiment strongly depending on the correct experimental approach. In return SELEX experiments have led to nucleic acids that catalyze a broad range of chemical transformations, ranging from cleavage of amide (Dai et al., 1995) or carboxylic ester bonds (Piccirilli et al., 1992) to amide-bond- (Wiegand et al., 1997), C-C- and C-S-bond forming reactions (Seelig and Jschke, 1999; Sengle et al., 2001; Tarasow et al., 1997) The Aptamer Handbook. Edited by S. Klussmann Copyright c 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-31059-2

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9 Catalytically Active RNA Molecules: Tools in Organic Chemistry

or the catalysis of s-bond rotation involved in isomerization reactions (Prudent et al., 1994). The history of ribozymes, in the true sense of the word, is connected to the socalled “RNA world” (Gilbert, 1986; Yarus, 1999), a prebiotic stage in the evolution of life, where the majority of catalytic functions were effected by RNA, and proteins did not yet exist (Woese, 2002). The idea of an RNA world alone implies that RNA should be practically omnipotent in the diversity of its catalytic potential, despite having been outperformed by proteins later on. This catalytical omnipotence necessarily includes stereoselectivity, a property of outstanding interest to the organic chemist. While intuition predicts stereoselectivity because of the chirality of RNA monomers, it was not until 2000 that enantioselectivity was described as a property of an in vitro selected ribozyme. The ribozyme described catalyzes the Diels–Alder reaction between anthracene and maleimide (Seelig et al., 2000) and is therefore called a Diels-Alderase. Previously, nucleic acids had been of interest to the organic chemist mainly as target molecules for synthesis, sometimes including more or less complicated modifications. Since the discovery of Diels-Alderase other reactions of significance to the organic chemist have been shown to be catalyzed by ribozymes, including for example Michael additions (Sengle et al., 2001) and even redox reactions (Tsukiji et al., 2003). At present, laboratories are looking for ribozymes accelerating yet other important reactions for organic chemistry (including for example aldol reactions and cycloadditions). In the light of the latest developments in the field, we might soon see the situation reversed, with particular nucleic acids used as customized catalysts in complex organic synthesis. In what follows, we will discuss the accomplishments, requisites and prerequisites, strategies, and possible applications of catalytically active RNAs. Biomedical applications and related issues are discussed elsewhere (Bramlage et al., 1998; Zinnen et al., 2002; Steele et al., 2003; Trang et al., 2004), as are the latest developments in the field of allosteric regulation and conformational rearrangements of ribozymes (Breaker, 2002; Silverman, 2003). The focus of the present discussion will hence be on the significance of ribozymes to the organic chemist.

9.2 Catalytic Biopolymers

By definition, a true catalyst accelerates the transformation of the reactant molecules without being either consumed or permanently modified. Fundamental aspects of true catalysts are intermediate binding of reactants and stabilization of the transition state, either aspect implying some sort of molecular recognition. The concept of catalysis by molecular recognition of the transition state has led scientists to look for molecules that bind transition state analogs (TSAs), resulting in the discovery of catalytic antibodies. It is relevant to note that these so-called abzymes are the result of a combinatorial approach, generated from a self-opti-

9.3 De Novo Creation of Ribozymes

mizing library by the mammalian immune system (e.g. Shi et al., 2002; Toker et al., 2000, reviewed by Hilvert, 2000; Xu et al., 2004). The development of aptamers by the SELEX technique and the demonstration of catalytic properties in RNA clearly showed that RNAs, like proteins, are biopolymers with the potential to form binding pockets and catalytic centers. This analogy and the success in the field of catalytic antibodies have likely spawned a similar combinatorial approach using nucleic acids to target TSAs. This approach, despite the development of several true catalysts with moderate activity, has meanwhile been outperformed by combinatorial approaches using the desired catalytic activity itself as a criterion for selection in vitro. Prior to a discussion of the most exciting developments in RNA catalysis, we will analyze these approaches in more detail.

9.3 De Novo Creation of Ribozymes

The general concept of SELEX and its use for the isolation of aptamer sequences is described elsewhere in this volume. The crucial step in this technique is selection – an effective distinction between desired and undesired species. It is in the selection step that the most decisive advances in ribozyme discovery are made. The standard selection event in SELEX is a binding event, and consequently the desired molecules can be distinguished from the rest by physical separation, namely by affinity chromatography. Conceptually starting from the basic SELEX protocol, several strategies to create RNA constructs with catalytic properties have been developed. As already mentioned, the TSA approach has been emulated from catalytic antibodies. Aptamers against TSAs of different reactions have been raised and tested for catalytic activity. Prudent et al. (1994) reported an aptamer with catalytic properties in biphenyl isomerization. An aptamer with porphyrin metallation properties was reported by the Schultz group (Conn et al., 1996). Cholesterol esterase activity of another RNA isolated by the TSA approach was found by Chun et al. (1999). Interestingly, the generation of catalytic antibodies failed in the latter case possibly as a result of poor immunogenicity of the hapten. While all these RNAs act as catalysts, the rate acceleration was limited, at about 88-fold (Prudent et al., 1994), 110-fold (Chun et al., 1999), and 460-fold (Conn et al., 1996), as compared with 103 –105 for TSA antibodies (Lolis and Petsko, 1990; Stewart and Benkovic, 1995) and up to 1017 for natural enzymes (Griffiths and Tawfik, 2000; Hilvert, 2000). Other attempts to isolate catalytic RNAs via TSA, for example for the Diels– Alder reaction, have thus far remained at the stage of aptamer binding (Morris et al., 1994; Arora et al., 1998). Better acceleration rates and catalysis of more complex reactions were obtained by a different strategy, termed direct selection. In this strategy, selection is not based on a binding event; rather it is based directly on the desired catalytic event. For example, if the reaction to be catalyzed is phosphodiester hydrolysis,

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the RNA library is immobilized on solid support. The selection event is thus directly tailored to hydrolysis: active molecules cleave their covalent linkage to the solid support and thus dissociate from it. The catalytic event thus enables spatial separation of active and inactive molecules. The use of reactants other than RNA in direct selection allows targeting of more complex reactions than hydrolysis or RNA ligation. As stated before, a reactant X can, for example, be conjugated to the RNA library as initiator nucleotide. Assuming a desired RNA catalyst of the chemical reaction XpZ, which may include simple addition as in X + YpZ, the selection has to be designed in such a way as to isolate all RNA library members carrying the target reaction product Z. This can be through chemical trapping or affinity purification of Z directly, or by designing Y in such a way that the final product contains a functional moiety of Y, which allows for easy affinity purification. An exciting example for the former case is the ribocatalytic oxidation of benzyl alcohol to benzaldehyde using NAD+ as a cofactor: the desired aldehyde product was trapped chemically by a biotin-conjugated hydrazine derivative (Tsukiji et al., 2003). This case will be more explicitly discussed below. The latter case has been successfully demonstrated by several labs employing a biotin-conjugated reactant Y thus allowing purification of the reactive species by streptavidin–agarose (Ekland and Bartel, 1996; Williams and Bartel, 1996; McGinness and Joyce, 2002). Our lab has developed yet another, further refined strategy, named direct selection with linker-coupled reactants. This design, tailored to find catalysts for the chemical addition of compound Y to compound X to yield the addition product Z, is depicted in Fig. 9.1. Reactant X is coupled to the RNA pool by transcription initiation, or by subsequent ligation to the 3l end. However, as opposed to direct selection, the reactant X is now separated from the RNA by a polyethylene glycol (PEG) linker, allowing it to interact with potential catalytic pockets in the RNA, which are spatially remote from either extremity. In our lab, using linkercoupled reactants, a ribozyme was selected that catalyzes a Diels–Alder reaction. Anthracene tethered to RNA via a PEG linker corresponds to reactant X, in this case a diene, and biotin-maleimide was employed as reactant Y, the free dienophile (Seelig and Jschke, 1999). This design allowed easy isolation of the reaction product Z by streptavidin–agarose. Depending on the reactivity of reactant Y towards the functional groups contained in unmodified RNA, its conjugation to species of the RNA library will occur with more or less site specificity to the conjugated X-reactant. More reactive Ys may add to RNA at positions other than the desired reactant X, thus also fulfilling the practical requirements for selection and amplification. Results of such selections are occasionally reported in the literature (e.g. Wilson and Szostak, 1995). A strategy to circumvent non-specific addition involves the incorporation of a chemically orthogonal cleavage site between reactant X and the RNA library. After immobilization as a primary selection event, a secondary selection event is created by a treatment targeted specifically to the cleavage site. If immobilization of a given RNA sequence is based on addition of reactant Y to reactant X with correct site specificity, the cleavage will release the desired sequence into solution for amplification. However, addition of Y to sites other than X anywhere in the

9.4 The Catalytic Spectrum of Ribozymes

Fig. 9.1 Direct selection with linker-coupled reactants for the reaction of X with Y to yield product Z. The DNA pool is transcribed and linked with reactant X by conjugation with a functionalized initiator X or by ligation of the transcript to a functionalized dinucleotide. After the reaction the reactive RNA is isolated by immobilization, on a solid phase through an

anchor group at the former reactant Y and further purified by washing the solid phase. Subsequent reverse transcription and polymerase chain reaction (PCR) result in an enriched DNA library, which is used as input for the next round of selection (Seelig and Jschke, 1999).

RNA will result in persistent immobilization during the secondary selection event, thus effectively removing unwanted species from the amplification cycle. The chemistry used for the cleavage event in the secondary selection step should not, of course, interact in any way with the rest of the conjugated library. To our knowledge, two such orthogonal approaches have been described in literature. In one case, reductive cleavage of a disulfide is used (Sengle et al., 2000). In our lab, a dinucleotide was designed containing a 5l-pCC ligation site, a PEG linker with an embedded photocleavable o-nitrophenyl group, and a terminal attachment site which can be derivatized for coupling to a desired reactant X (Hausch and Jschke, 1998). Such photocleavable linker has been successfully applied by the Famulok lab in the selection of a ribozyme catalyzing a Michael addition (Sengle et al., 2001).


Seven natural types of oligonucleotides with catalytic properties are known: hammerhead, hairpin, hepatitis delta virus, ribonuclease P, varkud satellite ribozyme, group I intron, and group II intron (Doudna and Cech, 2002). Catalysis through natural ribozymes is restricted to hydrolysis and transesterification reactions at in-

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ternucleotide phosphodiester bonds, whereas artificial ribozymes can also accelerate miscellaneous types of chemical reactions. These reactions include acylation (Illangasekare et al., 1995; Lohse and Szostak, 1996; Jenne and Famulok, 1998), alkylation (Wilson and Szostak, 1995), formation of amide bonds (Wiegand et al., 1997; Zhang and Cech, 1997), C-C bonds (Diels–Alder reaction) (Tarasow et al., 1997; Seelig and Jschke, 1999; Seelig et al., 2000; Stuhlmann and Jschke, 2002), C-S bonds by Michael addition (Sengle et al., 2001), N-glycosidic bonds (Unrau and Bartel, 1998), and even a redox reaction (Tsukiji et al., 2003). In the following, the scope of in vitro selected ribozymes will be pointed out with special regard to organic chemistry. Several highlights will be discussed in detail. In Table 9.1 the variety of reactions catalyzed by ribozymes, including some interesting DNAzymes, is summarized. Table 9.1 Catalytic activities of ribozymes Reaction type

Equation

References

C-C bonds Diels–Alder reaction

Seelig and Jschke, 1999; Seelig et al., 2000; Stuhlmann and Jschke, 2002; Tarasow et al., 2004; Tarasow et al., 1997

+

Biphenyl isomerization

Prudent et al., 1994 O

O

O

O

C-N bonds Amide bond formation

N-Glycosidic bond formation

R

+

R1O

R

R2

N H

R2

+

Baskerville and Bartel, 2002; Sun et al., 2002; Wiegand et al., 1997; Zhang and Cech, 1997

OH

R

R1 P

H

+

H

OH

P

OH

+

N

R

HN R2

P

P

H

Hal

R2 R1

P

H

H

OH

OH

H

O

Wilson and Szostak, 1995 + HalR2

Sheppard et al., 2000a P

R1

O

H2O

H H

P

H

N+

R

Chapple et al., 2003; Unrau and Bartel, 1998

R2 N

O H

R1

R2 N

O

P

O

R1

N-Alkylation

P

R1

O H

DNA depurination

O

O NH2

H

H

OH

HN R2

H P


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Table 9.1 Catalytic activities of ribozymes (continued) Reaction type

Equation

References

C-S bonds S-Michael reaction

O R

O NHR1

N H

+ HS

R

R2

O

SR2

O

S-Acylation R

O

Jadhav and Yarus, 2002 R1

R2

O-

S-Alkylation

RS

P

+

OH

R2

O-

O S-

OH

O

+ R1O

SH

Sengle et al., 2001 NHR1

N H

Hal

R1

OR

O

P

O

Wecker et al., 1996

S

R1

OR

+ Hal -

C-O bonds O

O

Transesterification R

OH +

R1 O

RO

R2

R2

+ R1

Illangasekare et al., 1995; Illangesekare and Yarus, 1999 Lohse and Szostak, 1996 Jenne and Famulok, 1998 Lee et al., 2000; Murakami et al., 2003

OH

R1 = small oligomer, AMP

O

Carbonate ester hydrolysis

Chun et al., 1999

H2O R1O

R1

OR2

OH + CO2

OH + R2

P-O bonds Phosphoanhydride formation

R1

Phosphorylation

R

OH

RNA ligation

R

OH

RNA/DNA cleavage

R

P

P

P

P

P

R2

R1 P

+

+

NTP P

R1

R

R1

P

R

R

OH

+

+

NDP

P

R1

P

P

R2

Li et al., 2000a

P

R1

P

Lorsch and Szostak, 1994 Johnston et al., 2001 Ekland et al., 1995; Kuhne and Joyce, 2003; Landweber and Pokrovskaya, 1999; McGinness and Joyce, 2002; Roger and Joyce, 1999 Carmi et al., 1996a Landweber and Pokrovskaya, 1999; Lazarev et al., 2003 Feldman and Sen, 2001; Santoro and Joyce, 1997; Santoro and Joyce, 1998; Santoro et al., 2000a Kong et al., 2002b

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Table 9.1 Catalytic activities of ribozymes (continued) Reaction type

Equation

References

C-metal bonds Porphyrin metallation

R2

R2 N

N

R1

N

M

HN

Redox reactions

a

N

M 2+

R1

b

Conn et al., 1996 Kawazoe et al., 2001; Li and Sen, 1996a

R2 NH

N

Oxidation and reduction

R2

R1

N

R1

O R

OH R

H

Tsukiji et al., 2003; Tsukiji et al., 2004

R generally indicates nucleic acid containing residue; P, phosphate; R1–2, various moieties; Hal, halogen; X NDP, nucleoside diphosphate; NTP, nucleoside triphosphate; DNA enzymes, RNA-DNA enzymes.

The catalytic spectrum of RNA cannot be discussed without the background of a hypothetical prebiotic RNA world. As stated in the introduction, the concept of an RNA world means that RNA, at one point, may have performed almost all catalytic functions necessary for survival. As Woese points out (Woese, 2002), the term “survival” does not necessarily apply to a discrete, living entity or even to distinct species, be they single molecules or single cells. Therefore, and because it is unclear exactly which chemical reactions might have been catalyzed by RNA or other matter such as catalytic surfaces (Miyakawa and Ferris, 2003), a complete picture of the RNA’s catalytic potential does not come with the RNA world hypothesis. Yet, there are a large number of chemical reactions and properties that have been predicted to occur in an RNA world. For many of the most important reactions, proof of the catalytic potential has been given, including for example ribonucleotide polymerization, aminoacylation, and peptide bond formation. Although the RNA world is thought to have existed almost four billion years ago, X-ray-structures of the ribosome (e.g. Nissen et al., 2000; Moore and Steitz, 2003) seem to exhibit fragments of this ancestral era. The structures revealed that RNA-mediated catalysis plays an important role in the peptide synthesis of the ribosome. The key step in translation is catalyzed only by the ribonucleic acid component of the ribosome, without any direct contribution of proteins from the spatial vicinity. This impressively demonstrates the catalytic potential of RNA in a biochemical reaction that may arguably be called the most important ever. An artificial ribozyme has been shown to mimic this translation step of the ribosome (Zhang and Cech, 1997). The specificity of this selected ribozyme is based on the recognition of an adenosine moiety of the amino acid ester and allows the


utilization of leucine- and phenylalanine- as well as methionine-derivatized substrates. This tolerance for various amino acids indicates the possibility of selecting more general ribozymes for protein synthesis. Furthermore, a recently characterized ribozyme efficiently catalyzes the synthesis of 30 different dipeptides from an aminoacyl-adenylate substrate. Ribozyme-mediated synthesis of uncoded peptides may have been an important step in the transition from a RNA to a peptide world before the emergence of the ribosome (Sun et al., 2002). Further hints supporting the existence of an RNA world could be the isolation of ribozymes which perform and accelerate intermolecular ligation of the 3l-hydroxyl group of various oligonucleotides to the 5l-triphosphate of an RNA hairpin (McGinness and Joyce, 2002) and the in trans-aminoacylation of a tRNA with specific recognition of an activated amino acid (Lee et al., 2000; Murakami et al., 2003). Another important development was the isolation of a ribozyme which performs nucleotide synthesis by forming a glycosidic linkage from activated ribose (pRpp) (Unrau and Bartel, 1998) in a way similar to the modern biosynthesis of nucleotides. Before the discovery of catalytic RNA, the principal indications of a possible RNA world had been the role of tRNA and ribosomal RNA in translation, the use of RNA as genetic material in retroviruses, and the ubiquitous occurrence of RNA-related enzymatic cosubstrates such as GTP, ATP, AMP, cAMP, SAM, FADH2, and NAD+ in all major metabolic pathways. Clearly, the ability of RNA to employ these and other ubiquitous cosubstrates in catalysis must be expected. A recent highlight in ribozyme research is the in vitro evolution of a ribozyme that oxidizes an alcohol in a NAD+-dependent manner. The resulting RNA-aldehyde was trapped via a chemoselective modification with biotin hydrazide. The function of this ribozyme is analogous to the natural alcohol dehydrogenase enzyme (ADH) and depends on the same cofactors (Tsukiji et al., 2003). Furthermore this ribozyme was coupled with an electron transfer process between NADH and FAD. Thus a NAD+ regeneration system is constituted. Interestingly, the reverse reaction, the RNA-catalyzed reduction of the aldehyde, is also possible in the presence of NADH (Fig. 9.2) (Tsukiji et al., 2004). This is the first clear-cut demonstration of a typical redox reaction catalyzed by RNA. Further artificial ribozymes are known to react with cosubstrates, for example acylating the thiol group of tethered coenzyme A with the AMP-activated biotin. These ribozymes also produce the crucial metabolic intermediates acetyl-CoA and butyryl-CoA at substantial reaction rates. For the selection of this ribozyme, the employed RNA pool had been coupled at its 5l-end to CoA by a previously isolated capping ribozyme (Jadhav and Yarus, 2002). The current hypothesis predicts evolution of the RNA world into the modern DNA–RNA–protein world, with DNA taking over the role of storage of genomic information. One advantage of DNA over RNA as genetic material is the better chemical and enzymatic stability of deoxynucleic acids. That same advantage is also shown by deoxyribozymes, a wide range of which have now been selected. Despite this, there is little evidence that ribozymes from the RNA world have

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Fig. 9.2 Reactions performed by an in vitro selected redox-active ribozyme. The redox reaction is NAD+/NADH-dependent, similar to natural alcohol dehydrogenase enzymes. The

oxidation of the benzyl alcohol can also be coupled to a spontaneous electron transfer between NADH and FAD (Tsukiji et al., 2003, 2004).

been replaced by deoxyribozymes. Rather, proteins have taken over the vast majority of catalytic functions. Because deoxyribozymes have little relevance to organic chemistry, these will not be discussed here in detail. The intrinsically restricted functionality of nucleic acids compared with that of proteins is a serious shortcoming for the expression of catalytic potential. A possible remedy is the introduction of additional, non-natural functional groups via the incorporation of modified nucleotides (reviewed by Verma et al., 2003). The tolerance of the employed RNA polymerase towards the modifications limits the general use of this technique for the generation of modified ribozymes. Many such ribozymes with modified bases catalyze RNA cleavage or ligation (Beaudry et al., 2000; Dai and Joyce, 2000; Santoro et al., 2000; Teramoto and Joyce, 2000). Further catalytic activities concern metallation of N-methylmesoporphyrin (Kawazoe et al., 2001), formation of a phosphodiester bond with a deoxynucleotide (Teramoto and Joyce, 2000), and cleavage of phosphodiester in trans under simulated physiological conditions (Zinnen et al., 2002). The incorporation by T7 RNA polymerase and successful use in SELEX of a number of uridine derivatives has been reported by Eaton’s laboratory (Vaught et al., 2004), resulting in the development of a cupric ion-dependent modified ribozyme with Diels-Alderase activity (Tarasow et al., 1997, 1999, 2004). As far as we know, the Diels–Alder reaction does not play a major role in any biochemical pathway, and the use of modified nucleotides to expand the catalytic repertoire of RNA was certainly not undertaken with the RNA world as principal motivation. Rather, these approaches underscore the potential for application of catalytic RNA in modern fields of biotechnology and organic synthesis. The use of proteic enzymes in organic synthesis has become a commonplace strategy where applic-


able, the limiting factor being the choice of suitable enzymes and their respective substrate specificities. From this perspective, several ribozymes display catalytic activity of high interest to the organic chemist (see Table 9.1). Importantly, most of these ribozymes have been obtained using a direct selection protocol, where the choice of reactants has dictated the substrate specificity of the resulting ribozymes. Thus, the direct selection method holds the alluring possibility of engineering substrate-specific ribozymes that are tailored to the particular reaction an organic chemist might wish to carry out. Since nucleic acids are chiral, one can even anticipate stereoselectivity in the would-be custom-made catalysts. Although this remains a vision yet to be accomplished, several cases of ribozymes with promising properties have been reported, accelerating certain redox reactions, Michael additions, and cycloadditions. The oxidation of a benzyl alcohol to the corresponding aldehyde has already been mentioned, and the reverse reaction from Suga’s laboratory. The redox ribozymes still require their substrate to be covalently bound, meaning they act in cis, thus performing neither true catalysis nor multiple turnover (Tsukiji et al., 2003). In the Famulok laboratory, a ribozyme has been isolated that promotes a reaction corresponding to the first step of the formation of dTMP from dUMP in proteic thymidylate synthases. This ribozyme mediates Michael-adduct formation at a Michael-acceptor substrate. The reaction is accelerated by a factor of nearly 105. The selected ribozyme could be engineered to act in an intermolecular reaction on a substrate tethered to an RNA oligomer. The demonstration of RNA catalysis of this reaction has bearing on the RNA world hypothesis, as well as implications for possible synthetic applications (Eisenfhr et al., 2003; Sengle et al., 2001). Among the several routes for the creation of ribozymes for a Diels–Alder reaction, a minimal conserved motif (Fig. 9.3b) from a direct selection with linkercoupled reactants conducted in our laboratory so far displays the most promising properties for a potential application in organic synthesis. This construct, a 49-mer RNA, accelerates the formation of C-C bonds between RNA-tethered anthracene and biotinylated N-alkylmaleimides by a factor of up to 18 500. The 49-mer motif features 11 conserved nucleotides in a bulge region and variable helical stems. In contrast to other ribozymes that accelerate bond-forming reactions (Illangasekare et al., 1995; Jenne and Famulok, 1998; Tarasow et al., 1997; Wiegand et al., 1997), this Diels-Alderase behaves like a typical protein enzyme displaying Michaelis–Menten kinetics and works in a bimolecular manner on two reactants that are not attached to any RNA. True catalysis with multiple turnover is performed with a kcat of about 20 min–1. This Diels-Alderase was employed in the first-ever demonstration of RNA-catalyzed enantioselective transformations. The minimal motif yields the Diels–Alder product as a single enantiomer with an enantioselectivity of up to 95% ee, depending on the substitution pattern of the anthracene. Use of the mirror-image l-ribozyme leads to the opposite enantiomer, whereas the uncatalyzed reaction yields a racemic mixture (Seelig et al., 2000) (Fig. 9.3b). Systematic variation of diene and dienophile showed that the DielsAlderase distinguishes between different enantiomers of achiral substrates and

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Fig. 9.3 Ribozyme-catalyzed Diels–Alder reaction in trans. (a) Reaction equation. (b) Reciprocal enantioselectivity in the Diels–Alder reactions catalyzed by the natural d-ribozyme and the spiegelmer (l-ribozyme). Proposed secondary structure motif of the Diels-Alderase ribozymes. The chromatograms show the analysis by chiral high-performance liquid

chromatography (HPLC) of reaction products generated by d-RNA (left) and by l-RNA (right) (R1 = (C2H4O)6 -H, R2 = (CH2)5COOCH3). The two peaks correspond to the two enantiomers. The ribozyme-catalyzed reaction produces one enantiomer in a 20-fold excess whereas the uncatalyzed background (light gray line) shows a racemic (1:1) mixture (Seelig et al., 2000).

hence displays diastereoselectivity (Stuhlmann and Jschke, 2002). The dienophile has to be a five-membered maleimidyl ring without substitution at the double bond. A hydrophobic side chain contributes to ribozyme binding.


Fig. 9.4 Architecture of the Diels–Alder ribozyme (Keiper et al., 2004). (a) Refined secondary structure. (b) Modeled three-dimensional structure.

Some insight into the tertiary structure has been obtained from a variety of probing experiments and a comprehensive mutation analysis. The postulated working model features a Y-shaped framework of double helical arms including an asymmetric internal loop. In an unprecedented manner, nucleotides of the 5l-terminus clamp the opposite sides of the bulge, forming a double pseudoknot. These investigations also suggest that in this rigid structure no significant conformational changes occur upon binding of substrates or products during the catalytic process (Keiper et al., 2004) (Fig. 9.4). A feature of interest to both biotechnology and organic synthesis is the persistence of catalytic activity upon immobilization, which has been unambiguously demonstrated using both enantiomers of the ribozyme. Products of the respective stereochemistry have been obtained after incubation of the reactants with solid phase onto which either enantiomeric ribozyme had been conjugated. The resins remained active even after storage and use for several months, opening up the possibility of the development of a ribozyme reactor prototype for economic synthesis of Diels–Alder products (Schlatterer et al., 2003). Similar stability of an immobilized RNA was reported for an aminoacyl-tRNA synthesis ribozyme, which retained its activity after five cycles (Murakami et al., 2002).

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9.5 Summary and Outlook

We have described early and recent developments in the field of ribozymes with the particular perspective of their potential application in organic synthesis. Today, ribozymes are still much further away from practical application than the organic chemist would like it. It has become clear that more basic research is required before organic synthesis with customized RNA catalysts can be developed into a standard method. However, as illustrated by the case of the Diels-Alderase, most basic requirements have been met in proof-of-principle experiments. Several often-cited shortcomings of RNA have actually been overcome or are on the verge of being solved, or key experiments pointing to a general solution have been reported. Despite their limited set of functional groups, ribozymes can accelerate complex organic transformations (like Diels–Alder reactions) between small molecules in a way similar to that of proteic enzymes or traditional chemical catalysts featuring multiple turnover, substrate specificity, and stereoselectivity. While RNA structure would intuitively be considered incompatible with organic solvents, it has been found that the natural hepatitis delta virus (HDV) ribozyme is structured and active in a 95% formamide solution (Duhamel et al., 1996; FerreD’Amare et al., 1998). Similarly, many supposed incompatibilities of RNA might be overcome by proper choice of selection conditions. Thus, for example, the pH range of ribozyme activity has been expanded to between pH 6.0 and 9.5 by continuous evolution (Kuhne and Joyce, 2003). Heterogeneous catalysis, including catalysis with immobilized ribozymes, presents a promising technique to further improve ribozyme performance (Murakami et al., 2002; Okumoto et al., 2002; Schlatterer et al., 2003).

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Bramlage, B., Luzi, E., Eckstein, F. (1998). Designing ribozymes for the inhibition of gene expression. Trends Biotechnol 16, 434– 438. Breaker, R. R. (2002). Engineered allosteric ribozymes as biosensor components. Curr Opin Biotechnol 13, 31–39. Carmi, N., Shultz, L. A., Breaker, R. R. (1996). In vitro selection of self-cleaving DNAs. Chem Biol 3, 1039–1046. Chapple, K. E., Bartel, D. P., Unrau, P. J. (2003). Combinatorial minimization and secondary structure determination of a nucleotide synthase ribozyme. RNA 9, 1208– 1220.

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10 Deoxyribozymes: Catalytically Active DNA Molecules

10 Deoxyribozymes: Catalytically Active DNA Molecules Kenny Schlosser, Simon A. McManus, and Yingfu Li

10.1 Initial Demonstration of DNA’s Catalytic Ability

Proteins were once regarded as the sole molecular format for biological catalysis. In the early 1980s, however, this perception was challenged by the revelation that certain natural RNAs could function as enzymes to mediate biological catalysis (Kruger et al., 1982; Guerrier-Takada et al., 1983). More natural ribozymes were discovered in subsequent years (Cech and Bass, 1986; Altman et al., 1989; Symons, 1992; Blount and Uhlenbeck, 2002; Cech, 2002; Ferre-D’Amare, 2004; Lilley, 2004), lending support to the “RNA world” hypothesis (Gilbert, 1986; Orgel, 1986; Joyce, 1991). Before long, natural ribozymes were being supplemented by novel artificial ribozymes that catalyzed an even broader range of chemical transformations (Breaker, 1997; Chapman and Szostak, 1994; Jaschke, 2001; Joyce, 1994; Lorsch and Szostak, 1996; Wilson and Szostak, 1999). Of course, the success of both natural and artificial ribozymes could not be fully appreciated without piquing one’s curiosity: if RNA could perform catalysis, might DNA also serve in this capacity? In nature, DNA is predominantly found as a duplex of complementary strands – a condition that prohibits the formation of more complex secondary and tertiary structures. If structural sophistication is a prerequisite for catalytic function, as is commonly observed with ribozymes and protein enzymes, then a naturally occurring DNA enzyme may be no more tangible than our dreams of finding one. Indeed, no DNA enzyme of natural origin has ever been reported. Nevertheless, scientists were not deterred from considering an artificial alternative. DNA could be chemically synthesized as a single-stranded polymer, which like RNA, should possess the freedom to fold into elaborate structures. Thus, it only remained to be asked whether the absence of a 2l-hydroxyl group, and the substitution of one nitrogenous base (uracil in RNA) for another (thymine in DNA), would preclude catalytic activity by single-stranded DNA? This question was ultimately answered in 1994 when the first DNA enzyme was created by in vitro selection techniques (Breaker and Joyce, 1994). The Aptamer Handbook. Edited by S. Klussmann Copyright c 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-31059-2


10.1.1 DNAzymes that Cleave RNA

Inspired by the catalytic achievements of natural and artificial ribozymes, and motivated by attractive characteristics such as great stability and ease of synthesis, Joyce and Breaker sought to develop an enzyme composed entirely of deoxyribonucleotide building blocks. A few prior experiments had provided circumstantial evidence to suggest that single-stranded DNA might conceivably support catalysis. For instance, the observation that a tRNA and its DNA analog could form very similar structures suggested that single-stranded DNA possessed adequate flexibility for complex tertiary structure formation (Paquette et al., 1990). Even more promising was the demonstration that a large fraction of the hammerhead ribozyme could be replaced with deoxyribonucleotides without significant loss of catalytic activity (Perreault et al., 1990). Using the in vitro selection concept originally conceived for the purpose of isolating nucleic acid aptamers (Ellington and Szostak, 1990; Tuerk and Gold, 1990), Breaker and Joyce devised a novel selection strategy to isolate DNA molecules that could catalyze intramolecular cleavage of an embedded ribonucleotide within a DNA chain (Breaker and Joyce, 1994). As an initial endeavor, they reasoned that an RNA cleavage reaction would represent a good starting model system, given the variety of natural and synthetic RNA enzymes that perform the same reaction. Furthermore, they opted in favor of including a divalent metal cofactor to facilitate the reaction, since nearly all ribozymes were known to be dependent on divalent metal ion(s), or display enhanced activity in their presence. Divalent lead was the preferred cofactor in light of Pan and Uhlenbeck’s prior success in isolating Pb2+-dependent RNA-cleaving ribozymes from a random-sequence RNA pool (Pan and Uhlenbeck, 1992). They began with a vast combinatorial library composed of Z1014 different single-stranded DNAs, each containing a stretch of 50 random-sequence nucleotides. This DNA library was subjected to the in vitro selection scheme shown in Fig. 10.1a, which would serve to partition the catalytically active DNA sequences away from the inactive, and then enrich this surviving population by subsequent polymerase chain reaction (PCR) amplification. Each successive round of selective amplification would effectively increase the ratio of active to inactive molecules in the DNA pool. Ultimately, by progressively increasing the selection stringency over the course of multiple rounds of selection, the trillions of DNA sequences in the original starting pool would be narrowed down to just one or a few of the most efficient catalytic sequences. It was and indeed still is, a highly presumptuous endeavor. Nevertheless, their efforts were rewarded. After five rounds of in vitro selection, conducted in just 4 days, the composite population had acquired the ability to cleave 50% of the embedded RNA linkage, with the aid of 1 mmol/L Pb2+ (Breaker and Joyce, 1994). Based on the sequence of 20 clones isolated from this population, a simplified version of the catalytic domain was designed to operate in an intermolecular format (Fig. 10.1b), just like a true enzyme. Exhibiting a turnover rate of Z1 min–1 (at 23 hC and pH 7.0), this 38-nt DNA en-

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Fig. 10.1 Isolation of Pb2+-dependent RNAcleaving DNAzymes. (a) In vitro selection scheme. Double-stranded DNA that contained a 50-nt random-sequence domain (N50) was amplified by PCR using a DNA primer containing a 5l-biotin moiety (labeled as “B” in the figure) and a 3l-terminal adenosine ribonucleotide (labeled as “rA”). The resulting DNA product contained a single embedded ribonucleotide as the target cleavage site, and was immobilized on a streptavidin column. The unbiotinylated DNA strand was washed away with 0.2 N NaOH. A selection buffer containing 1 mmol/L Pb(OAc)2 was applied to the column

and used to promote Pb2+-dependent selfcleavage. The resulting cleavage products were released from the column, collected in the eluate, and amplified by PCR for use in the next round of selective amplification. (b) Intermolecular complex between a Pb2+-dependent RNA-cleaving DNAzyme and its RNA/DNA chimera substrate. A 38-nt DNAzyme binds a 19-nt substrate via two recognition arms that are complementary to portions of the substrate. The substrate contains a single adenosine ribonucleotide within an otherwise allDNA substrate.

zyme provided an approximate 105 fold rate enhancement over the corresponding uncatalyzed reaction – a modest achievement by protein standards, but a bold step forward for the newly emergent field of DNA enzymes. The following year, Breaker and Joyce successfully isolated a Mg2+-dependent RNA-cleaving DNA enzyme (Breaker and Joyce, 1995). This enzyme was no faster than its predecessor, but was nonetheless significant because it suggested that in vitro selected DNAzymes could be compatible with intracellular conditions, and therefore DNAzymes might one day find gainful employment in vivo. It was not too difficult to imagine RNA-cleaving DNAzymes as therapeutic agents that could slice up disease-causing RNAs. The demonstration of a Mg2+-dependent RNA-cleaving DNAzyme would foreshadow a flurry of activities in subsequent years, as molecular biologists were eager to turn this idea into a reality. In the meantime, new representative DNAzymes would continue to emerge from random-sequence libraries and expand DNA’s catalytic repertoire.


10.1.2 Deoxyribozymes that Join DNA

Less than a year after the first reported DNA enzyme, Cuenoud and Szostak described the isolation of yet another. This time, however, DNA ligation was the target reaction (Cuenoud and Szostak, 1995). The initial pool used for in vitro selection was composed of Z1014 different sequences, each containing 116 random-sequence nucleotides flanked by two PCR primer-binding sites. These DNA molecules were incubated with a special DNA substrate bearing an activated 3lphosphorimidazolide to facilitate the ligation reaction and a 5l-biotin tag for selection convenience. DNA sequences that catalyzed the ligation of their own 5l-hydroxyl to the activated substrate would subsequently be retained on a streptavidin–agarose affinity column, while inactive sequences would be removed during washing steps (Fig. 10.2a). An excess of free biotin was added to elute the bound molecules, followed by a PCR step to enrich the remaining population for use in

Fig. 10.2 Isolation of Zn2+/Cu2+-dependent DNA-ligating DNAzymes. (a) In vitro selection scheme. A single-stranded combinatorial DNA pool containing 116 random-sequence (N116) nucleotides was incubated with activated substrate DNA molecules bearing a 5l-biotin moiety and a 3l-phosphorimidazolide (P-Im). The proposed ligation reaction was carried out in a selection buffer containing Mg2+ and Zn2+ for a designated period of time. The ligation mixture was subsequently passed over a streptavidin column where ligated molecules were retained on the column by virtue of their biotin tag. Unligated molecules were washed away under denaturing conditions (3 mol/L urea and 150 mmol/L NaOH), and the ligated molecules specifically eluted with excess free biotin. A selective PCR amplification was con-

ducted by employing a primer with the same sequence as the substrate and a second primer complementary to the 3l constant region. In this way, only DNA molecules ligated to the substrate could be efficiently amplified. The PCR product was used in a second nested PCR reaction to regenerate DNA molecules of the correct size using a biotinated reverse primer, and then passed over a streptavidin column and treated with NaOH to obtain singlestranded molecules for use in the next round of selection. (b) Proposed secondary structure of the E47 DNA ligase DNAzyme. The 47-nt E47 DNAzyme catalyzes the formation of a phosphodiester bond between a 3l-phosphorimidazolide substrate S2, and the 5l-hydroxyl substrate S1.

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the next round of selection. Ultimately, nine rounds of in vitro selection were performed, at which point the ligation activity was observed to plateau. The terminal population was cloned and sequenced to reveal a common consensus sequence in most of the clones. As a result, the researchers were able to design a 47-nt catalyst, E47, which could ligate two entirely separate DNA substrates (Fig. 10.2b). This DNAzyme operated with multiple-turnover kinetics, exhibiting a kcat of 0.07 min–1 at 25 hC. Like its RNA-cleaving predecessors, E47 was a metalloenzyme, which required either divalent zinc or copper for activity. The three preceding DNAzymes were isolated through “direct selection” techniques, in which catalytic DNA molecules were selected based on their ability to perform a self-modification reaction. The next DNAzyme, however, was isolated through an entirely different in vitro selection strategy. 10.1.3 Catalytic DNA for Porphyrin Metallation

In an effort to further expand the list of reactions catalyzed by DNA, Li and Sen enlisted the aid of a transition state analog to find potential DNA catalysts that could promote porphyrin metallation (Fig. 10.3a). This strategy had previously been used to obtain antibodies that could catalyze the metallation of mesoporphyrin IX (Cochran and Schultz, 1990), and so Li and Sen reasoned the same strategy could be exploited to find analogous catalytic DNAs. A large pool of random-sequence DNAs were chemically synthesized and screened for molecules that could bind to N-methylmesoporphyrin IX (NMM), a transition-state analog for the insertion of metal ions into mesoporphyrin IX (MPIX, Fig. 10.3a). A number of NMM-binding aptamers were isolated using the in vitro selection scheme shown in Fig. 10.3b (Li et al., 1996). Two of these DNA aptamers were chosen at random and tested for their ability to catalyze the insertion of Zn2+ or Cu2+ into MPIX. A 33-nt oligomer corresponding to the NMM-binding site of one parent aptamer, designated as PS5.ST1 (Fig. 10.3c), was found to have the most robust catalytic activity. This guanine-rich DNAzyme exhibited a catalytic rate constant of 0.23 min–1 (a 1600-fold rate enhancement over the uncatalyzed reaction) and a Michaelis–Menten constant (KM) of 2.9 mmol/L (Li and Sen, 1996). In subsequent years, an optimal version known as PS5.M was shown to consist of only 24 nucleotides (Fig. 10.3c) and boasted a substantially improved KM of 12.3 mmol/L (Li and Sen, 1997).

10.2 A Tale of Two Deoxyribozymes that Cleave RNA

Fig. 10.3 Isolation of porphyrin metallating DNAzymes. (a) Porphyrin metallation reaction scheme. Insertion of a divalent metal ion (M2+) into the flat ring of mesoporphyrin IX (MPIX) is believed to proceed via a transition state in which the porphyrin core is bent. N-methylmesoporphyrin IX (NMM), with a permanently bent porphyrin core due to the existence of a bulky methyl group in the center of its ring, is a known transition state analog. (b) In vitro selection scheme. A single-stranded combinatorial DNA pool containing a total of 228 random nucleotides (N228) was initially passed through a column lacking NMM derivatized beads to remove DNA molecules that nonspecifically bound to the column beads. The unbound DNA molecules subsequently under-

went positive selection on an NMM-derivatized column. This time, the unbound molecules were washed off and discarded, while the NMM-binding DNA molecules were specifically eluted with free NMM. The eluate was PCR amplified using two suitable primers, one of which contained a 5l-biotin tag. The resulting double-stranded PCR product was then subjected to an avidin column, followed by a denaturing treatment with NaOH to release single-stranded DNA molecules for use in the next round of selection. After 12 rounds of selection, the population was cloned and sequenced, and individual NMM aptamers were randomly tested for their ability to catalyze the metallation of MPIX. (c) Nucleotide sequences of two porphyrin-metallating DNAzymes.


The first few examples of DNA enzymes were remarkable for the sheer fact that they were enzymes made of deoxyribonucleic acid. Aside from their novelty, however, these early representatives had little to offer in the way of utility. Although the Mg2+-dependent RNA-cleaving DNAzyme by Breaker and Joyce (1995) alluded to some potential therapeutic applications, its inability to cleave an all-RNA substrate made such prospects somewhat questionable. The situation would change with the isolation of two general-purpose RNA-cleaving DNA enzymes, referred to as 10-23 and 8-17 (Santoro and Joyce, 1997). Despite an ever-increasing catalogue

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of DNA enzymes, no other DNA motifs would receive as much attention and recognition in the years to come. 10.2.1 In Vitro Selection and Secondary Structures of 10-23 and 8-17

As with all in vitro selection experiments, the isolation of 10-23 and 8-17 began with a library of random-sequence DNA molecules (Santoro and Joyce, 1997). Each member of this library contained a 5l-biotin moiety, followed in turn by a short deoxyribonucleotide spacer, 12 ribonucleotides, and 50 random-sequence deoxyribonucleotides flanked on either side by fixed primer-binding sequences. The in vitro selection scheme was similar to the one used for the creation of the very first deoxyribozyme (Fig. 10.1a). The pool of DNA molecules were immobilized on a streptavidin-coated matrix by virtue of their biotin tag, and subsequently eluted with a reaction buffer containing 10 mmol/L MgCl2 at pH 7.5 and 37 hC. Under these conditions, a small fraction of bound molecules were able to cleave the embedded RNA substrate. The 3l-cleavage products were collected from the eluate and amplified by PCR. The 5l biotin, deoxynucleotide spacer, and substrate ribonucleotides were reintroduced by a primer extension reaction, and the resulting molecules were used to initiate the next round of selection. Two sites within the RNA substrate emerged as the most favorable for cleavage, one dominating during rounds 6–8 and the other during rounds 9 and 10. Individual molecules from the 8th and 10th rounds were cloned and sequenced to reveal a large variety of catalytic motifs. Two of these motifs designated as 10-23 and 8-17 because they were derived from the 23rd and 17th clones after the 10th and 8th round of selection, respectively, were selected for further analysis. Both motifs possessed special qualities that set them apart from all other clones isolated. In addition to exhibiting superior catalytic activity, each motif showed extensive Watson–Crick base pairing to substrate nucleotides on either side of the cleavage site – a characteristic that suggested they could be tailor-made to target other RNA sequences. The 10-23 (Fig. 10.4a) and 8-17 (Fig. 10.4b) motifs were converted into an intermolecular cleavage format by separating the DNA catalytic domain from the RNA substrate. Both DNAzymes promoted multiple-turnover cleavage under simulated physiological conditions (2 mmol/L MgCl2 and 150 mmol/L KCl, pH 7.5, 37 hC). The catalytic core of the 8-17 and 10-23 enzymes consisted of only 13 and 15 deoxynucleotides, respectively, which in turn were situated between two substrate binding arms of 7–8 nucleotides (nt) each. The sequence of the RNA substrate could be changed without compromising catalytic activity as long as the substrate-binding arms of the enzyme were also changed to maintain complementarity (Santoro and Joyce, 1997, 1998). Cleavage occurred on the 3l side of a single unpaired nucleotide. The proposed secondary structure of the 8-17 catalytic core consisted of a short internal stem–loop followed by a single-stranded region of 4–5 nt. The stem contained three base pairs and the loop region was represented by the invariant sequence 5l-AGC-3l. Only the 5l-CG-3l residues were absolutely


Fig. 10.4 Secondary structures of the 10-23 and 8-17 DNAzymes. The DNAzyme (bottom strand) binds the RNA substrate (top strand) through Watson–Crick base pairing. Cleavage occurs at the position indicated by the arrow. R = A or G; Y = U or C. (a) 10-23. The catalytic core of 10-23 consists of 15 highly conserved

nucleotides. The eighth nucleotide position (indicated by italicized T) may occur as a T, C, or A. (B) 8-17. The catalytic core of 8-17 contains 13–14 nt and is highly variable. Only the AG in the loop region of the stem–loop, and the CG in the single-stranded bulge (underlined letters) are absolutely conserved.

conserved in the single-stranded region. By contrast, the 10-23 deoxyribozyme consisted of a single loop which was largely intolerant of variation (Santoro and Joyce, 1997). As an initial demonstration of the potential utility of 10-23, several deoxyribozyme constructs were tested for their ability to cleave a number of biologically important RNAs. Synthetic RNA substrates corresponding to 15–17 nt surrounding the translation initiation site of HIV-1 gag/pol, env, vpr, tat, and nef, were all cleaved at the expected position by a 10-23 motif containing the appropriate substrate-binding arms. Under simulated physiological conditions, all reactions proceeded at a rate of approximately 0.1 min–1 (Santoro and Joyce, 1997). These discoveries paved the way for studies using 10-23 to target full-length biological RNAs, and tests of 10-23 for in vivo applications. 10.2.2 10-23 as Gene Therapeutics

With the discovery of RNA-cleaving DNAzymes, it was only a matter of time before molecular biologists began to investigate whether these DNA-based catalysts could be exploited as therapeutic agents. In general, DNA possesses some advantageous qualities that make it quite attractive as a potential therapeutic tool. In addition to being highly stable to both chemical and thermodynamic stresses, DNA can be synthesized quickly, easily, and relatively inexpensively. When coupled with the ability to cleave RNA with catalytic turnover, DNA-based enzymes represent a promising alternative to traditional antisense therapy. The 10-23 RNA-cleaving DNAzyme, in particular, was considered to be a prime candidate for two very good reasons. Even today, it is the fastest known DNAzyme with a kcat of 10 min–1 under optimized conditions and a reasonably fast catalytic rate under simulated physiological conditions. Equally important, the substrate binding arms of 10-23 (Fig. 10.4a) have been shown to be highly tolerant of base changes. Consequently, 10-23 variants can be engineered to cleave essentially

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any RNA molecule at a specified purine–pyrimidine junction (Santoro and Joyce, 1997, 1998). In practice, some important factors need to be considered when designing a 10-23 construct for therapeutic applications. First is the selection of an appropriate cleavage site within an RNA of interest. Although 10-23 is known to cleave any purine–pyrimidine junction in small (unstructured) RNA molecules, larger RNAs have a tendency to form secondary and tertiary interactions that may prevent 10-23 from accessing the targeted cleavage site. An effective way for identifying a suitable cleavage site is to perform cleavage experiments using long RNA transcripts. Several successful methods have been reported (Cairns et al., 1999; Sriram and Banerjea, 2000; Kurreck et al., 2002). For example, Sriram and Banerjea used a screening method to identify accessible cleavage sites in the HIV-1 gag RNA (Sriram and Banerjea, 2000). They made two pools of DNAzymes both containing the essential catalytic motif but differing in the nature of their substrate binding arms. One pool contained DNAzymes with fully randomized substratebinding arms, while the other contained randomized binding arms with a constant CA preceding the catalytic core. The latter design was intended to favorably target the A-U site of any AUG (methionine) codons. The cleavage products obtained with these two pools of DNAzymes were then amplified by reverse transcriptase PCR (RT-PCR). This approach allowed the authors to pick two cleavage sites for the effective design of DNAzymes that were efficient in suppressing gag gene expression. The intracellular stability of DNAzymes is another factor that needs to be addressed before they can be used productively in vivo. Although DNA is chemically more stable than RNA and proteins, standard DNA oligonucleotides can still be degraded quickly during and after administration in serum and under cellular conditions. Special DNA modifications have been implemented in order to improve the cellular stability of DNAzymes. In nearly all studies reported to date, 10-23 has been modified to contain either phosphorothioate linkages, 3l-3l inverted nucleotides, or 2l-O-methyl-nucleotides. The strategy of using locked nucleic acids to construct the binding arms of 10-23 has also been reported (Vester et al., 2002). In addition, expression vectors capable of producing DNAzymes in bacterial and mammalian cells have been reported as well (Chen et al., 2000; Chen and McMicken, 2003; Tan et al., 2004). To date, many 10-23 variants have been examined for the cleavage of a variety of biologically relevant RNAs. These RNA targets include viral RNAs as well as bacterial and eukaryotic mRNAs involved in disease processes (Table 10.1). 10-23 has been studied as a tool for the inactivation of a host of HIV protein-coding RNAs. For example, Chakraborti and Banerjea have recently described a study in which several DNAzymes were designed to target a stem–loop region of the HIV-1 genome known as TAR (Chakraborti and Banerjea, 2003). Tat and several other cellular proteins are known to bind to the TAR region and regulate transcription of viral genes. This makes TAR an attractive target because cleavage at this site could potentially downregulate the expression of all viral genes. In vitro tests with RNA transcripts followed by examinations using cultured cells cotransfected

10.2 A Tale of Two Deoxyribozymes that Cleave RNA Table 10.1 A list of studies examining therapeutic potential of the 10-23 deoxyribozyme Results/Implication of study

References

Viral and bacterial RNAs HIV-1 HXBR

Inhibition of HIV-1 replication by cleavage of envelope protein mRNA

Zhang et al., 1999

HIV-1 Tat/Rev

Inhibition of mRNAs for regulatory proteins, resulted in inhibition of HIV-1 replication

Unwalla and Banerjea, 2001a,b

HIV-1 env

Cleavage HIV-1 env RNA and inhibition of HIV-1 envelope-CD4 mediated cell fusion

Dash et al., 1998

HIV-1 gag

Inhibition of viral gene expression and replication by cleavage of gag mRNA

Basu et al., 2000; Sriram and Banerjea, 2000

HIV-1 Tat

Inhibition of viral gene expression by cleavage of HIV-1 Tat RNA

Chakraborti and Banerjea, 2003

Influenza A PB2 mRNA

Inhibition of viral replication by cleavage of the PB2 mRNA at translation initiation site

Takahashi et al., 2004

Bacterial ftsZ mRNA

Inhibition of bacterial cell proliferation by an intracellularly expressed DNAzyme

Tan et al., 2004

Eukaryotic mRNAs Protein kinase Ca Induction of apoptosis

Sioud and Leirdal, 2000

TWIST protein

Induction of apoptosis

Hjiantoniou et al., 2003

Human platelettype 12-lipoxygenase

Inhibition of tumor angiogenesis

Liu et al., 2001

LG-BCR-ABL fusion

Cleavage at fusion site of CML-specific hybrid mRNA

Warashina et al., 1999; Wu et al., 1999

Integrins

Inhibition of endothelial cell capillary tube formation

Cieslak et al., 2002

c-myc

Suppression of smooth muscle cell proliferation Sun et al., 1999

Huntington protein

Suppression of HD protein expression

Yen et al., 1999

C-raf kinase

Reduction of C-raf mRNA by up to 36% and inhibition of apoptosis

Chen et al., 2000, 2003

Vanilloid receptor Cleavage of vanilloid receptor subtype 1 mRNA subtype 1

Kurreck et al., 2002

Laminins

Inhibition of mossy fiber axon regeneration

Grimpe et al., 2002

Early growth response factor 1

Inhibition of vascular smooth muscle cell proliferation, neointima formation, breast carcinoma proliferation, migration, and solid tumor growth; abrogating cellular regrowth after mechanical injury

Fahmy and Khachigian, 2004; Lowe et al., 2001, 2002; Mitchell et al., 2004; Santiago et al., 1999

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10 Deoxyribozymes: Catalytically Active DNA Molecules Table 10.1 A list of studies examining therapeutic potential of the 10-23 deoxyribozyme (continue) Results/Implication of study

References

TNF-a

Improving hemodynamic performance

Iversen et al., 2001

VEFG receptor 2

Inhibition of angiogenesis

Zhang et al., 2002

c-JUN

Inhibition of angiogenesis and vascular smooth muscle cell proliferation

Khachigian et al., 2002; Zhang et al., 2004

Raf-1 mRNA

Inhibition of myelomonocytic leukemia

Iversen et al., 2002

with HIV-1 and DNAzyme showed that a 10-23 variant was capable of instigating a significant reduction in HIV-1 replication (i 80%). Among the examples of 10-23 variants that target endogenous eukaryotic mRNAs, one such deoxyribozyme was designed to inhibit angiogenesis by cleaving the mRNA coding for the vascular endothelial growth factor receptor 2 (VEGFR2) (Zhang et al., 2002). Angiogenesis refers to the creation of new blood vessels, which is a process critical for tumor growth. VEGFR2 is usually expressed in embryonic cells and downregulated in adult cells. During induction of angiogenesis, however, VEGFR2 is upregulated. A 10-23 DNAzyme targeting the mRNA of VEGFR2 was created and tested in vitro, with the intended cleavage site between the U and G residues of the AUG initiation codon. The tests showed that the DNAzyme was capable of cleaving an RNA oligonucleotide and a long RNA transcript, producing cleavage fragments consistent with cleavage at the UG site in the initiation codon. When tested in BAEC cells, the DNAzyme caused a 90% reduction in VEFGR2 mRNA production 24 h after administration. DNAzyme treatment of null mice with pre-established human MDA-MB-435 breast carcinomas resulted in a 75% reduction in tumor size, compared with a 35% reduction when treated with a deactivated DNAzyme as a control. Confirmation of angiogenesis inhibition was shown by the reduction of blood vessel density in DNAzyme-treated mice tumors as compared with untreated mice. Another DNAzyme targeted to c-Jun mRNA displayed similar angiogenic inhibition properties (Zhang et al., 2004). In addition to showing that the DNAzyme was capable of reducing the size of certain tumors, this study also demonstrated that c-Jun has a pro-angiogenic role, which was not previously characterized. Another recently designed construct has been found to aid in ganglia axon regeneration at lesions in the spinal cord (Grimpe and Silver, 2004). This is achieved by cleavage of an mRNA for glycoaminoglycan (GAG), an inhibitory proteoglycan. The preceding examples demonstrate the wide range of RNA targets for which 10-23 has been studied, and illustrates the potential utility of these DNAzymes as possible treatment for a variety of diseases and afflictions.


10.2.3 Other Uses of 10-23

10-23 has also been used to construct an autonomous DNA nanomotor (Chen et al., 2004). Like natural protein motors, this DNA motor extracts chemical energy from covalent bonds and converts it into mechanical motion. The DNA motor is composed of two 15-bp helical domains that are connected to opposite ends of the same 10-23 DNA enzyme, and directly to each other via a singlebase hinge (Fig. 10.5a). This DNA motor adopts an open or closed conformational state, depending on its substrate-binding status. In the absence of bound substrate, the presence of divalent metal ions cause the single-stranded DNA enzyme to collapse into a compact coil due to entropic forces, and assume a “closed state.”

Fig. 10.5 A 10-23-based DNA nanomotor. (a) The initial design. The DNA motor is composed of two single strands. One strand contains the catalytic domain of the 10-23 DNAzyme, and the other strand contains a fluorophore (F) and quencher (Q) on opposing ends. The motor is fuelled by an RNA substrate, which is susceptible to cleavage by 10-23. When the RNA substrate hybridizes to the DNAzyme by conventional Watson–Crick base pairing, the DNA motor adopts an open conformation that produces a fluorescent signal. Upon substrate cleavage and dissociation, the DNA motor adopts a closed conformation that quenches the fluorescent signal. The motor continues to cycle between the open and closed states as long as the RNA substrate is

available. (b) The revised design. The improved design incorporated a braking mechanism for the inactivation–reactivation of the DNA motor. The brake strand is an all-DNA strand that preferentially binds to the 10-23 DNAzyme by virtue of additional complementary segments that extend into the catalytic domain, thereby excluding RNA substrate molecules from binding to the same DNAzyme. However, the motor can be reactivated by a strand-displacement mechanism, which exploits another DNA oligonucleotide (i.e. the removal strand) that is fully complementary to the brake strand. The removal strand forms a long duplex with the brake strand, allowing the DNA motor to bind new RNA substrate molecules and resume motion.

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Conversely, when a substrate molecule binds to the DNAzyme it forms a bulged duplex structure, which pushes the two helical domains apart to yield an “open state.” However, subsequent cleavage of the RNA substrate by 10-23 causes dissociation of the resulting lower-affinity cleavage fragments, and the DNA motor returns to its closed state. Thus, the DNA motor will cycle between the open and closed states as long as RNA substrate “fuel” is available. The same group has also engineered a reversible braking mechanism for their DNA nanomotor (Chen and Mao, 2004). It consists of the 10-23 DNAzyme, an RNA substrate, a brake strand and a brake removal strand (Fig. 10.5b). A cycle of substrate binding, cleavage, and dissociation from the DNAzyme provides power to the motor, which is manifested in the form of mechanical motion. Addition of the all-DNA brake strand serves to displace the substrate from the DNAzyme and halts the action of the motor. A brake removal strand can then be introduced to sequester the brake strand away from the DNAzyme, and restart the DNAzyme nanomotor. This technology could potentially be exploited in a number of fields including nanorobotics, molecular computation, dynamic nanomaterials, and biosensing. 10.2.4 Utilities of 8-17

Unlike its 10-23 sibling, the 8-17 DNAzyme has not been examined as a potential therapeutic tool. It has, however, found employment in vitro as a biosensing agent. An 8-17 variant known as 17E was found to have strong Pb2+-dependent RNA cleavage activity (Li and Lu, 2000). Li and Lu reasoned that this lead dependence could be used to create a biosensor for this highly toxic metal ion. They developed a lead biosensor by labeling the 5l end of a substrate oligonucleotide with a fluorophore, and the 3l end of the 17E DNAzyme with a fluorescence quencher (Fig. 10.6). The substrate strand is simply a chimeric DNA/RNA oligomer containing a single embedded RNA linkage as the proposed cleavage site. In the uncleaved state, the substrate forms a duplex with the DNAzyme thereby positioning the fluorophore and quencher in close proximity to yield maximal fluorescence quenching. When Pb2+ is added to a solution containing the substrate and DNAzyme, the lead-dependent catalytic activity of 17E is manifested by cleavage of the RNA linkage. The resulting cleavage fragments have lower affinity for the DNAzyme and therefore dissociate. Separation of the fluorophore from the quencher leads to a fluorescent signal. This biosensor exhibits Z80-fold discrimination against other divalent metal ions and has a large detection range from 10 nmol/L to 4 mmol/L (Li and Lu, 2000). In addition to this fluorogenic biosensor, an analogous colorimetric biosensor for lead has also recently been developed by the same group (Liu and Lu, 2003). This system utilizes an extra DNA oligomer functionalized with gold nanoparticles, which can hybridize to portions of the substrate. In the absence of lead, the DNAzyme, substrate, and gold nanoparticles assemble into large aggregates characterized by a blue color. When the DNAzyme cleaves the substrate in response to a Pb2+ stimulus, the aggregates disperse into separate gold nanoparti-


Fig. 10.6 A catalytic DNA biosensor for Pb2+ ions. The 5l end of the substrate and the 3l end of the 17E DNAzyme are labeled with a fluorophore (F) and quencher (Q), respectively. The DNAzyme binds the substrate through conventional Watson–Crick base pairing, which positions the quencher close to the fluorophore to limit fluorescence emission. In the presence of Pb2+ ions, the DNAzyme becomes

active and cleaves the single ribonucleotide linkage (rA, adenosine ribonucleotide) embedded in the otherwise all-DNA substrate. The individual cleavage fragments have lower affinity for the DNAzyme, and therefore dissociate to produce a concomitant increase in the fluorescence intensity as the fluorophore is separated from the quencher.

cles characterized by a red color. This system is able to detect Pb2+ between 100 nmol/L and 4 mmol/L, and is even tunable to a detection range of 10– 200 mmol/L by simply adding a certain proportion of inactive enzyme strands into the gold nanoparticle network. Nucleic acid sensing represents another vocation of the 8-17 DNAzyme. Recently, Sando et al. designed target-assisted self-cleavage (TASC) probes as a new type of reagent-free nucleic acid sensor (Sando et al., 2003). A TASC probe contains three essential structural elements that are linked together in a single chain: a target-binding sequence, the 8-17 DNAzyme, and a single RNA cleavage site (Fig. 10.7). Hybridization between the target and its corresponding binding sequence on the probe leads to stabilization of an internal hairpin structure which positions the RNA cleavage site in proximity to the DNAzyme domain. In this orientation, the ribo-linkage embedded in the probe is more susceptible to cleavage by 8-17. The resulting two cleavage fragments release the target, and dissociate from one another to yield a fluorescent signal by virtue of the fluorophore–quencher pair located on opposite sides of the cleavage site. Meanwhile, the target is free to bind new probes in a multiple turnover fashion, leading to signal amplification. TASC probes exhibit only a modest rate enhancement of Z16-fold, and a fluorescence enhancement of Z10-fold, upon target activation (Sando et al., 2003). 10.2.5 Recurrence of 8-17 from Several In Vitro Selection Experiments

Despite its small size, 8-17 has had a very large influence on the outcome of in vitro selections for RNA-cleaving DNAzymes. To date, this simple motif has been identified in five independent in vitro selection experiments – a remarkable demonstration of convergent evolution. After its initial introduction in 1997 (Santoro and Joyce, 1997), the 8-17 motif emerged again in 2000, reincarnated

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Fig. 10.7 Nucleic acid signal amplification by a target-assisted self-cleavage (TASC) probe. The TASC probe consists of a target binding sequence, an 8-17 DNAzyme, and a single RNA cleavage site flanked by a fluorophore and quencher. Target binding stabilizes an internal hairpin structure which brings the RNA cleavage site in proximity to the 8-17 DNAzyme. Upon cleavage the resulting two probe frag-

ments dissociate from the target, which is then free to bind another TASC probe in a multiple turnover fashion. A fluorescent signal is concurrently generated via separation of the fluorophore-quencher pair located on opposite probe fragments, and is subjected to amplification since one target can process multiple TASC probes.

as the 17E lead-dependent DNAzyme isolated by J. Li et al. (2000). Interestingly, it was the clever detective work of Peracchi in the same year that led to the discovery of yet another 8-17-yielding selection (Peracchi, 2000). He provided evidence for the existence of 8-17 within the proposed catalytic domain of the Mg5 deoxyribozyme – an enzyme that had been isolated in 1996 (Faulhammer and Famulok, 1996, 1997) prior to the original discovery and characterization of 8-17 by Santoro and Joyce! In 2004, two more reports of 8-17 yielding selections surfaced (Cruz et al., 2004; Schlosser and Li, 2004), one of which provided substantial insight into the reasons behind its prevalence (Cruz et al., 2004). Starting from a common DNA library, Cruz et al. conducted 16 parallel in vitro selection experiments in search of RNA-cleaving DNA enzymes that together could cleave all 16 possible dinucleotide junctions. Ultimately, they observed hundreds of sequences that could form 8-17-like secondary structures with various base alterations (Fig. 10.8). Five representative 8-17 variants were chemically synthesized (Fig. 10.9a), and collectively they were able to cleave 14 different dinucleotide junctions with variable rate constants (Fig. 10.9b). All of the above experiments started with different random-sequence DNA libraries and differed from each other in at least one or more aspects of their in vitro selection protocols, including the type of selection method, library design, and reaction conditions, and yet the same structural solution was ultimately derived. In light of prevailing information, however, the recurrence of 8-17 is not so surprising, and nowadays should even be anticipated. Its relatively large


Fig. 10.8 Structural variations of the 8-17 DNA enzyme. The proposed secondary structure of the original 8-17 DNA enzyme is dissected into six structural domains (denoted SDA to SDF), and individual boxes illustrate the observed variations in structural domains A–D. Structural domain A (SDA) is a trinucleotide loop (triloop), SDB is a 3-bp stem, SDC is the single-stranded region located opposite the cleavage site, SDD corresponds to three nucleotides (the dinucleotide cleavage

site on the substrate strand and one nucleotide on the catalytic strand), and SDE and SDF are two substrate binding arms. The existence of 2–10 alternatives in each structural domain means there are many potential structural permutations and even more sequence permutations for the 8-17 motif. Reprinted from Cruz, R.P.G., Withers, J.W., Li, Y. (2004). Dinucleotide junction cleavage versatility of 8–17 deoxyribozyme. Chem Biol 11, 57–67. Copyright (2004), with permission from Elsevier.

catalytic rate coupled with its ability to function under various metal ion conditions and cleave multiple dinucleotide junctions, allows 8-17 to readily survive and propagate under typical selection pressures. Moreover, the catalytic core of 8-17 is only 13–14 nt in length and the base composition is highly variable, which means that this motif is expected to occur at a relatively high frequency in any given library (Schlosser and Li, 2004). It seems the true significance of the 8-17 DNAzyme is not its catalytic competence, but rather its ability to “hijack” the course of in vitro selection. Indeed, this motif probably represents a kind of evolutionary trap (Lehman, 2004). In order to expand the current collection of RNA-cleaving DNA enzymes, future in vitro selections will have to be wary of this motif, and try to employ novel experimental measures to prevent its emer-

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Fig. 10.9 Catalytic activity of 8-17 variant motifs. (a) Sequences of five chemically synthesized 8-17 variants. The five synthetic DNAs were designed to represent some of the combinations of the most frequently observed structural domains. Each DNA enzyme is denoted by four numerals corresponding to a specific combination of the four structural domains in the order A–D. (b) Catalytic rate constants of five 8-17 variants versus 16 different dinucleotide cleavage junctions. Collec-

tively, these five 8-17 variants can cleave 14 of the 16 possible dinucleotide junctions. The kcat values are expressed in min–1. The cleavage reactions were performed in a buffer containing 7.5 mmol/L MgCl2 and 7.5 mmol/L MnCl2, under single-turnover conditions. Reprinted from Cruz, R.P.G., Withers, J.W., Li, Y. (2004). Dinucleotide junction cleavage versatility of 8– 17 deoxyribozyme. Chem Biol 11, 57–67. Copyright (2004), with permission from Elsevier.

gence. One such measure might be to select for DNA enzymes that can cleave CrT or UrT junctions, both of which were not found to be susceptible to 8-17 mediated cleavage (Cruz et al., 2004).

10.3 Other Deoxyribozymes

10.3 Other Deoxyribozymes 10.3.1 Other RNA-cleaving Deoxyribozymes

As mentioned earlier, the 8-17 motif has been discovered in several independent in vitro selection experiments and has a tendency to “commandeer” selections and become the dominating sequence class, due to its efficiency and small size. Despite this fact, other RNA-cleaving deoxyribozymes with interesting properties have been isolated. One example is a pair of “bipartite” DNAzymes, so called because of the observed segregation of purine and pyrimidine bases in their catalytic core (Feldman and Sen, 2001). Bipartite I was isolated from a pool of DNA molecules each containing a 40-nt random-sequence region, and a single internal ribonucleotide phosphodiester linkage. A dominant class of DNAzymes with a 22-nt catalytic region was found, and a representative sequence was able to cleave the attached substrate at a rate of 1.7 min–1 under optimized conditions. Further rounds of selection with a partially randomized library containing an extended RNA region produced bipartite II (Fig. 10.10a), a deoxyribozyme that is capable of cleaving an all-RNA substrate. This DNAzyme was also shown to have multiple-turnover kinetics using an HIV-1-derived RNA substrate under physiological conditions, demonstrating its potential for RNA cleavage in vivo.

Fig. 10.10 Proposed secondary structures of three other RNA-cleaving DNAzymes. (a) The “bipartite II” deoxyribozyme. (b) A histidinedependent DNAzyme. (c) The fluorescencesignaling DNAzyme DET22-18. Lines between

strands or bases represent Watson–Crick basepairing interactions. F and Q in the structure of DET22-18 are deoxythymidines modified with fluorescein (as the fluorophore) and DABCYL (as the quencher), respectively.

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Divalent metal ions are known to be critical for activity of the majority of nucleic acid catalysts. Nevertheless, an RNA-cleaving DNAzyme has been isolated that mediates catalysis in the absence of any divalent metal ions or other known cofactors (Geyer and Sen, 1997). This DNAzyme has been shown to function completely independent of divalent metal ions, as reflected by its activity in the presence of the metal chelator EDTA, and by the fact that its cleavage rate does not increase in the presence of divalent metal ions. At the other end of the spectrum, an RNA-cleaving DNAzyme that utilizes histidine as a cofactor has also been isolated (Roth and Breaker, 1998) (Fig. 10.10b). The pH profile of this DNAzyme shows half maximal activity at pH 6, which corresponds precisely with the pKa of the imidazole group of histidine. This observation is consistent with the argument that the imidazole may act as a general base in the deprotonation of the 2l-hydroxyl group. The fact that an amino acid can be used as the catalytic moiety by a DNAzyme demonstrates that the breadth of DNA catalyzed reactions could be extended by the exploration of other chemically useful organic molecules as cofactors. DNAzymes have also been developed that combine RNA cleavage with fluorescence signaling (Liu et al., 2003; Mei et al., 2003). This arrangement allows the cleavage reaction to be monitored in real time. These DNAzymes have incorporated a fluorophore and a quencher, which are attached to the nucleotides immediately flanking the cleavage site. Before cleavage, fluorescence is largely quenched by the close proximity of the fluorophore and quencher. Upon cleavage, the fluorophore and quencher are separated and an increase in fluorescence ensues. Through in vitro selection, a fluorescence-signaling DNAzyme, named DET22-18 (Fig. 10.10c), was isolated that cleaves a single ribonucleotide within a DNA substrate at a rate of 7 min–1 (Mei et al., 2003). A group of fluorescencesignaling DNAzymes have also been isolated that cleave the same substrate under different pH conditions (Liu et al., 2003). The DNAzymes that cleave at acidic pH are of particular interest since the protonation states of the nitrogenous bases are different than at neutral pH, and hence they must form interactions different from those used by other DNAzymes that operate at neutral pH. 10.3.2 RNA-ligating Deoxyribozymes

RNA ligation is the reverse reaction of RNA cleavage. Due to the high cost of producing long RNA polymers, ligation of smaller RNA molecules is critical in synthesizing large RNA constructs necessary for studies of RNA structure and function. Traditionally, RNA ligation has been accomplished enzymatically using DNA or RNA ligases and a splint RNA complementary to the 3l and 5l ends of the two ribonucleotide fragments to be ligated. Although efficient in some cases, this procedure has consistency problems and tends to give low yields when linking some RNAs, making it difficult to obtain sufficient quantities of certain RNA molecules. DNAzymes capable of performing this ligase function could potentially be exploited as an alternative to this protein based approach. Starting


from a random-sequence DNA library, several RNA-ligating DNAzymes were isolated that can ligate an RNA fragment with a terminal 2l,3l-cyclic phosphate to the 5l-hydroxyl of a second RNA fragment (Fig. 10.11a) (Flynn-Charlebois et al., 2003b). Interestingly, upon testing individual catalysts all were found to exclusively produce 2l-5l linkages at the ligation site, in contrast to the 3l-5l linkage that is normally observed in RNA. In an attempt to isolate DNAzymes with 3l5l ligase activity, a selection was carried out in which the 8-17 and 10-23 catalytic motifs were partially randomized (Flynn-Charlebois et al., 2003a). Since 8-17 and 10-23 catalyze the cleavage of 3l-5l phosphodiester linkage, it was thought that variants may be isolated that catalyze the reverse, 3l-5l ligation reaction. However, this selection ultimately yielded a small but catalytically efficient ligase deoxyribozyme, 7Q10 (Fig. 10.11b), that catalyze 2l-5l ligation. The same group carried out a similar selection for RNA ligases that produce branched and lariat RNA molecules (Wang and Silverman, 2003). In this case the 2l-5l ligation product is formed by connecting the 2l-hydroxyl of an internal adenosine in one strand to the 5l end of a second RNA strand. The reactants differ from those used by the aforementioned ligase deoxyribozymes, as a 2l-hydroxyl and a 5l-triphosphate are involved, as opposed to a 2l,3l-cyclic phosphate and a 5l-hydroxyl group. This study provided the first synthetic means of producing branched RNA molecules, which should facilitate the study of naturally branched RNA molecules. Since branching is the first step in RNA splicing, the ability to perform this reaction synthetically should enable researchers to learn more about the splicing process (Coppins and Silverman, 2004).

Fig. 10.11 DNAzymes for RNA ligation. (a) Proposed mechanism of DNAzyme catalyzed RNA ligation. The reaction proceeds by nucleophilic attack of the 5l-hydroxyl of one strand of RNA on the phosphorus of a 2l,3lcyclic phosphate on the 3l end of a second

strand of RNA. Of the two possible ligation products, the isolated DNAzymes only catalyzed the formation of a 2l-5l linkage, as shown in the figure. (b) The secondary structure model of 7Q10, a small but efficient ligase DNAzyme.

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10.3.3 DNA-cleaving DNA Enzymes

After the discovery of RNA-cleaving DNAzymes, studies were carried out to see whether in vitro selection could also be used to isolate DNAzymes that cleave DNA. All known RNA-cleaving DNAzymes cleave phosphodiester bonds through a transesterification mechanism, using the 2l-hydroxyl (in the form of a 2l-oxyanion) as a nucleophile. The fact that DNA lacks this 2l-hydroxyl makes cleavage by a hydrolytic mechanism far more complex, since an exogenous nucleophile (such as a water molecule) would be required. Breaker and his co-workers sought to create deoxyribozymes that could cleave a DNA strand using an oxidative mechanism involving Cu2+ and the reducing agent ascorbate (Carmi et al., 1996). Their in vitro selection effort yielded two groups of DNA catalysts. Investigation of some individual catalysts revealed that they contained a major cleavage site, flanked by minor cleavage sites. This pattern is consistent with a diffusible hydroxyl radical acting to cleave the DNA, with the major site being in close proximity to the site of radical formation. Catalysts were also found that cleaved at a slow rate in the presence of Cu2+ and the absence of ascorbate. Another selection conducted on a partially randomized pool produced efficient Cu2+-dependent, DNAcleaving DNAzymes that function independently of any reducing agent. Structural analysis of some catalysts, including bimolecular “substrate” and “enzyme” systems (Fig. 10.12a), revealed that substrate recognition is achieved by duplex and triplex formation (Carmi et al., 1996, 1998). The adaptability of this recognition makes these DNAzymes attractive as possible restriction enzymes.

Fig. 10.12 Two deoxyribozymes that cleave a DNA strand. (a) The secondary structure of the Cu(II)-dependent class II DNA-cleaving deoxyribozyme. Y, R, and N represent a pyrimidine, purine, and any nucleotide, respectively. The major cleavage site is shown with an arrow. Covariation experiments have shown the existence of two stems in the active structure. Similar experiments involving one of the stems and a polypyrimidine tract have revealed that the DNAzyme structure contains a triplex

helical region (triplex interactions represented by *). (b) Proposed secondary structure model for the 10-28 N-glycosylase DNAzyme. The model was made from analysis of sequences obtained from a reselection of 10-28 in which there was a 10% mutation rate per nucleotide. The site of depurination is shown as a hollow letter. The * shows the point where the DNAzyme and substrate portions can be separated to form a trans construct.


Deoxyribozymes have also been isolated that catalyze the cleavage of DNA through an alternative mechanism: N-glycosylation of a guanine base followed by b-elimination (Sheppard et al., 2000). In this case, the lack of a 2l-hydroxyl, which makes the phosphodiester bonds of DNA less susceptible to hydrolysis compared with RNA, actually helps to make the N-glycosyl bonds within DNA more susceptible to hydrolysis. After hydrolysis of the N-glycosyl bond, the 3lphosphate group becomes more susceptible to b-elimination. DNAzymes with this N-glycosylase activity were first discovered during an in vitro selection designed to isolate DNAzymes that cleave b-1,4-O-glycosidic bonds between the galactose and glucose subunits of lactose (Sheppard et al., 2000). It was found that the cleavage products did not correspond to cleavage at the glycosidic bond within lactose, but rather to cleavage at the deoxyguanine residue downstream of the lactose. The fact that cleavage activity was not inhibited by DTT indicated that the cleavage reaction was not proceeding by an oxidative mechanism. Addition of spermidine or piperidine enhanced the cleavage rate. Since these chemical agents excise deoxyribose from apurinated sites, the enhanced reaction rate supports a cleavage mechanism that operates via N-glycosylation and b-elimination. A further selection was carried out to select for N-glycolyase DNAzymes with enhanced catalytic activity. One deoxyribozyme, named “10-28,” has the proposed secondary structure shown in Fig. 10.12b. The model contains two hairpin structures, near the 5l and 3l ends. Between these hairpins are two stems of 9 and 18 base pairs. Support for these stems comes from sequence analyses, which show some base covariations in certain sequences seen in the selected population. 10.3.4 DNA-modifying DNA Enzymes

In addition to their ability for self-cleavage, DNAzymes have been isolated for other self-modification reactions. Several of these DNAzymes catalyze reactions that mimic DNA modifications traditionally carried out by protein enzymes. One example is the 5l phosphorylation of DNA molecules by polynucleotide kinases (PNKs). Two separate in vitro selection experiments have yielded many self-phosphorylating DNAzymes that catalyze the transfer of a g-phosphate from a ribonucleotide triphosphate (NTP) or deoxyribonucleotide triphosphate (dNTP) to their own 5l end (Li and Breaker, 1999; Wang et al., 2002) (Fig. 10.13a). The first selection was designed to isolate self-phosphorylating DNAzymes that could utilize different NTPs/dNTPs as a phosphate source, and show discrimination between these molecules (Li and Breaker, 1999). Using a series of parallel selections which incorporated different (d)NTPs, numerous catalysts were ultimately isolated with wide ranging properties. When supplied with all four standard NTPs or dNTPs, selection from a random DNA library produced many unique catalytic sequences. Two major functional classes were identified in both the NTP and dNTP selection. One class was dependent on (d)GTP as a substrate while the other class could use any of the four (d)NTPs as a source of phosphate. When selection was continued with only a spe-

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Fig. 10.13 DNAzymes for DNA phosphorylation. (a) DNA phosphorylation reaction. The 5l-OH performs a nucleophilic attack on the g phosphorous of an ATP molecule. This results in phosphate transfer and the formation of 5lphosphorylated DNA and an ADP molecule. (b) The secondary structure of NTP-A1. The four GGG elements are proposed to form a

guanine quartet structure. Boxed regions were shown to be highly conserved in reselection experiments. The 3l region of the NTP-A1 shown with a dashed line can be truncated without significant loss of activity. A bimolecular DNAzyme system shows activity with the site of separation shown by the * in the figure.

cific (d)NTP, DNAzymes were eventually isolated that were specific for each of the remaining (d)NTPs, except dTTP. The fact that these DNAzymes could selectively use one (d)NTP as a substrate over another, demonstrates that DNA is capable of discriminating between chemically similar molecules. This selectivity is further exemplified by the isolation of a pair of DNAzymes that can discriminate between GTP and dGTP, which differ only in the presence of a 2l-hydroxyl group in the former. A reselection was conducted on a representative ATP-utilizing DNAzyme named NTP-A1. This led to the isolation of an improved catalyst exhibiting a kcat of Z0.01 min–1, corresponding to a rate enhancement of 109 -fold. The proposed secondary structure for NTP-A1 is shown in Fig. 10.13b. This DNAzyme is one of many catalytic DNAs whose structure appears to have a guanine quartet-containing structure (see more discussion below). In another experiment by Wang and co-workers, self-phosphorylating DNAzymes with different divalent metal ion requirements were isolated by conducting a series of parallel selections each utilizing a different divalent metal cofactor (Wang et al., 2002). When Mn2+ (or Cu2+) was used as the sole divalent cofactor, they discovered many unique catalysts that were Mn2+ (or Cu2+)-dependent but could not utilize other divalent metal ions as cofactors. On the other


hand, in selections where Ca2+ or Mg2+ were used, only a few unique catalytic sequences emerged, and these catalysts could function using any one of four different divalent metal ions (Ca2+, Cu2+, Mg2+, Mn2+). These results suggest that DNAzymes with specific or broad cofactor requirements can be isolated, and that the level of divalent cofactor specificity is likely dependent on the type of metal ions initially supplied in the selection. The same study resulted in two Mn2+-dependent self-phosphorylating deoxyribozymes, named Dk1 and Dk2, that displayed a kcat of 2.8 and 0.8 min–1, respectively. Self-capping (or self-adenylating) DNAzymes represent another functional class of self-modifying DNA, which mimic the first reaction catalyzed by T4 DNA ligase. In this reaction, T4 DNA ligase catalyzes the addition of AMP to the 5lphosphorylated end of a DNA molecule to form an AppDNA. The reaction proceeds via nucleophilic attack of a phosphate oxygen against the a-phosphorus atom of an ATP molecule, resulting in a DNA molecule with an adenylyl 5l-5lpyrophosphate cap (Fig. 10.14a). Selection for these DNAzymes was carried out using a combinatorial library of 5l-phosphorylated DNA molecules (Y. Li et al., 2000b). After incubation for the desired capping activity, the pool was supplied with T4 DNA ligase as well as a template and acceptor DNA strand. In the absence of ATP, only molecules possessing an App cap could be ligated to the acceptor DNA molecule by T4 DNA ligase and subsequently separated by gel electrophoresis. Ultimately, 12 distinct classes of self-capping DNAzymes were isolated and the dominant class denoted as “class-I capase deoxyribozyme.” The self-capping ability of the class I capase deoxyribozyme, which boasted a rate enhancement of 1010 -fold, was confirmed by the observation that periodate oxidation/belimination treatment was necessary for dephosphorylation of the reacted DNAzymes. Additional evidence was provided by experiments using different radiolabeled ATPs, indicating the DNAzymes were indeed catalyzing the transfer

Fig. 10.14 DNAzymes for DNA capping. (a) 5l-Self-capping reaction scheme. The reaction proceeds via nucleophilic attack of an oxygen from a 5l-phosphorylated DNA on the a-phosphate of an ATP molecule. This results in a DNA strand with a 5l pyrophosphate “cap.” (b) A secondary structure model for the class I

capase deoxyribozyme. It contains a four-tier guanine quartet (three complete and one incomplete) predicted from guanine N7 methylation interference assays. The p indicates the phosphate group at the 5l end of the DNAzyme.

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Fig. 10.15 DNA enzymes for DNA ligation. (a) DNAzyme-catalyzed DNA ligation reaction scheme. The 3l-hydroxyl at the terminus of a DNA strand performs nucleophilic attack on a phosphate in the pyrophosphate cap of a 5l-

capped DNA strand. This results in formation of a phosphodiester bond between the two strands. (b) A secondary structure model for a trimolecular DNA ligating DNAzyme.

of AMP to the 5l end of the phosphorylated DNAzyme. The class I capase has the proposed guanine quartet-based secondary structure shown in Fig. 10.14b. DNAzymes have also been isolated that mimic the second reaction catalyzed by T4 DNA ligase (Sreedhara et al., 2004). In this reaction, a DNA molecule with an App moiety (AppDNA) is joined to an acceptor DNA molecule to form a 3l-5l phosphodiester linkage (Fig. 10.15a). This new selection effort utilized the previously mentioned class I capase deoxyribozyme as the source of AppDNA, which was then incubated with a randomized DNA library. The selection yielded DNAzymes capable of forming a 3l-5l linkage between the two DNA molecules, as reflected by the fact that PCR amplification can only proceed in the presence of 3l-5l-linked DNA. Thus, only DNAzymes that could catalyze this reaction would be amplified during each round of in vitro selection. The isolated DNAzyme was configured into a trimolecular DNA ligating system (Fig. 10.15b). The three internal stems and the helical interactions between the DNAzyme and 3l-substrate (AppDNA) are supported by covariation and mismatch experiments. 10.3.5 DNA Enzymes that Catalyze the Formation of Phosphorothioester Bond

In addition to the formation of 3l-5l phosphodiester linkages, DNAzymes have been isolated that catalyze the formation of a non-natural bond between DNA molecules (Levy and Ellington, 2001, 2002). It was known that two DNA fragments, one with a 3l-phosphothioate and the other with a 5l-iodine, became ligated in the presence of a “splint” DNA fragment to form a 5l-phosphothioester linkage (Xu and Kool, 1999) (Fig. 10.16). Since the uncatalyzed reaction is relatively slow, in vitro selection was conducted to identify DNAzymes that could increase the rate of this transformation. Although DNA molecules that catalyzed the reaction were eventually obtained, the rate could not be improved beyond about two per hour despite multiple rounds of selection (Levy and Ellington, 2001). Using the

10.3 Other Deoxyribozymes Fig. 10.16 DNA enzymes catalyzing the formation of phosphorothioester bond. In this reaction a DNAzyme with a 5l-iodo group is reacted with a DNA molecule with a terminal 3l phosphothioate. This results in nucleophilic displacement of the iodine with the generation of a DNA ligation product containing one bridging sulfur atom instead of an oxygen at the ligation junction.

same general system, a second selection was carried out to isolate DNAzymes that could form phosphothioester linkages with different substrates (Levy and Ellington, 2002). This was achieved by alternating between one of five different substrates during each round of selection, where each substrate shared six nucleotides in common adjacent to the 3l-phosphothioate. DNAzymes were isolated that could catalyze the ligation of all five different substrates, suggesting that DNAzymes could have secondary structures capable of accommodating different base sequences, and that future DNAzymes might be developed that could act as universal DNA ligases or replicases. 10.3.6 Deoxyribozymes for Thymine Dimer Repair

Exposure to UV light produces damaging lesions in DNA, of which the thymine dimer is the most predominant. Many different strategies have been developed over the course of evolution to repair these damaging lesions. One such solution is the existence of photolyase enzymes that use lower energy light (compared with the light that produces thymine dimers) to excite chromophores, which donate electrons to the thymine dimer leading to its destabilization and reversion to thymine monomers. An in vitro selection was conducted to isolate DNAzymes that mimic this photoreaction (Chinnapen and Sen, 2004) (Fig. 10.17a). Serotonin was used as a cofactor because the indole group is an adequate sensitizer in the tryptophan residues of protein-catalyzed photoreactions. The thymine dimer was produced between two DNA fragments containing a 5l-thymine and a 3l-thymine, along with a splint DNA that was complimentary to the dimerization site and the flanking 5l and 3l regions of the DNA molecules. UV light of wavelength 337 nm was the stimulus for dimerization. The dimerized constructs were then used as PCR primers to amplify the random-sequence DNA library, allowing their incorporation into the DNA pool. DNA molecules that catalyze self-repair at their thymine dimer site could be isolated by virtue of the fact that dimer synthesis produced molecules lacking a phosphodiester linkage at their dimerization

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10 Deoxyribozymes: Catalytically Active DNA Molecules Fig. 10.17 DNAzymes for thymine dimer repair. (a) Thymine dimer repair reaction scheme. Absorption of light energy results in electron transfer to the thymine dimer initiating the cycloinversion repair process, which transforms the dimer into two adjacent thymine residues. (b) Proposed secondary structure for UV1C, a thymine dimer repair DNAzyme.

site. Therefore, after repair the remaining dimerized fragments could be separated from the repaired DNAs based on size. Serotonin-dependent and serotonin-independent selections were performed, which led to the isolation of one dominant sequence class in the serotonin-independent selection and two major sequence classes in the serotonin-dependent selection. The fact that a serotonin-independent selection (originally a negative selection) yielded DNA catalysts was quite surprising, since neither thymine dimers nor DNA absorb at the longer wavelengths (>300 nm) used in that selection. A bimolecular experiment performed with the dominant sequence, UV1C, from the serotonin-independent selection, showed that the repair DNAzyme could successfully work in “trans” (in which the dimer fragment was physically separated from the enzyme domain). To determine whether the DNAzyme was acting solely to position the dimer in a photoreactive orientation, tests were done with dimercontaining DNA molecules and splint DNA. These tests did not show activity above the background reaction, indicating that the DNAzyme was performing the intended catalytic repair reaction. The proposed secondary structure for UV1C is shown in Fig. 10.17b. Only a few Watson–Crick interactions (shown with lines) have been identified in the structure, indicating that extensive tertiary interactions are used by this deoxyribozyme. Several guanine bases are proposed to be involved in the formation of guanine quartets (shown as hollow letters in Fig. 10.17b). Evidence for the existence of guanine quartets includes the requirement of the DNAzyme for potassium, and methylation protection of the N7 position of the concerned guanines. In addition, this DNAzyme has spectral properties that are consistent with the presence of guanine quartets. 10.3.7 DNA Enzymes with Foreign Functionalities

One of the major criticisms of DNA in terms of its potential as a catalyst is the fact that it is limited in the number of different interactions it can form, since it is composed of only four chemically similar building blocks. Although numer-


ous DNAzymes have been isolated that can perform efficient catalysis despite this limitation, the addition of extra chemical moieties to a strand of DNA could increase the number of interactions DNA can form with itself, ligands, or substrate molecules. To date, three in vitro selection studies have been conducted to derive RNA-cleaving DNAzymes utilizing different base modifications. In the first study, a random-sequence library of DNA molecules containing C5-imidazole deoxyuridine residues were used for in vitro selection (Santoro et al., 2000). One isolated catalyst, denoted 16.2–11, was subsequently minimized to a 12-nt catalytic core, containing three critical imidazole deoxyuridine residues (Fig. 10.18a). The fact

Fig. 10.18 RNA-cleaving DNAzymes with foreign functionanities. (a) The secondary structure model for 16.2-11, a DNAzyme containing C5-imidazole deoxyuridine residues. The modified deoxyuridines critical for catalysis are shown with hollow U’s and their chemical structure is shown on the right side of the figure. (b) Proposed secondary structure of 925 -11, a DNAzyme capable of cleavage of an

embedded RNA linkage in a strand of DNA. The modified bases 5-aminoallyl-dU and 8-histaminyl-dA are shown with italic U’s and A’s respectively, and are critical for activity of this modified DNAzyme. (c) An RNA-cleaving DNAzyme with C5-imidole deoxyuridine (hollow U) and 3-(aminopropynyl)-7-deazadeoxyadenosine (hollow A). Cleavage sites are indicated by arrows.

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that removal of these three residues resulted in loss of activity indicated that these modified bases were critical for folding or catalysis. Another group has isolated a modified RNA-cleaving deoxyribozyme, termed 925 -11 (Fig. 10.18b), which contains two foreign functionalities: an imidazole group placed on deoxyadenosine (italicized “A” in the structure of 925 -11) and an alkylamine attached to deoxyuridine (italicized “U” in the same structure) (Perrin et al., 2001; Lermer et al., 2002). A third selection simultaneously used C5-imidazole deoxyuridine and 3(aminopropynyl-)-7deaza-deoxyadenosine-modified DNA (Sidorov et al., 2004) (Fig. 10.18c). These modifications mimic the metal-independent reaction catalyzed by RNase A, which contains a catalytic histidine and lysine residue. The fact that these two modified bases represent histidine and lysine analogs, respectively, and the fact that the reaction rate decreases by two orders of magnitude when unmodified bases are used, indicate that the modified bases are likely playing an active role in catalysis. These examples illustrate how modified bases can be used advantageously to create DNAzymes, which may fold differently or catalyze reactions through different mechanisms not possible with native DNA.

10.4 Outlook

A decade ago, even after the discovery of catalytic RNA, an enzyme made from DNA would have been regarded as a fanciful notion with little merit. Rather than abandon the idea, however, a few researchers decided to abandon DNA’s complementary strand. What they found was a single-stranded polymer with a remarkable aptitude for catalysis. Since then, the field of DNA enzymes has come a long way in a surprisingly short period of time. In just the first 10 years alone, hundreds of DNA enzymes have been created in vitro, catalyzing more than a dozen different chemical transformations, and boasting rate enhancements as large as 10 billion fold. The next 10 years promise to be just as exciting as researchers continue to challenge DNA with new functional problems, and aspire for rate enhancements that rival protein enzymes. To date, nearly all DNA enzymes catalyze chemical reactions involved in the processing or modification of nucleic acids. Future in vitro selection efforts will no doubt aim to expand the existing collection to include reactions of both greater difficulty and diversity, such as the hydrolytic cleavage of DNA, peptide synthesis, and carbon–carbon bond formation. These reactions have so far eluded catalysis by DNA, but are not necessarily beyond its reach. A marriage between synthetic organic chemists and in vitro selection specialists would likely facilitate their creation, since specially modified substrates have to be attached to combinatorial DNA pools in order to apply “direct selection” techniques. Improvements in the selection methodology would also be highly beneficial toward this end. Although in vitro selection has been used successfully many times in the past, this does not guarantee future success. During the formative years of DNAzyme research, the majority of time was naturally spent in the pursuit of new catalytic

References

DNA molecules. By comparison, little time was ever taken to understand the route by which they were created, and consequently, the underlying processes that govern the outcome of in vitro selection remain poorly characterized. The recurrence of 8-17 serves as a subtle indication that the existing in vitro selection approach, rather than DNA itself, may be a limiting factor in the isolation of new, more efficient, and functionally diverse DNAzymes. For instance, the catalytic rate of 8-17 coincides closely with the minimum reaction time that can be practically applied through manual quenching methods. Unless greater selection stringency can be imposed, the truly fast but exceedingly rare DNAzymes will have no selective advantage over motifs like 8-17, which are distributed more densely through sequence space. The isolation of exceptionally fast DNAzymes, therefore, will depend on our ability to raise the “speed limit” associated with manual quenching. Better ways of exploring sequence space must also be developed (as an added measure of security against future 8-17 recurrence). The reality of this problem is more apparent when you consider it would take around 30 000 tons of DNA to screen all the possible sequence permutations of a 50-nt combinatorial DNA library. Given the sheer magnitude of sequence space, the 8-17 motif likely represents only a local optimum, albeit one that occurs frequently along the catalytic fitness landscape. However, even with the current in vitro evolution protocols, we can only “walk” to neighboring regions of sequence space. To expedite our search for that elusive global optimum, we may need to implement ideas like non-homologous random recombination (Bittker et al., 2002, 2004), which will allow us to “jump” to non-contiguous stretches of sequence space. Recognizing the limitations of in vitro selection will no doubt be the first step toward correcting them. The utility of DNAzyme has become more and more evident over the years, as the focus of research has largely shifted from proving DNA’s catalytic ability to proving its practical utility. DNAzymes have already found employment as biosensors, diagnostic tools, and probable therapeutic agents. However, their rugged durability, facile synthesis, and amenability to surface immobilization and chemical modification, are enduring characteristics that will continue to make DNAzymes attractive targets for innovative applications above and beyond our current imagination. References Altman, S., Baer, M. F., Bartkiewicz, M., Gold, Bittker, J. A., Le, B. V., Liu, D. R. (2002). Nucleic acid evolution and minimization by H., Guerrier-Takada, C., Kirsebom, L. A., nonhomologous random recombination. Lumelsky, N., Peck, K. (1989). Catalysis by Nat Biotechnol 20, 1024–1029. the RNA subunit of RNase P – a minireBittker, J. A., Le, B. V., Liu, J. M., Liu, D. R. view. Gene 82, 63–64. (2004). Directed evolution of protein enBasu, S., Sriram, B., Goila, R., Banerjea, A. C. zymes using nonhomologous random re(2000). Targeted cleavage of HIV-1 corecepcombination. Proc Natl Acad Sci USA 101, tor-CXCR-4 by RNA-cleaving DNA-enzyme: 7011–7016. inhibition of coreceptor function. Antiviral Res 46, 125–134.

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Joyce, G. F. (1991). The rise and fall of the RNA world. New Biol 3, 399–407. Joyce, G. F. (1994). In vitro evolution of nucleic acids. Curr Opin Struct Biol 4, 331–336. Khachigian, L. M., Fahmy, R. G., Zhang, G., Bobryshev, Y. V., Kaniaros, A. (2002). c-Jun regulates vascular smooth muscle cell growth and neointima formation after arterial injury. Inhibition by a novel DNA enzyme targeting c-Jun. J Biol Chem 277, 22985–22991. Kruger, K., Grabowski, P. J., Zaug, A. J., Sands, J., Gottschling, D. E., Cech, T. R. (1982). Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena. Cell 31, 147–157. Kurreck, J., Bieber, B., Jahnel, R., Erdmann, V. A. (2002). Comparative study of DNA enzymes and ribozymes against the same full-length messenger RNA of the vanilloid receptor subtype I. J Biol Chem 277, 7099–7107. Lehman, N. (2004). Assessing the likelihood of recurrence during RNA evolution in vitro. Artif Life 10, 1–22. Lermer, L., Roupioz, Y., Ting, R., Perrin, D. M. (2002). Toward an RNaseA mimic: A DNAzyme with imidazoles and cationic amines. J Am Chem Soc 124, 9960–9961. Levy, M., Ellington, A. D. (2001). Selection of deoxyribozyme ligases that catalyze the formation of an unnatural internucleotide linkage. Bioorg Med Chem 9, 2581–2587. Levy, M., Ellington, A. D. (2002). In vitro selection of a deoxyribozyme that can utilize multiple substrates. J Mol Evol 54, 180–190. Li, J., Lu, Y. (2000). A highly sensitive and selective catalytic DNA biosensor for lead ions. J Am Chem Soc 122, 10466–10467. Li, J., Zheng, W., Kwon, A. H., Lu, Y. (2000). In vitro selection and characterization of a highly efficient Zn(II)-dependent RNAcleaving deoxyribozyme. Nucleic Acids Res 28, 481–488. Li, Y., Breaker, R. R. (1999). Phosphorylating DNA with DNA. Proc Natl Acad Sci USA 96, 2746–2751. Li, Y., Sen, D. (1996). A catalytic DNA for porphyrin metallation. Nat Struct Biol 3, 743–747. Li, Y., Sen, D. (1997). Toward an efficient DNAzyme. Biochemistry 36, 5589–5599.

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10 Deoxyribozymes: Catalytically Active DNA Molecules Li, Y., Geyer, C. R., Sen, D. (1996). Recognition of anionic porphyrins by DNA aptamers. Biochemistry 35, 6911–6922. Li, Y., Liu, Y., Breaker, R. R. (2000). Capping DNA with DNA. Biochemistry 39, 3106– 3114. Lilley, D. M. (2004). The Varkud satellite ribozyme. RNA 10, 151–158. Liu, C., Cheng, R., Sun, L. Q., Tien, P. (2001). Suppression of platelet-type 12-lipoxygenase activity in human erythroleukemia cells by an RNA-cleaving DNAzyme. Biochem Biophys Res Commun 284, 1077–1082. Liu, J., Lu, Y. (2003). A colorimetric lead biosensor using DNAzyme-directed assembly of gold nanoparticles. J Am Chem Soc 125, 6642–6643. Liu, Z., Mei, S. H., Brennan, J. D., Li, Y. (2003). Assemblage of signaling DNA enzymes with intriguing metal-ion specificities and pH dependences. J Am Chem Soc 125, 7539–7545. Lorsch, J. R., Szostak, J. W. (1996). Chance and necessity in the selection of nucleic acid catalysts. Acc Chem Res 29, 103–110. Lowe, H. C., Fahmy, R. G., Kavurma, M. M., Baker, A., Chesterman, C. N., Khachigian, L. M. (2001). Catalytic oligodeoxynucleotides define a key regulatory role for early growth response factor-1 in the porcine model of coronary in-stent restenosis. Circ Res 89, 670–677. Lowe, H. C., Chesterman, C. N., Khachigian, L. M. (2002). Catalytic antisense DNA molecules targeting Egr-1 inhibit neointima formation following permanent ligation of rat common carotid arteries. Thromb Haemost 87, 134–140. Mei, S. H., Liu, Z., Brennan, J. D., Li, Y. (2003). An efficient RNA-cleaving DNA enzyme that synchronizes catalysis with fluorescence signaling. J Am Chem Soc 125, 412–420. Mitchell, A., Dass, C. R., Sun, L. Q., Khachigian, L. M. (2004). Inhibition of human breast carcinoma proliferation, migration, chemoinvasion and solid tumour growth by DNAzymes targeting the zinc finger transcription factor EGR-1. Nucleic Acids Res 32, 3065–3069. Orgel, L. E. (1986). RNA catalysis and the origins of life. J Theor Biol 123, 127–149.

Pan, T., Uhlenbeck, O. C. (1992). In vitro selection of RNAs that undergo autolytic cleavage with Pb2+. Biochemistry 31, 3887–3895. Paquette, J., Nicoghosian, K., Qi, G. R., Beauchemin, N., Cedergren, R. (1990). The conformation of single-stranded nucleic acids tDNA versus tRNA. Eur J Biochem 189, 259–265. Peracchi, A. (2000). Preferential activation of the 8-17 deoxyribozyme by Ca(2+) ions. Evidence for the identity of 8-17 with the catalytic domain of the Mg5 deoxyribozyme. J Biol Chem 275, 11693–11697. Perreault, J. P., Wu, T. F., Cousineau, B., Ogilvie, K. K., Cedergren, R. (1990). Mixed deoxyribo- and ribo-oligonucleotides with catalytic activity. Nature 344, 565–567. Perrin, D. M., Garestier, T., Helene, C. (2001). Bridging the gap between proteins and nucleic acids: a metal-independent RNAseA mimic with two protein-like functionalities. J Am Chem Soc 123, 1556–1563. Roth, A., Breaker, R. R. (1998). An amino acid as a cofactor for a catalytic polynucleotide. Proc Natl Acad Sci USA 95, 6027–6031. Sando, S., Sasaki, T., Kanatani, K., Aoyama, Y. (2003). Amplified nucleic acid sensing using programmed self-cleaving DNAzyme. J Am Chem Soc 125, 15720–15721. Santiago, F. S., Lowe, H. C., Kavurma, M. M., Chesterman, C. N., Baker, A., Atkins, D. G., Khachigian, L. M. (1999). New DNA enzyme targeting Egr-1 mRNA inhibits vascular smooth muscle proliferation and regrowth after injury. Nat Med 5, 1264–1269. Santoro, S. W., Joyce, G. F. (1997). A general purpose RNA-cleaving DNA enzyme. Proc Natl Acad Sci USA 94, 4262–4266. Santoro, S. W., Joyce, G. F. (1998). Mechanism and utility of an RNA-cleaving DNA enzyme. Biochemistry 37, 13330–13342. Santoro, S. W., Joyce, G. F., Sakthivel, K., Gramatikova, S., Barbas, C. F., 3rd (2000). RNA cleavage by a DNA enzyme with extended chemical functionality. J Am Chem Soc 122, 2433–2439. Schlosser, K., Li, Y. (2004). Tracing sequence diversity change of RNA-cleaving deoxyribozymes under increasing selection pressure during in vitro selection. Biochemistry 43, 9695–9707.

References Sheppard, T. L., Ordoukhanian, P., Joyce, G. F. (2000). A DNA enzyme with N-glycosylase activity. Proc Natl Acad Sci USA 97, 7802– 7807. Sidorov, A. V., Grasby, J. A., Williams, D. M. (2004). Sequence-specific cleavage of RNA in the absence of divalent metal ions by a DNAzyme incorporating imidazolyl and amino functionalities. Nucleic Acids Res 32, 1591–1601. Sioud, M., Leirdal, M. (2000). Design of nuclease resistant protein kinase C alpha DNA enzymes with potential therapeutic application. J Mol Biol 296, 937–947. Sreedhara, A., Li, Y., Breaker, R. R. (2004). Ligating DNA with DNA. J Am Chem Soc 126, 3454–3460. Sriram, B., Banerjea, A. C. (2000). In vitro-selected RNA cleaving DNA enzymes from a combinatorial library are potent inhibitors of HIV-1 gene expression. Biochem J 352, 667–673. Sun, L. Q., Cairns, M. J., Gerlach, W. L., Witherington, C., Wang, L., King, A. (1999). Suppression of smooth muscle cell proliferation by a c-myc RNA-cleaving deoxyribozyme. J Biol Chem 274, 17236–17241. Symons, R. H. (1992). Small catalytic RNAs. Annu Rev Biochem 61, 641–671. Takahashi, H., Hamazaki, H., Habu, Y., Hayashi, M., Abe, T., Miyano-Kurosaki, N., Takaku, H. (2004). A new modified DNA enzyme that targets influenza virus A mRNA inhibits viral infection in cultured cells. FEBS Lett 560, 69–74. Tan, X. X., Rose, K., Margolin, W., Chen, Y. (2004). DNA enzyme generated by a novel single-stranded DNA expression vector inhibits expression of the essential bacterial cell division gene ftsZ. Biochemistry 43, 1111–1117. Tuerk, C., Gold, L. (1990). Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505–510. Unwalla, H., Banerjea, A. C. (2001a). Inhibition of HIV-1 gene expression by novel macrophage-tropic DNA enzymes targeted to cleave HIV-1 TAT/Rev RNA. Biochem J 357, 147–155. Unwalla, H., Banerjea, A. C. (2001b). Novel mono- and di-DNA-enzymes targeted to

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Part 4 Applications and Outlook


11.2 Target Validation with Aptamers as Pharmacological Probes

11 In Vivo and In Vitro Target Validation with Nucleic Acid Aptamers as Pharmacological Probes P. Shannon Pendergrast and David M. Epstein

11.1 Introduction

Aptamers are small nucleic acid molecules that function as direct protein inhibitors. Over the last several years, research and development has led to the preclinical evaluation of serum-stabilized aptamers in a variety of in vitro and in vivo systems. Not only have aptamers now demonstrated their broad utility as an exciting class of compounds for use as pharmacological tools for target validation within drug discovery, they have also demonstrated broad potential as therapeutic candidates in drug development.

11.2 Target Validation with Aptamers as Pharmacological Probes

Aptamers are synthetically derived oligonucleotides that bind with high affinity and specificity to protein targets. Serum-stabilized aptamers suitable for in vivo studies can be developed rapidly, usually within 9–14 months (Fig. 11.1). The aptamer generation process is divided into four stages (Fig. 11.1). Identifying a biologically functional and potent aptamer “hit” is a rapid process, taking from five to six months. This target-to-hit step is made highly efficient because the selection of target binders is coupled iteratively to function-based screening (biochemical or cellular assay), such that aptamers identified throughout the selection process are readily characterized for desired potency and functional properties (e.g. specificity) and mechanism-of-action. Over two to three months (stage 2), the targetto-hit aptamer is minimized to elucidate its smallest functional unit, usually from 15 to 35 nucleotides. Over several months medicinal chemistry efforts are aimed at incorporating site-specific backbone modifications to stabilize the minimized aptamer against serum nucleases. Finally, the lead aptamer is readied for use in vivo proof-of-concept studies through conjugation to give the final in vivo aptamer lead its desired pharmacokinetic properties. The combination of backThe Aptamer Handbook. Edited by S. Klussmann Copyright c 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-31059-2

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11 In Vivo and In Vitro Target Validation with Nucleic Acid Aptamers as Pharmacological Probes

Fig. 11.1 Overview of the timeline and the process of discovery and development of aptamers for use as in vivo pharmacological probes.

bone modifications within the aptamer core and the final conjugation (e.g. 20 kDa, 30 kDa, 40 kDa polyethylene glycol (PEG)) provide control of and the ability to tune the pharmacokinetic and distribution properties of the in vivo lead molecule (Fig. 11.2). Aptamers as pharmacological probes have proven to be versatile reagents for in vivo target validation and proof-of-concept studies in that they provide direct evidence for the effect of target inhibition in vivo. We describe the use of aptamers as both intracellular (in vitro) and extracellular (in vitro and in vivo) pharmacological probes. Importantly, these same pharmacological probes are also being developed into therapeutic agents for the treatment of acute, subacute, and chronic indications (Longman, 2004). In vivo target validation determines whether a drug target is involved in disease pathology, and therefore, is a point of intervention for new therapeutics. For most of the pharmaceutical industry’s targets, which are now genomically derived, as well as for many targets that are biochemically and genetically validated, there

11.2 Target Validation with Aptamers as Pharmacological Probes Fig. 11.2 Overview of the combined effects of aptamer backbone composition and aptamer 5l- or 3l-terminal conjugation on in vivo pharmacokinetic halflife (measured in rodents and primates).

may be little, if any, in vivo biological data that can be used to guide drug discovery and development decisions. Traditionally, target validation in preclinical discovery research has relied on biochemical (in vitro) or genetic (in vivo) means for linking a target’s biological mechanism to disease pathology. In this review we discuss the merits of biochemical, or pharmacological, based approaches to target validation that utilize nucleic acid aptamers as in vivo agents to probe pharmacology and mechanism- of-action in animal models of disease. We contrast the aptamer-based target validation approach to gene or message-directed methods for assessing a target’s biological function. Aptamers are highly specific and target-selective pharmacological probes, and as such, we argue that aptamer-based methods have the advantage of allowing the researcher to directly interrogate the pharmacological effect of target blockade in vivo, in a dose-dependent manner. Hence, an aptamer-based in vivo validation approach provides direct information on target biology, while also providing two distinct routes to drug discovery: one route utilizes the aptamer itself as the drug development candidate and the second route uses the aptamer as the pharmacological probe against which small molecule or biologic agents are screened and subsequently developed.

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11.3 Limitations of Target Validation by Gene or mRNA Knockout

A biological approach for determining the relevance of a protein to a particular disease is to block expression of the gene in a model organism. As such, target validation methods have been devised that attack gene expression at the DNA and mRNA level (Fig. 11.3). Blocking gene expression at the genetic level is accomplished by constructing a knockout animal. The gene knockout approach completely abolishes gene expression such that the function of a drug target can be examined in every system of the mammalian species simultaneously. In our view, however, one is forced to make relatively risky drug discovery decisions if target validation is based solely on a gene knockout approach, because the results observed in the model organism often do not translate to understanding human disease. It is worrying that transgenic knockouts, which otherwise have been well-characterized in cellular assays, often exhibit phenotypes that are inconsistent with those observed in previous work (Pich and Epping-Jordan, 1998). These conflicting phenotypic effects can manifest either as a very mild phenotype, possibly as a result of an additional redundant gene, or as an animal that has developmental defects resulting in lethality (Ihle, 2000). More importantly, however, since knockout approaches result in the complete removal of a protein target of interest, it is likely impossible to predict target-specific pharmacological effects from the phenotypic effects of the knockout. Alternatively, in vivo aptamerbased target validation studies afford a direct link between dose level and efficacy.

Fig. 11.3 The levels of gene expression and target validation techniques that target each.

11.3 Limitations of Target Validation by Gene or mRNA Knockout

Gene-level target validation approaches include techniques that knock out gene expression at the mRNA level. RNA interference (RNAi) (Hannon, 2002) and antisense (Taylor et al., 1999) are two such approaches. RNAi uses double-stranded RNA to induce homology-dependent degradation of the cognate mRNA, and therefore block the expression of the desired protein. Initially, a major drawback of RNAi was that when applied to mammalian cells it triggered non-specific responses which obscured sequence-specific silencing. One of these non-specific responses was the activation of the RNA-dependent protein kinase (PKR) pathway, which phosphorylates EIF-2a and non-specifically arrests translation. Due to their small size, short inhibitory RNAs (siRNAs) can effectively block protein expression, without inducing PKR (Elbashir et al., 2001). Although some non-specific effects via the interferon response (Sledz et al., 2002; Bridge et al., 2003) and possibly other pathways (Persengiev et al., 2004) have been reported more recently. Furthermore, there still remain significant technical and scientific hurdles to the broad, systemic use of functional siRNA molecules in vivo in animal models of disease. Indeed, the delivery and uptake of functional siRNA molecules into mammalian model organisms has been achieved by either local injection (intravitreal) or injection at high pressure and volume. So far, for instance, there have only been a few examples of their use in mouse models for disease (Buckingham et al., 2004; Tompkins et al., 2004; Xia et al., 2004). In the case of antisense, base pairing of an antisense strand of RNA (or DNA) with its corresponding mRNA blocks translation of the mRNA by marking the RNA for degradation by RNase or by blocking the protein translation machinery. Antisense reagents have a number of disadvantages for use in in vivo target validation studies, including poor pharmacokinetic properties and concerns about specificity. Advances in the chemical modification of antisense RNA backbones have alleviated some of these concerns (Gewirtz, 1999; Bennett, 2002), but do not address fundamental issues of intracellular uptake and cellular targeting of a oligonucleotide agent, nor do they address the broad utility of antisense or siRNA as pharmacological probes of target function. In addition to validating the role of the protein in disease pathology, it is also important to validate the protein as a target for therapeutic intervention. Since proteins sometimes have more than one function and are part of more than one multiprotein complex, deletion of the protein through altering mRNA levels can lead to the disruption of numerous pathways and regulatory cascades, some of which may not be relevant to the disease model in question. Finally, from the aptamer-centric view, validation techniques which function at the gene level do not afford predictable pharmacology, such as that based on dose-dependent inhibition of protein function. Validating drug targets at the protein level with aptamers yields direct evidence on pharmacology and mechanism-of-action (Shaw et al., 1995; Ruckman et al., 1998), and may lead to predictions of what may be expected of a small molecule or biological drug directed at the same target. Antibodies, which bind proteins with high affinity and specificity, are also a powerful way to inactivate protein targets (Lichtlen et al., 2002). Some progress has been made towards the use of single-chain antibody fragments (scFvs) for intracellular applications, however even

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for these molecules the reducing conditions within the cytoplasm lead to misfolding and aggregation (Lichten et al., 2002).

11.4 Target Validation Using Nucleic Acid Aptamers

The interrogation of target function in vivo using nucleic acid aptamers, probes the mechanism-of-action of target blockade and the pharmacology related to target inhibition. Aptamers have many attributes that make them suitable for use as pharmacological probes in in vivo proof-of-concept studies and, more broadly as reagents for in vivo target validation. They bind with high affinity (James, 2000) and exhibit a degree of specificity that allows them to distinguish between closely related molecules and includes the ability to discriminate between the phosphorylated forms of a MAP kinase (Fig. 11.4). Like small-molecule therapeutics, their dosage can be easily adjusted and they exhibit a dose response (Reyderman and Stavchansky, 1998). Importantly, an aptamer can inhibit target function in vivo by blocking or knocking out a single domain of a protein while leaving the remainder of the protein functional. Small-molecule drugs are also well known for this capability (Aramburu et al., 1999). Small molecules and aptamers share another characteristic in that they both, potentially, can act as agonists. Along with the above conceptual advantages, there are also practical advantages to working with aptamers. Being nucleic acids, aptamers are easy to produce, store and modify. Large amounts of aptamers can be produced in vitro either enzymatically or synthetically. They can be easily delivered intracellularly by standard transfection techniques (Chan et al., 2006) or produced in vivo with the appropriate expression vectors. Lyophilized aptamers can be stored for years without loss of activity and once they are reconstituted, they can be boiled or subjected to numerous freeze–thaw cycles. Their stability both in vitro and in vivo can be further enhanced by various chemical modifications such as 2l-fluoro and 2l-Omethyl substitution (Brody and Gold, 2000).

Fig. 11.4 Aptamer specificity is demonstrated through binding to only the phosphorylated form, a MAP kinase, pERK. Positive and negative selection methods were used to drive drives preferential recognition of pERK (squares) over that of native ERK (circles).


11.4.1 In Vitro Target Validation with Aptamers against Intracellular Targets

A number of studies have demonstrated the use of aptamers as intracellular target validation tools. Aptamers have been selected against HIV-1 Rev and Tat, two RNA-binding proteins that are necessary for HIV-1 replication. Rev interacts with the Rev binding element (RBE) and functions to transport the viral RNA from the nucleus to the cytoplasm. Tat is an RNA-binding transcription factor that is required for viral replication. When expressed intracellularly anti-Rev or anti-Tat aptamers have inhibited HIV-1 production in cell culture (Lee et al., 1995; Symensma et al., 1996). Further studies have demonstrated that aptamers can recognize proteins that mediate the mammalian signal transduction processes. For example, Blind et al. (1999) selected aptamers that recognize the cytoplasmic domain of aLb2 -integrin. b2 -Integrins have been demonstrated to play a critical role in leukocyte cell adhesion. Anti-b2 aptamers were demonstrated to inhibit aLb2 -integrin activity, as measured by a decrease in cell adhesion (Blind et al., 1999). Mayer et al. (2001) have extended these observations by knocking out cytohesin 1, a signaling protein downstream of aLb2 -integrin. The anti-cytohesin 1 aptamer was demonstrated to inhibit the guanine nucleotide exchange factor (GEF) activity of cytohesin 1. Moreover, cytohesin 1 inhibition translated into a biological effect as measured by cell adhesion and cytoskeletal rearrangement. Another consequence of working at the protein level is that aptamers can compliment gene-level validation approaches. Recently, we have reduced expression of another signal transduction protein, NFkB, by overexpression of an aptamer specific for its p50 subunit (Chan et al., 2006). NFkB activity was best inhibited by transfecting a vector that expressed a transcript consisting of the aptamer and

Fig. 11.5 Diagram of theoretical transcript generated from aptamer expression constructs. Transcript generated from 7SL-NFkB. 7SL sequences are in black, aptamer sequence in red and terminator sequence in blue.

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Fig. 11.6 NFkB activity is most significantly inhibited in the presence of both p50-specific siRNA and p50-specific aptamer. Bar graph of results of NFkB-dependent luciferase assay of cells transfected with NFkB-dependent reporter plasmid along with control plasmids for both siRNA and aptamer expressors (siRNA CON + 7SL), p50-specific siRNA and aptamer control

plasmid (siRNA2 + 7SL), p50-specific aptamer + siRNA control plasmid (siRNA CON + 7SL-ap50), and plasmids expressing both p50-specific siRNA and aptamer (siRNA 2 + 7SL-ap50). Shown experiment is representative of multiple experiments. Error bars generated by Microsoft Excel.

the stabilizing natural RNA, 7SL (Fig. 11.5). The aptamer reduced NFkB activity by 64%, about the same as could be achieved using siRNA (Fig. 11.6). Interestingly, cotransfection of vectors expressing the NFkB-specific aptamer with vectors expressing the NFkB-specific siRNA resulted in nearly complete inhibition NFkB (Fig. 11.3). This result is consistent with the idea that attacking gene expression at multiple levels (in this case at the mRNA and the protein levels) might be the most effective way to reduce activity. Given that both siRNA and aptamers can be delivered via an RNA transcript, it may be possible to express them simultaneously from one vector. 11.4.2 In Vivo Target Validation with Aptamers against Intracellular and Extracellular Targets

Transgenic aptamer-expressing Drosophila have been used to demonstrate the efficacy of aptamers as target validation tools in whole animals. Shi et al. (1999) showed that an anti-B52 aptamer could function in vivo to inhibit B52 function. B52 is a member of the Drosophila SR protein family, a group of nuclear proteins that are essential for pre-mRNA splicing. Shi et al. (1999) developed an RNA aptamer that specifically bound to B52 with high affinity and configured a multiva-


lent aptamer for expression in cells and in flies. Previous work has shown that the level of B52 expression is critical for normal Drosophila development. B52 deletion results in death (Ring and Lis, 1994), and overexpression of B52 is associated with lethality and morphological defects including missing bristles and the absence of salivary glands (Kraus and Lis, 1994). Shi et al. (1999) used anti-B52 aptamers to suppress the effects of B52 overexpression in Drosophila. They observed the reversal of abnormal bristle, wing, abdominal sternite, and salivary gland developmental associated with B52 overexpression. Moreover, an increase in the survival rate was observed for flies coexpressing B52 and the anti-B52 aptamer as compared with B52 expressers alone. These results demonstrate that aptamers can be used to inhibit intracellular target function in a non-mammalian, whole animal systems. More relevant to drug discovery and development is the ability to use serum-stabilized aptamers in vivo as pharmacological probes. Over the last several years, research and technology enhancements, including facile synthesis and conjugation chemistries, have led to the preclinical evaluation of serum-stabilized aptamers in a variety of in vitro and in vivo systems, as summarized in Table 11.1. Thus, aptamers have now demonstrated their broad utility as a novel class of compounds for use as pharmacological tools for in vivo target validation and as unique agents for drug discovery. The ability to synthesize appropriate quantities of stabilized lead aptamers and to subsequently couple such aptamers with high molecular weight PEG polymers (termed “PEGylation,” see below) without abrogating biological function allows serum-stabilized aptamers to survive and remain pharmacologically active in vivo, rendering them exceptionally specific tools for extracellular target validation (Fig. 11.1). A nice illustration of the use of such molecules for extracellular target validation is a series of experiments from Pietras et al., utilizing an anti-PDGF-B aptamer (Pietras et al., 2001, 2002). Platelet-derived growth factor (PDGF) belongs to the cysteine-knot growth factor family and was originally isolated from platelets for promoting cellular mitogenic and migratory activity. The binding of PDGF isoforms to their cognate receptors induces the phosphorylation of specific residues in the intracellular tyrosine kinase domain of the receptors and activation of the signaling pathway. In general, PDGF isoforms are potent mitogens and thus are targeted for proliferative diseases such as cancer, diabetic retinopathy, glomerulonephritis, and restenosis. PDGF-B has been implicated in the regulation of interstitial fluid pressure (IFP) in human tumors. Elevated IFP is one of the physiologically distinctive properties of solid tumors that differ from healthy connective tissue and is considered to be a primary obstacle limiting free diffusion of therapeutic agents into solid tumors. IFP increases as a function of tumor size and malignancy. In general, high IFP in cancer patients is associated with poor prognosis. Notably, solid tumors, including those that are treated with standard chemotherapy regimens, exhibit high IFP. In addition, the stromal tissue into which the tumor epithelial cells proliferate contains PDGF-B responsive fibroblasts. Current data indicate that tensile strength and mechanical stiffness of connective tissue are regulated by a complex interaction between cells such as fibroblasts with extracel-

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Aptamer enhances tumor immunity in a B16 melanoma mouse tumor model

Aptamer increases clotting times ACT and aPTT but not PT, when administered by infusion in pig and murine models. In vivo anticlotting activity of the aptamer can be reversed by addition of the antidote sequence In a castrated rat model, GnRH-mediated luteinizing hormone (LH) levels are reduced, upon aptame treatment, to those observed in intact animals

KD = 30 nmol/L Aptamer blocks CTLA-4 mediated negative regulation of stimulated T-cellsleading to enhanced proliferation

KD = 5 nmol/L

KD = 10 nmol/L Aptamer increases clotting times (ACT, aPTT and PT) in plasma from rat, primate, human

KD = 650 pmol/L Aptamer increases clotting times aPTT but not PT, in human plasma. In vitro anti-clotting activity of the aptamer can be reversed by addition of antidote sequence

KD = 20 nmol/L Aptamer GnRH-mediate calcium flux in Chinese hamster ovary cells expressing the GnRH receptor

CTLA-4 (Santulli-Marotto et al., 2003)

Tenascin C (Hiche et al., 2001)

Thrombin (Bock et al., 1992; Griffin et al., 1993; DeAnda et al., 1994)

Factor IXa (Rusconi et al., 2004)

GnRH (Wlotzka et al., 2002)

VEGF, vascular endothelial growth factor PDGF, platelet-derived growth factor; CTLA, cytotoxic T lymphocyte antigen; GnRH, gonadotropin-releasing hormone; TNF, tumor neurosis factor; ACT, activated clotting time; aPTT, activated partial thromboplastin time; PT, prothrombin time.

Aptamer increases clotting times (ACT, aPTT and PT) during intravenous infusion and reverses rapidly without antidote in dog cardiopulmonary bypass, sheep hemodialysis and, primate infusion models

Tc-labeled aptamer specifically localizes to tumors

99

Aptamer reduces tumor interstitial fluid pressure and increases taxol uptake and efficacy Aptamer blocks experimental glomerulonephritis in rat model Aptamer blocks restenosis in rat model

KD = 100 pmol/L Aptamer blocks PDGF-BB-mediated proliferation

PDGF-B (Pietras et al., 2003)

Aptamer binds equivalently to cellsurface target

Aptamer blocks Wilms’ tumor growth in mouse xenograft model Aptamer blocks A673 rhabdomyosarcoma growth Aptamer blocks neovascularization in corneal angigenesis and ROP models

VEGF-A KD = 300 pmol/L Aptamer blocks VEGF165-mediated proliferation (Ruckman et al., 1998; Drolek et al., 2000; Ishida et al., 2003)

Aptamer blocks Ang1, Ang2-mediated inhibiAptamer inhibits neovascularization in a bFGF-mediated corneal tion of apoptosis in TNF-a treated HUVEC cells nicropocket angiogenesis assay in rat

KD = 10 nmol/L

Angiopoietin-2 (White et al., 2003)

In Vivo disease model

In Vitro cell-based activity

Biochemical

Target

Table 11.1 Overview of in vitro and in vivo pharmacological properties of select functional aptamers: The protein target of interest is listed along with key results on the in vitro mechanism and in vivo proof-of-concept studies

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lular matrix components such as collagen and hyaluronan. PDGF is known to upregulate synthesis of collagen and to mediate interactions of anchor proteins such as integrins with extracellular matrix components. Thus, it has been hypothesized that tumor cell-derived PDGF-B secretion leads to proliferation of tumor stromal fibroblasts and deposition of collagen, thereby leading to reduced fluid flow within the tumor and elevated IFP levels. Pietras et al. sought to test the hypothesis that local PDGF-B secretion within the tumor microenvironment leads to high IFP levels in human tumors. To test this hypothesis Pietras examined the ability of the PDGF-B aptamer to block PDGF-B function in vivo, and whether PDGF-B inhibition was specific for PDGF-B receptors within the tumor stroma, and finally whether the PDGFB inhibition leads to decreased IFP levels and concomitantly increased levels of chemotherapeutic agents within the tumor (Pietras et al., 2001). Using a KAT-4 thyroid carcinoma xenograft model these researchers demonstrated that the KAT-4 tumor cells in vivo do not express PDGF b-receptors and thus are not responsive to PDGF-B inhibition. Further, they demonstrated that PDGF b-receptor expression is localized to the tumor stroma in this xenograft model and that labeled PDGF-B bound only to the stromal component of the KAT-4 xenograft. To examine the relationship between PDGF-B signaling and IFP in the KAT-4 model, STI571, a small-molecule tyrosine kinase inhibitor was administered by oral gavage once daily at a dose of 100 mg/kg per day. Daily STI571 treatment significantly decreased tumor IFP in vivo (Fig. 11.7a), leading to increased uptake of Taxol. Most significantly, STI571 treatment also enhanced the antiproliferative effect of Taxol (Fig. 11.8a). These data, along with previously published data, indicates that PDGF-B is likely a good target for lowering IFP and concomitantly increasing chemotherapeutic efficacy. However, STI571, like all small-molecule

Fig. 11.7 Treatment with platelet-derived growth factor (PDGF) receptor antagonists lowers interstitial fluid pressure (IFP) in KAT-4 tumors. Tumor IFP was measured 1–2 h after last administration of PDGF receptor inhibitor in KAT-4 tumors grown s.c. in SCID mice.

PDGF receptor inhibitors were administered for a total of 4 days. (a) mice were treated with phosphate-buffered saline (PBS) (n = 8) or STI571 (n = 9). (b) Mice were treated with control aptamer (n = 8) or PDGF aptamer (n = 8). From Pietras et al. (2002).

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Fig. 11.8 Treatment with platelet-derived growth factor (PDGF) receptor antagonists enhances the effect of Taxol on KAT-4 tumors in vivo. Growth curves of KAT-4 tumors grown s.c. in SCID mice. (a) Mice received no treatment (solid square, n = 8), STI571 (solid circle, n = 6), Taxol (solid triangle, n = 4), or STI571 and Taxol (cross, n = 8). (b) Mice received polyethylene glycol (PEG) (solid square, n = 8),

PEG-conjugated PDGF aptamer (solid circle, n = 8), PEG and Taxol (solid triangle, n = 8), or PDGF aptamer and Taxol (cross, n = 8). *P I 0.05, PDGF receptor antagonist + Taxol versus Taxol alone, Student’s t-test, **P I 0.01 PDGF receptor antagonist + Taxol versus Taxol alone, Student’s t-test. From Pietras et al. (2002).

kinase inhibitors, is known to affect other tyrosine kinases, it was impossible to know if the effect was due to PDGF-B blockade alone. This dilemma was solved by using a highly specific aptamer to block PDGF-B in similar experiments. The PDGF-B aptamer (Fig. 11.9) was isolated through single-stranded DNA SELEX (Green et al., 1996). The aptamer has an affinity of 100 pmol/L for PDGF-B and no appreciable affinity for the PDGF-A isoform. As with STI571, treatment of KAT-4 xenograft mice with PEG-conjugated PDGF-B aptamer lowered IFP (Fig. 11.7b) and significantly increased tumor uptake of Taxol. Most importantly, aptamer treatment strongly enhanced Taxol’s ability to inhibit tumor growth (Fig. 11.8b). Given the high specificity of aptamers, these experiments further validate the concept of blocking PDGF-B as a means of enhancing the uptake and efficacy of chemotherapeutics. The rapid generation and utilization of the anti-PDGF-B aptamer to rapidly obtain in vivo proof-of-concept data illustrates the utility of aptamers for target validation. Furthermore, because aptamers in general and the anti-PDGF-B aptamer in particular, already have many of the attributes required for a therapeutic, the anti-PDGF-B aptamer can directly enter into a therapeutic development program.

References

Fig. 11.9 Anti-PDGF-B specific aptamer, ARC127.

11.5 Summary

High-affinity, target-specific aptamers for use as both in vitro and in vivo pharmacological probes can be generated within one year. As such, aptamers provide versatile tools for the validation of intracellular and extracellular targets. In the case of extracellular targets, such as vascular endothelial growth factor (VEGF), thrombin, and PDGF discussed here, aptamer-based validation affords a direct path to therapeutic development. Therapeutic aptamer leads can be readily stabilized or shielded from renal filtration by chemical or compositional modification for evaluation in in vivo preclinical discovery programs. Aptamers appear poised to make a significant contribution to the treatment of acute and chronic diseases.

References Aramburu, J., Yappe, M. B., Lopez-Rodriguez, C., Cantley, L. C., Hogan, P. G., Rao, A. (1999). Affinity-driven peptide selection of an NFAT inhibitor more selective than cyclosporin A. Science 285, 2129–2133. Bennett, C. F. (2002) Efficiency of antisense oligonucleotide drug discovery. Antisense Nucleic Acid Drug Dev 12, 215–224. Blind, M., Kolanus, W., Famulok, M. (1999). Cytoplasmic RNA modulators of an insideout signal-transduction cascade. Proc Natl Acad Sci USA 96, 3606–3610. Bock, L. C., Griffin, L. C., Latham, J. A., Vermaas, E. H., Toole, J. J. (1992). Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature 355, 564–566.

Bridge, A. J., Pebernard, S., Ducraux, A., Nicoulaz, A. L., Iggo, R. (2003). Induction of an interferon response by RNAi vectors in mammalian cells. Nat Genet 34, 263–264. Brody, E. N., Gold, L. (2000). Aptamers as therapeutic and diagnostic agents. J Biotechnol 74, 5–13. Buckingham S. D., Esmaeili, B., Wood, M., Sattelle, D. B. (2004). RNA interference: from model organisms towards therapy for neral and neuromuscular disorders. Hum Mol Genet 13, R275–R288. Chan, R., Gilbert, M., Marshal, K., Marsh, N., Epstein, D., Pendergrast, P. S. (2006). Co-expression of anti-NFkB RNA aptamers and siRNAs leads to maximal suppresion of NFkB activity in mammalian cells. NAR Methods in press.

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11 In Vivo and In Vitro Target Validation with Nucleic Acid Aptamers as Pharmacological Probes DeAnda, A., Jr. Coutre, S. E., Moon, M. R., Vial, C. M., Griffin, L. C., Law, V. S., Komeda, M., Leung, L. L., Miller, D. C. (1994). Pilot study of the efficacy of a thrombin inhibitor for use during cardiopulmonary bypass. Ann Thorac Surg 58, 344–350. Drolet, D. W., Nelson, J., Tucker, C. E., Zack, P. M., Nixon, K., Bolin, R., Judkins, M. B., Farmer, J. A., Wolf, J. L., Gill, S. C., Bendele, R. A. (2000). Pharmacokinetics and safety of an anti-vascular endothelial growth factor aptamer (NX1838) following injection into the vitreous humor of rhesus monkeys. Pharmac Res 17, 1503–1510. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498. Gewirtz, D. A. (1999). A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin. Biochem Pharmacol 57, 727–741. Green, L. S., Jellinek, D., Jenison, R., Ostman, A., Heldin, C. H., Janjic, N. (1996). Inhibitory DNA ligands to platelet-derived growth factor B-chain. Biochemistry 35, 14413–14424. Griffin, L. C., Tidmarsh, G. F., Bock, L. C., Toole, J. J., Leung, L. L. (1993). In vivo anticoagulant properties of a novel nucleotide-based thrombin inhibitor and demonstration of regional anticoagulation in extracorporeal circuits. Blood 81, 3271–3276. Hannon, G. J. (2002). RNA interference. Nature 418, 244–251. Hicke, B. (2001). Tenascin-C nucleic acid ligands. United States Patent No. US 6232071. Ihle, J.N. (2000). The challenges of translating knockout phenotypes into gene function. Cell 102, 131–134. Ishida, S., Usui, T., Yamashiro, K., Kaji, Y., Amano, S., Ogura, Y., Hida, T., Oguchi, Y., Ambati, J., Miller, J. W., Gragoudas, E. S., Ng, Y. S., D’Amore, P. A., Shima, D. T., Adamis, A. P. (2003). VEGF164-mediated inflammation is required for pathological, but not physiological, ischemia-induced retinal neovascularization. J Exp Med 198, 483–489.

James, W. (2000). Aptamers. Encyclopedia of Analytical Chemistry, pp. 4848–4871. John Wiley & Sons, Chichester (UK). Kraus, M. E., Lis, J. T. (1994). The concentration of B52, an essential splicing factor and regulator of splice site choice in vitro, is critical for Drosophila development. Mol Cell Biol 14, 5360–5370. Lee, S. W., Gallardo, H. F., Gaspar, O., Smith, C., Gilboa, E. (1995). Inhibition of HIV-1 in CEM cells by a potent TAR decoy. Gene Ther 2, 377–384. Lichtlen, P., Auf der Maur, A., Barberis, A. (2002). Target validation through proteindomain knockout – applications of intracellularly stable single-chain antibodies. Targets 1, 37–44. Longman, R. (2004). Finding the opportunities in aptamers. In Vivo March, 37–45. Mayer, G., Blind, M., Nagel, W., Bohm, T., Knorr, T., Jackson, C. L., Kolanus, W., Famulok, M. (2001). Controlling small guanine-nucleotide-exchange factor function through cytoplasmic RNA intramers. Proc Natl Acad Sci USA 98, 4961–4965. Persengiev, S. P., Zhu, X., Green, M. R. (2004). Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs). RNA 10, 12–18. Pich, E. M., Epping-Jordan, M. P. (1998). Transgenic mice in drug dependence research. Ann Med 30, 390–396. Pietras, K., Ostman, A., Sjoquist, M., Buchdunger, E., Reed, R. K., Heldin, C. H., Rubin, K. (2001). Inhibition of platelet-derived growth factor receptors reduces interstitial hypertension and increases transcapillary transport in tumors. Cancer Res 61, 2929–2934. Pietras, K., Rubin, K., Sjoblom, T., Buchdunger, E., Sjoquist, M., Heldin, C. H., Ostman, A. (2002). Inhibition of PDGF receptor signaling in tumor stroma enhances antitumor effect of chemotherapy. Cancer Res 62, 5476–5484. Reyderman, L., Stavchansky, S. (1998). Pharmacokinetics and biodistribution of a nucleotide-based thrombin inhibitor in rats. Pharmac Res 15, 904–910. Ring, H. Z., Lis, J. T. (1994). The SR protein B52/SRp55 is essential for Drosophila development. Mol Cell Biol 14, 7499–7506.

References Ruckman, J., Green, L. S., Beeson, J., Waugh, S., Gillette, W. L., Henninger, D. D., Claesson-Welsh, L., Janijic, N. (1998). 2l-Fluoropyrimidine RNA-based aptamers to the 165amino acid form of vascular endothelial growth factor (VEGF165). Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain. J Biol Chem 273, 20556–20567. Rusconi, C.P., Roberts, J. D., Pitoc, G. A., Nimjee, S. M., White, R. R., Quick, G. Jr., Scardino, E, Fay, W. P., Sullenger, B. A. (2004). Antidote-mediated control of an anticoagulant aptamer in vivo. Nat Biotechnol 22, 1423–1428. Santulli-Marotto, S., Nair, S. K., Rusconi, C., Sullenger, B., Gilboa, E. (2003). Multivalent RNA aptamers that inhibit CTLA-4 and enhance tumor immunity. Cancer Res 63, 7483–7489. Shaw, J.P., Fishback, J. A., Cundy, K. C., Lee, W. A. (1995). A novel oligodeoxynucleotide inhibitor of thrombin. I. In vitro metabolic stability in plasma and serum. Pharmac Res 12, 1937–1942. Shi, H., Hoffman, B. E., Lis, J. T. (1999). RNA aptamers as effective protein antagonists in a multicellular organism. Proc Natl Acad Sci USA 96, 10033–10038. Sledz, C. A., Holko, M., de Veer, M. J., Silverman, R. H., Williams, B. R. (2002). Activation of the interferon system by short-interfering RNAs. Nat Cell Biol 5, 834–839.

Symensma, T. L., Giver, L., Zapp, M., Takle, G. B., Ellington, A. D. (1996). RNA aptamers selected to bind human immunodeficiency virus type 1 Rev in vitro are Rev responsive in vivo. J Virol 70, 179–187. Taylor, M. F., Wiederholt, K., Sverdrup, F. (1999). Antisense oligonucleotides: a systematic high-throughput approach to target validation and gene function determination. Drug Discov Today 4, 562–567. Tompkins, S. M., Lo, C. Y., Tumpey, T. M., Epstein, S. L. (2004). Protection against lethal influenza virus challenge by RNA interference in vivo. Proc Natl Acad Sci USA 101, 8682–8686. White, R.R., Shan, S., Rusconi, C. P., Shetty, G., Dewhirst, M. W., Kontos, C. C., Sullenger, B. A. (2003). Inhibition of rat corneal angiogenesis by a nuclease-resistant RNA aptamer specific for angiopoietin-2. Proc Natl Acad Sci USA 100, 5028–5033. Wlotzka, B., Leva, S., Eschgfaller, B., Burmeister, J., Kleinjung, F, Kaduk, C., Muhn, P., Hess-Stumpp, H., Klussmann, S. (2002) In vivo properties of an anti-GnRH Spiegelmer: An example of an oligonucleotidebased therapeutic substance class. Proc Natl Acad Sci USA 99, 8898–8902. Xia, H., Mao, Q., Eliason, S. L., Harper, S. Q., Martins, I. H., Orr, H. T., Paulson, H. L., Kotin, R. M., Davidson, B. L. (2004). RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia. Nat Med 10, 816–820.

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12 Intramers for Protein Function Analysis and Drug Discovery

12 Intramers for Protein Function Analysis and Drug Discovery* Michael Famulok and Gnter Mayer

12.1 Introduction

One of the biggest challenges in today’s life sciences is to gain a detailed understanding of the function and interplay of the numerous proteins in different organisms, tissues, cell types, and conglomerate protein complexes. One of the most efficient ways of studying protein function in the context of the living cell or organism is the application of a small molecule drug that exhibits high specificity, affinity, and inhibitory activity to the protein under investigation. However, such inhibitors are available only for a minority of the estimated total number of proteins of a higher vertebrate organism (Claverie, 2001; Harrison et al., 2002). Therefore, it is becoming increasingly important to develop functional screening assays compatible with massively parallel screening formats that can be applied independent of the nature and function of a given target. This would allow for a rapid identification of lead or optimized small-molecule compounds for largescale chemical genomics applications to assess the function of a growing number of functionally unexplored gene products in the postgenomics era. Inhibiting a protein or subdomain directly (i.e. without altering the genetic or mRNA status of an organism) allows immediate insight into questions such as drugability, or the functional role of an individual subdomain or post-translational modification. This requires direct recognition and inhibition of protein targets by inhibitory molecules that need to fulfill certain criteria: they should be routinely obtained and applied independent of the target, act at low concentrations, with high specificity, and in an intracellular context. A class of molecules that fulfill these requirements are aptamers – short, single-stranded oligonucleotides that fold into distinct three-dimensional structures capable of binding their targets with high affinity and specificity, basically mediated by complementary shape interactions (Gold et al., 1995; Famulok et al., 2000). Historically, aptamers have * A large body of this chapter has already appeared in print in: Famulok, M., Mayer, G. Intramers and aptamers: applications in pro-

tein-function analyses and potential for drug screening. Chem Bio Chem 2005, 6, 19–26.


12.2 Intramers: Intracellular Aptamers

been and still are isolated from vast combinatorial sequence libraries by the SELEX (systematic evolution of ligands by exponential enrichment) process (Ellington and Szostak, 1990; Tuerk and Gold, 1990). Many examples summarized in this book show that SELEX can be applied to a variety of different targets ranging from small organic molecules (Famulok, 1999) to large proteins (Gold et al., 1995) or whole viruses (Pan et al., 1995; Wang et al., 2000). In most cases, anti-protein aptamers not only bind their cognate protein but also inhibit its function efficiently. Owing to the increasing demand for protein inhibitors in the postgenome era, selection routines compatible with automation have been established that allow us to perform highly parallel aptamer selections to several targets at once within a few days (Cox et al., 1998, 2002; Cox and Ellington, 2001). Aptamers thus represent an interesting class of protein inhibitors that can be easily obtained and used for assessing the function of a defined protein target (Famulok and Mayer, 2005). Many aptamers target extracellular proteins or protein epitopes because in that way they have direct access to their targets without having to pass through plasma or nuclear membranes. However, for widespread applications as inhibitory tools for functional genomics research, it will be necessary to provide methods that allow for targeting proteins that reside inside cells with inhibitory aptamers. Being nucleic acids, intracellular aptamers (or intramers) are intrinsically adapted to the reductive environment inside a cell, unlike, for example, intracellular antibodies (intrabodies) that require further engineering to tolerate the reductive milieu of the cytoplasm. The cellular delivery of aptamers can be accomplished either by direct transfection, by use of viral vectors, or through defined expression systems encoding for the aptamer sequence under the control of a highly active promoter.


The earliest examples demonstrating that aptamers can be functional inside cells included prokaryotic or nuclear targets, among them the special elongation factor SelB from Escherichia coli (Klug et al., 1997), RNA polymerase II from yeast (Thomas et al., 1997), the splicing factor B52 from Drosophila melanogaster (Shi et al., 1999), and the Rev protein from HIV-1 (Symensma et al., 1999). Mostly, aptamers targeting natural nucleic acid binding proteins were selected in vitro and intramer expression systems were engineered subsequently. Expressed intramers often acted as decoys for natural RNA-binding proteins or represented variants of natural transcripts. Their intracellular expression aimed to dissect functional aspects of nucleic acid binding proteins or of structural elements in natural transcripts (Famulok et al., 2001; Famulok and Verma, 2002; Burke and Nickens, 2002). A recent study reported the expression of an HIV-1 reverse transcriptase (RT)binding RNA aptamer inside human 293T cells (Chaloin et al., 2002). The cells were transiently transfected with a chimeric RNA expression system consisting

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of the human tRNAMet and the anti-RT aptamer pseudo-knot motif. Intracellular expression of the anti-RT intramer resulted in reduction of HIV particle release by more than 75% in cells cotransfected with proviral HIV-1 DNA. HIV-1 particles produced by 293T cells in the presence of the anti-RT intramer construct showed 75% reduction of infectivity in human T-lymphoid cells compared with control experiments. Virus replication was completely inhibited in stably transfected T-lymphoid cells over a period of 35 days after low-dose HIV infection. The feasibility of the intramer concept is underlined by targeting proteins in organisms in which they are not normally present. HIV-1 RT function can also be inhibited in E. coli mutants that were genetically engineered to depend on reverse transcriptase for growth at 37 hC (Nickens et al., 2003). In that way, expression of the anti-RT intramer inhibited growth complementation by the expressed exogenous HIV-1 RT. Another intramer target was the nucleocapsid (NC) protein of HIV-1 with the aim to inhibit HIV-1 replication (Kim and Jeong, 2004). An anti-NC aptamer was selected in vitro and shown to interfere with NC binding to the stable trans-activation response (TAR) hairpin and psi RNA stem–loops of HIV-1 RNA. In vivo, the aptamer abolished packaging of viral genomic RNA. Inhibition of viral replication by intramers was also achieved when proteins from viruses other than HIV-1 were targeted. For example, expression of intramers targeting the non-structural protein NS3 of hepatitis C virus (HCV), a protein with both helicase and protease activity, led to inhibition of protease activity (Nishikawa et al., 2003). An RNA aptamer that targets the transcription factor NFkB was shown to recognize NFkB inside cells and to inhibit its binding to its cognate DNA sequence, presumably by mimicking the double-stranded DNA motif (Cassiday and Maher, 2001; Huang et al., 2003). A yeast three-hybrid system was applied to reselect the RNA aptamer for improved NFkB interaction, to further optimize the formation of the NFkB–RNA complex in the eukaryotic nucleus (Cassiday and Maher, 2003). By using either a degenerated RNA library or sequences from earlier selection cycles, aptamer variants with substantially improved binding affinity in yeast cells were obtained. Furthermore, the improved aptamer variant inhibited the transcriptional activity of NFkB in vivo (Cassiday and Maher, 2003). These results underline the power of the combination of in vitro and in vivo genetic selections for the optimization of aptamer properties and their adaption to distinct conditions. Our research led us to the development of highly specific aptamers targeting cytoplasmic regulatory proteins and protein domains implicated in the leukocyte function associated antigen 1 (LFA-1)-mediated inside-out signaling cascade. The activation of LFA-1 by T-cell receptor stimulation or stimulation with phorbol esters results in T-cell adhesion to ICAM-1 presented on the surface of endothelial cells. Cytohesin-1, a cytoplasmic signaling molecule, participates in the mechanism of LFA-1 activation, presumably by direct interaction with the cytoplasmic tail of the b2-chain (CD18) of the LFA-1 integrin (Kolanus et al., 1996). Cytohesin-1 belongs to a family of highly homologous guanine nucleotide exchange factors


(GEFs) that act on ADP-ribosylation factors (ARFs). The small ARF-GEFs are known to be involved in integrin signaling, actin cytoskeleton remodeling, and vesicle transport. Today, four highly homologous members of the cytohesin family are known: cytohesin-1, ARNO/cytohesin-2, cytohesin-3, and cytohesin-4. They comprise an N-terminal coiled-coil domain, followed by a Sec7 and pleckstrin homology domain, and a polybasic C-domain. To dissect their individual functions, we isolated RNA aptamers that specifically interact with cytohesin proteins and/or their individual domains and thus allow for dissecting their functions in living cells. In an initial study we isolated and characterized the cytohesin-1 binding aptamers M69. M69 specifically recognized the Sec7 domain of cytohesin-1, which comprises the GEF activity and is thought to directly interact with the b2-cytoplasmic tail of LFA-1. Sec7 domains are widespread within the small and large families of ARF-GEFs. M69 distinguished between the Sec7 domains of the large and small GEF family members, by binding only to those of the small GEFs ARNO and cytohesin-1. However, it did not discriminate between the Sec7 domains of these two cytohesins (Mayer et al., 2001). An expression system based on transgenic vaccinia viruses (Blind et al., 1999) was used for cytoplasmic expression of M69 in Jurkat cells (Mayer et al., 2001). Intrameric expression of M69 caused inhibition of LFA-1-mediated adhesion. Furthermore, the intramer M69 inhibited reorganization of the cytoskeleton and cell spreading. Dominant negative expression of a GEF-deficient cytohesin1 (E157K) point mutant gave similar results confirming an important role of the GEF activity of cytohesin-1 in T-cell spreading. As mentioned above, M69 did not discriminate between the highly homologous cytohesin family members. To address the question whether the highly homologous cytohesins 1 and 2 exhibit different functions in T cells in which they are both expressed, we performed an in vitro selection using ARNO/cytohesin-2 as a target and cytohesin-1 in a counterselection, to isolate discriminatory aptamers. We obtained an RNA aptamer, dubbed K61, that bound ARNO/cytohesin-2 with a Kd of 115 nmol/L whereas cytohesin-1 was bound with at least 35-fold reduced affinity. This aptamer did not inhibit the GEF-activity of ARNO in vitro, presumably because it recognizes the coiled-coil-Sec7 interface of ARNO/cytohesin-2, exhibiting only weak affinity to the Sec7 domain alone (Theis et al., 2004). GTPases of the Rho and ARF families are thought to regulate membrane traffic and cytoskeletal remodelling (Santy and Casanova, 2002). Furthermore, the Rho family of small GTPases is involved in the stimulation of serum response factor transcriptional activation, induced by serum growth factors (Hill et al., 1995). We thus hypothesized that cytohesins may also play a role in transcriptional activation via the serum-response element (SRE) and investigated whether K61 affects the transcription of a luciferase reporter gene under the control of the SRE promoter in serum-stimulated HeLa cells. We found that K61 downregulates SREcontrolled luciferase expression to basal levels in a concentration-dependent manner. The inhibitory activity of K61 was fully reversed by overexpressed wild-type ARNO, confirming that the effect was aptamer-specific (Theis et al., 2004). In accordance with this novel activity of cytohesin-2, we found that both K61 and the

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non-discriminatory aptamer M69 led to a specific downregulation of MAPK activation, monitored by the phosphorylation status of Elk. An siRNA targeting cytohesin-2 also resulted in the downregulation of MAPK activation but interestingly, an siRNA that downregulated cytohesin-1 expression did not. These results suggested that transcriptional regulation of the SRE in HeLa cells could be assigned to ARNO rather than cytohesin-1. This study further demonstrated that intramers can be used to reveal insight into novel biological activities of target proteins and to assign specific biological functions to individual members or defined domains of a protein family. Intramers represent an alternative and complementary approach to siRNAs to elucidate the function of a protein within its natural context.

12.3 Aptamers as Probes for Inhibitor Screening

The results described above show that aptamer selections provide highly versatile sources for rapidly obtaining powerful inhibitors of intracellular proteins. Once an inhibitory aptamer is available, it is fairly straightforward to transform it into an intramer. In fact, the anti-ARNO aptamer K61 was directly transfected into cultured cells by lipofection without the need to stabilize it against nuclease degradation or to direct it into a particular cellular compartment (Theis et al., 2004). The question is how general this approach will be with respect to cell types, targets, or the aptamer sequence itself. The analysis of protein function in multicellular organisms or tissues will necessitate the generation of aptamerexpressing transgenic animals or the development of gene therapeutic approaches, at least in the case of vertebrate studies. For such purposes, drug-quality small-molecule inhibitors still would seem advantageous compared with any nucleic acid- or biopolymer-based inhibitor. An intriguing idea is to directly convert an aptamer/protein complex with verified intracellular functionality into lead compounds by developing assays that screen small-molecule libraries that displace the aptamer from its target and adopt its inhibitory activity. Indeed, aptamers would seem perfectly suited for functioning directly as competitive probes in high-throughput screening (HTS) assays. This would allow direct translation of information stored within an aptamer into a small molecule, which likely will be an inhibitor itself. As a first step towards this direction, we have developed what we called “reporter ribozymes” – chimeric RNA molecules that consist of an aptamer domain attached to a ribozyme (Hartig and Famulok, 2002; Hartig et al., 2002). Reporter ribozymes are potentially compatible with the parallel screening of large compound libraries since they report the displacement of protein-bound aptamer with a small molecule by a fluorescence signal. The detection principle relies on an intrinsic property of aptamers and many natural protein-binding RNAs: adaptive binding. Ever since the first NMR structures of aptamer/small molecule complexes were elucidated it became clear that the complexation of a ligand by an aptamer almost exclusively occurs by adaptive recognition. That is, for example,

12.3 Aptamers as Probes for Inhibitor Screening

aptamers often comprise unpaired loop or bulge regions, which are disordered in the free nucleic acid and acquire a defined conformation by adaptive folding around the ligand (Hermann and Patel, 2000). The target of the reporter ribozymes was the Rev protein of HIV-1. Binding of Rev to its cognate natural RNA element, the Rev-responsive element (RRE) in the reporter ribozyme rendered the ribozyme module inactive. Only when the aptamer was competed by a Rev-binding small molecule, does the ribozyme module undergo conformational changes, enabling it to cleave a substrate oligonucleotide, which has a fluorophore at one end and a quencher at the other. Fluorescence is detected only when the ribozyme is active. Conversely, an alternative ribozyme construct containing a Rev-binding aptamer (Giver et al., 1993) module showed exactly the opposite behavior, i.e. it was switched on in the presence of Rev, and switched off after Rev was competed with a small molecule. The Rev-responsive reporter ribozyme was used to screen a 96-member sample library of antibiotics for molecules that could disrupt the interaction between Rev and its cognate RNA. The screen identified three compounds as hits, one of which, the gyrase-inhibitor coumermycin A1, exhibited fairly specific binding with a Kd of 7.5 mmol/L. Moreover, cell culture experiments revealed that the coumermycin A1 inhibits the HIV-1 virus replication in a concentration-dependent fashion, indicating that the small molecule possesses the same characteristics as the aptamer from were it was derived. This study established that it is possible to identify novel small molecule inhibitors for a given protein by using interference with RNA/protein interactions as a basis for screening. In a similar study (Hartig and Famulok, 2002), we have fused an HIV-1 reverse transcriptase (RT) binding aptamer (Tuerk et al., 1992) to the hammerhead ribozyme. The presence of RT induces the formation of a different structure of the aptameric portion (i.e. a pseudo-knot structure) (Kensch et al., 2000). As in the study described above (Hartig et al., 2002), the binding of RT to the aptamer prevents the ribozyme from cleaving the small oligonucleotide substrate RNA, labeled with the fluorescent dye and the quencher. In the absence of RT the reporter ribozyme remains active, and substrate cleavage can be followed by an increase in fluorescence. This is highly specific for HIV-1 RT. The homolog RT of HIV-2 is not detected. Thus, the reporter ribozyme serves as a specific biosensor signaling the presence of HIV-1 RT. In this sense, reporter ribozymes supplement currently used antibody-based techniques like ELISA while being considerably more straightforward due to real-time readout in solution and other advantages discussed below. The assay is reversible: When the protein is displaced from the reporter ribozyme by interacting with another molecule, such as the primer/template complex that is mimicked by the aptamer, the reporter ribozyme can again cleave the substrate, resulting in a fluorescence signal. In other words, these systems can act as domain-specific sensors for screening purposes. As mentioned above, the reporter ribozyme binds HIV-1 RT at the same site where the protein recognizes the primer/template complex. Remarkably, RT-binding molecules that are specific for sites other than the primer/template binding site are ignored by the reporter ribozyme. This enables to configure an assay

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into a high-throughput screening mode that might allow for a highly focused search for inhibitors that target a distinct epitope or domain of a protein. Most of the HIV-1 RT inhibitors known today are nucleotide RT inhibitors (NRTIs) such as azidothymidine, and non-nucleotide RT inhibitors (NNRTIs), like nevirapine, which target other domains of the polymerase. As a perspective, our approach would now allow to search for completely novel classes of HIV-1 RT inhibitors which target the primer/template-binding site. This potential was tested by adding the free aptamer as a specific competitor for the reporter ribozyme. The aptamer was able to displace the protein from the reporter ribozyme, switching it on again. Thus, reporter ribozymes have three distinct advantages over many other assays or sensors: detection occurs in real-time, none of the actual reaction partners requires to be labeled and the format is highly modular and can be configured for any kind of protein for which aptamers can be selected. Further modularity of reporter ribozymes was achieved in another format, based on hairpin ribozyme variants that can be induced or repressed by external effector oligonucleotides. The key step here was to introduce a binding domain specific for a certain RNA sequence into the hairpin ribozyme. When the domain is bound by the cognate RNA, the reporter ribozyme undergoes conformational changes enabling it to cleave the fluorophore and quencher-labeled substrate oligonucleotide. Small sequence changes in the RNA-binding domain allowed targeted switching of ribozyme activity: the same effector oligonucleotide then serves either as an inducer or repressor. We applied this format to a hairpin variant fused to the complementary version of the trp leader mRNA (Najafi-Shoushtari et al., 2004), the RNA sequence tightly bound by l-tryptophan-activated trp-RNA-binding attenuation protein (TRAP) from Bacillus subtilis. TRAP only binds to trp leader mRNA in the presence of l-tryptophan (Gollnick et al., 1995). Ribozyme activity can be altered by annealing trp leader mRNA, then specifically reverted by its TRAP/tryptophan-mediated sequestration. Thus, these reporter ribozymes sense the activity status of a protein as a function of its metabolite molecule and could potentially be applied for the screening of TRAP-binding small molecules. Using the same format, we designed nine ribozyme variants that were activated by different microRNAs (Hartig et al., 2004). Each of them detected its cognate miRNA reliably and sensitively in a mix of other miRNA sequences. These reporter ribozymes join other ribozymes that report nucleic acids (Robertson and Ellington, 1999; Vaish et al., 2003; Vauleon and Muller, 2003; Kossen et al., 2004). They are entirely RNA based and thus could be expressed endogenously, requiring only the addition of the short substrate oligonucleotide to report the presence of a certain miRNA in an in vivo context. Taken together, these results illustrate the potential of aptamers and aptamerbased sensor systems for the identification of small molecule inhibitors.

References

12.4 Summary

Aptamers are easy-to-handle chemicals that can be isolated for various proteins by in vitro selection, which selectively bind a large variety of different targets, from proteins or individual domains of homologous proteins to small molecules, viruses, cells, and parasites. They can be used as both functional inhibitors to characterize proteins either inside or outside of a cell and tools to develop inhibitors for protein interference. Aptamers offer a valuable complement to loss-offunction phenotypic knockdown approaches and for assigning novel activities to members of highly homologous protein families. Moreover and besides their conventional use as diagnostic reagents, affinity matrices, or therapeutics, aptamers offer an exciting novel interface between target validation and drug screening, as a biologically active aptamer can be used directly to identify functionally equivalent small molecules in competitive high-throughput screening assays. We consider aptamers as a highly promising alternative to other tools for the loss-of-function phenotypic analysis of proteins to validate the biological activity of new proteins inside cells, and for the development of novel chemical entities for the rapid characterization of proteins in the context of whole cells or organisms.

Acknowledgments

Our work is supported by the Gottfried-Wilhelm-Leibniz program, and other grants from the Deutsche Forschungsgemeinschaft (SFB 645, GRK 804), by the Volkswagenstiftung, the BMBF, and the Fonds der Chemischen Industrie.

References Blind, M., Kolanus, W., Famulok, M. (1999). Cytoplasmic RNA-modulators of an insideout signal transduction cascade. Proc Natl Acad Sci USA 96, 3606–3610. Burke, D. H., Nickens, D. G. (2002). Expressing RNA aptamers inside cells to reveal proteome and ribonome function. Brief Funct Genomic Proteomic 1, 169–188. Cassiday, L. A., Maher, L. J., III (2001). In vivo recognition of an RNA aptamer by its transcription factor target. Biochemistry 40, 2433–2438. Cassiday, L. A., Maher, L. J., III (2003). Yeast genetic selections to optimize RNA decoys for transcription factor NF-kappa B. Proc Natl Acad Sci USA 100, 3930–3935.

Chaloin, L., Lehmann, M. J., Sczakiel, G., Restle, T. (2002). Endogenous expression of a high-affinity pseudoknot RNA aptamer suppresses replication of HIV-1. Nucleic Acids Res 30, 4001–4008. Claverie, J. M. (2001). Gene number. What if there are only 30,000 human genes? Science 291, 1255–1257. Cox, J. C., Ellington, A. D. (2001). Automated selection of anti-protein aptamers. Bioorg Med Chem 9, 2525–2531. Cox, J. C., Rudolph, P., Ellington, A. D. (1998). Automated RNA selection. Biotechnol Prog 14, 845–850. Cox, J. C., Rajendran, M., Riedel, T., Davidson, E. A., Sooter, L. J., Bayer, T. S., Schmitz-

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12 Intramers for Protein Function Analysis and Drug Discovery Brown, M., Ellington, A. D. (2002). Automated acquisition of aptamer sequences. Comb Chem High Throughput Screen 5, 289–299. Ellington, A. D., Szostak, J. W. (1990). In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822. Famulok, M. (1999). Oligonucleotide aptamers that recognize small molecules. Curr Opin Struct Biol 9, 324–329. Famulok, M., Mayer, G. (2005). Intramers and aptamers: applications in protein-function analyses and potential for drug screening. Chembiochem 6, 19–26. Famulok, M., Verma, S. (2002). In vivo-applied functional RNAs as tools in proteomics and genomics research. Trends Biotechnol 20, 462–466. Famulok, M., Mayer, G., Blind, M. (2000). Nucleic acid aptamers – from selection in vitro to applications in vivo. Acc Chem Res 33, 591–599. Famulok, M., Blind, M., Mayer, G. (2001). Intramers as promising new tools in functional proteomics. Chem Biol 8, 931–939. Giver, L., Bartel, D., Zapp, M., Pawul, A., Green, M., Ellington, A. D. (1993). Selective optimization of the Rev-binding element of HIV-1. Nucleic Acids Res 21, 5509–5516. Gold, L., Polisky, B., Uhlenbeck, O., Yarus, M. (1995). Diversity of oligonucleotide functions. Annu Rev Biochem 64, 763–797. Gollnick, P., Baumann, C., Yang, M., Otridge, J., Antson, A. (1995). Interaction of the 11subunit trp RNA-binding attenuation protein (TRAP) with its RNA target. Nucleic Acids Symp Ser 43–45. Harrison, P. M., Kumar, A., Lang, N., Snyder, M., Gerstein, M. (2002). A question of size: the eukaryotic proteome and the problems in defining it. Nucleic Acids Res 30, 1083–1090. Hartig, J. S., Famulok, M. (2002). Reporter ribozymes for real-time analysis of domainspecific interactions in biomolecules: HIV-1 reverse transcriptase and the primer-template complex. Angew Chem Int Ed Engl 41, 4263–4266. Hartig, J. S., Najafi-Shoushtari, S. H., Grune, I., Yan, A., Ellington, A. D., Famulok, M. (2002). Protein-dependent ribozymes report molecular interactions in real time. Nat Biotechnol 20, 717–722.

Hartig, J. S., Grune, I., Najafi-Shoushtari, S. H., Famulok, M. (2004). Sequence-specific detection of MicroRNAs by signal-amplifying ribozymes. J Am Chem Soc 126, 722– 723. Hermann, T., Patel, D. J. (2000). Adaptive recognition by nucleic acid aptamers. Science 287, 820–825. Hill, C. S., Wynne, J., Treisman, R. (1995). The Rho family GTPases RhoA, Rac1, and CDC42Hs regulate transcriptional activation by SRF. Cell 81, 1159–1170. Huang, D. B., Vu, D., Cassiday, L. A., Zimmerman, J. M., Maher, L. J., III, Ghosh, G. (2003). Crystal structure of NF-kappaB (p50)2 complexed to a high-affinity RNA aptamer. Proc Natl Acad Sci USA 100, 9268–9273. Kensch, O., Connolly, B. A., Steinhoff, H. J., McGregor, A., Goody, R. S., Restle, T. (2000). HIV-1 reverse transcriptase-pseudoknot RNA aptamer interaction has a binding affinity in the low picomolar range coupled with high specificity. J Biol Chem 275, 18271–18278. Kim, M. Y., Jeong, S. (2004). Inhibition of the functions of the nucleocapsid protein of human immunodeficiency virus-1 by an RNA aptamer. Biochem Biophys Res Commun 320, 1181–1186. Klug, S. J., Huttenhofer, A., Kromayer, M., Famulok, M. (1997). In vitro and in vivo characterization of novel mRNA motifs that bind special elongation factor SelB. Proc Natl Acad Sci USA 94, 6676–6681. Kolanus, W., Nagel, W., Schiller, B., Zeitlmann, L., Godar, S., Stockinger, H., Seed, B. (1996). aLb2 integrin/LFA-1 binding to ICAM-1 induced by cytohesin-1, a cytoplasmic regulatory molecule. Cell 86, 233–242. Kossen, K., Vaish, N. K., Jadhav, V. R., Pasko, C., Wang, H., Jenison, R., McSwiggen, J. A., Polisky, B., Seiwert, S. D. (2004). Highthroughput ribozyme-based assays for detection of viral nucleic acids. Chem Biol 11, 807–815. Mayer, G., Blind, M., Nagel, W., Bhm, T., Knorr, T., Jackson, C. L., Kolanus, W., Famulok, M. (2001). Controlling small guanine-nucleotide-exchange factor function through cytoplasmic RNA intramers. Proc Natl Acad Sci USA 98, 4961–4965.

References Najafi-Shoushtari, S. H., Mayer, G., Famulok, M. (2004). Sensing complex regulatory networks by conformationally controlled hairpin ribozymes. Nucleic Acids Res 32, 3212– 3219. Nickens, D. G., Patterson, J. T., Burke, D. H. (2003). Inhibition of HIV-1 reverse transcriptase by RNA aptamers in Escherichia coli. RNA 9, 1029–1033. Nishikawa, F., Kakiuchi, N., Funaji, K., Fukuda, K., Sekiya, S., Nishikawa, S. (2003). Inhibition of HCV NS3 protease by RNA aptamers in cells. Nucleic Acids Res 31, 1935– 1943. Pan, W., Craven, R. C., Qiu, Q., Wilson, C. B., Wills, J. W., Golovine, S., Wang, J. F. (1995). Isolation of virus-neutralizing RNAs from a large pool of random sequences. Proc Natl Acad Sci USA 92, 11509–11513. Robertson, M. P., Ellington, A. D. (1999). In vitro selection of an allosteric ribozyme that transduces analytes to amplicons. Nat Biotechnol 17, 62–66. Santy, L. C., Casanova, J. E. (2002). GTPase signaling: bridging the GAP between ARF and Rho. Curr Biol 12, R360-R362. Shi, H., Hoffman, B. E., Lis, J. T. (1999). RNA aptamers as effective protein antagonists in a multicellular organism. Proc Natl Acad Sci USA 96, 10033–10038. Symensma, T. L., Baskerville, S., Yan, A., Ellington, A. D. (1999). Polyvalent Rev decoys act as artificial Rev-responsive elements. J Virol 73, 4341–4349.

Theis, M. G., Knorre, A., Kellersch, B., Moelleken, J., Wieland, F., Kolanus, W., Famulok, M. (2004). Discriminatory aptamer reveals serum response element transcription regulated by cytohesin-2. Proc Natl Acad Sci USA 101, 11221–11226. Thomas, M., Chdin, S., Carles, C., Riva, M., Famulok, M., Sentenac, A. (1997). Selective targeting and inhibition of yeast RNA polymerase II by RNA aptamers. J Biol Chem 272, 27980–27986. Tuerk, C., Gold, L. (1990). Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505–510. Tuerk, C., MacDougal, S., Gold, L. (1992). RNA pseudoknots that inhibit human immunodeficiency virus type 1 reverse transcriptase. Proc Natl Acad Sci USA 89, 6988– 6992. Vaish, N. K., Jadhav, V. R., Kossen, K., Pasko, C., Andrews, L. E., McSwiggen, J. A., Polisky, B., Seiwert, S. D. (2003). Zeptomole detection of a viral nucleic acid using a target-activated ribozyme. RNA 9, 1058– 1072. Vauleon, S., Muller, S. (2003). External regulation of hairpin ribozyme activity by an oligonucleotide effector. Chembiochem 4, 220–224. Wang, J., Jiang, H., Liu, F. (2000). In vitro selection of novel RNA ligands that bind human cytomegalovirus and block viral infection. RNA 6, 571–583.

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13 Aptazymes: Allosteric Ribozymes and Deoxyribozymes as Biosensors

13 Aptazymes: Allosteric Ribozymes and Deoxyribozymes as Biosensors Scott M. Knudsen and Andrew D. Ellington

13.1 Introduction

Nucleic acids can perform a number of functions, including information storage, ligand recognition, and catalysis. It is well-known that information can be stored in primary sequence and “read” via base-pairing interactions. However, nucleic acids can also fold into more complex structures that recognize and bind to a diverse range of targets; natural RNA sequences which bind to particular proteins have long been known, and more recently a series of so-called “riboswitch” RNAs have been found that bind to cellular metabolites. Additionally, the process of in vitro selection has led to the isolation of an increasingly wider range of ligandbinding nucleic acids (both RNA and DNA) known as aptamers. The ability to form defined three-dimensional structures also allows RNA to catalyze chemical reactions. Such catalytic RNAs are known as ribozymes, and a number of distinct ribozyme motifs have been found in Nature. Again, in vitro selection has been used to generate RNA and DNA enzymes with novel structures and catalytic functions. The ligand-binding and catalytic features of nucleic acids can be combined to generate allosteric ribozymes or “aptazymes.” Just as allosteric protein catalysts undergo ligand-dependent changes in structure that lead to changes in catalytic activity, when ligands bind to an aptazyme, conformational changes in the ligand-binding domain are transduced to a change in the catalytic core and a concomitant modulation of catalytic activity. However, unlike proteins, the relative simplicity of nucleic acid secondary structure facilitates the design and engineering of allostery. For example, a number of aptazymes have been created by the simple expedient of appending known binding elements to the catalytic cores of ribozymes via connecting stems. In this chapter, we will highlight a number of specific aptazymes with exceptional characteristics and will outline the methods by which aptazymes are generated. We will also describe the use of aptazymes in diagnostic, sensor, and genetic engineering applications, and discuss potential future uses of aptazymes. The Aptamer Handbook. Edited by S. Klussmann Copyright c 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-31059-2

13.1 Introduction

13.1.1 Oligonucleotide-dependent Aptazymes

Given that changes in base pairing can lead to changes in catalysis, it should be unsurprising that a number of oligonucleotide-dependent aptazymes have been generated. Indeed, the first reported allosteric ribozyme was generated by Paul Lizardi and co-workers (Porta and Lizardi, 1995). The hammerhead ribozyme was extended with a sequence which caused an intentional misfolding of its known, functional secondary structure. The misfolded ribozyme was unable to efficiently catalyze the hydrolysis of its substrate. However, upon introduction of an oligonucleotide that could sequester the inhibitory extension, the catalytic structure was restored. Oligonucleotide-dependent allosteric aptazymes have also been isolated (sometimes serendipitously) via in vitro selection. For example, when David Bartel and Jack Szostak selected a series of novel ribozyme ligases, individual ribozymes were found to have 2- to 20-fold dependence on an oligonucleotide that was present during selection (Ekland et al., 1995). Similarly, a ligase ribozyme selected in the Ellington lab exhibited a remarkable dependence (10 000-fold) on an oligonucleotide sequence present during its selection (Robertson and Ellington, 1999). These examples illustrate that the activities of nucleic acid catalysts can be readily modulated by oligonucleotide effectors, in part because the simple base-pairing rules that impart secondary structural features can be rationally engineered. The mechanism for oligonucleotide modulation of catalysis is so simple that it should come as no surprise that natural as well as unnatural ribozymes exhibit dependence upon oligonucleotides. Both the Tetrahymena self-splicing group I intron and the genomic hepatitis delta virus (HDV) ribozyme have been shown to be inhibited by flanking sequences upstream of the catalytic structure (Cao and Woodson, 1998; Chadalavada et al., 2000). In both cases, the inhibitory sequences can also be sequestered by sequence elements further upstream, allowing formation of the active structure. These natural strategies can be mimicked by designed constructs. For example, the addition of an oligonucleotide complementary to known inhibitory sequences has been shown to activate a misfolded HDV ribozyme by over three orders of magnitude. Similarly, structural complementation studies have shown that inactive deletion mutants of the Tetrahymena ribozyme can be activated by adding the deleted portion in trans, thus allowing splicing to occur (van der Horst et al., 1991; Doudna and Cech, 1995). 13.1.2 Activation by Non-nucleic Acid Effectors

Nucleic acid binding species (also known as aptamers) can be selected from random sequence pools (reviewed in Brody and Gold, 2000; Hesselberth et al., 2000; Rimmele, 2003). Aptamers assume folded, tertiary structures that present chemical moieties that are complementary to a target. Ligand-bound aptamers frequently undergo conformational changes that maximize these interactions relative to unbound aptamers. In the same way that secondary structural changes

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have been engineered to generate oligonucleotide-dependent ribozymes, the ligand-dependent conformational changes seen in aptamers can be adapted to generate allosteric nucleic acid catalysts. Aptazymes have so far been designed and selected to respond to a variety of effector molecules: from small organics, to peptides, to proteins. In addition, a number of different ribozymes have been engineered to function as aptazymes. Multiple cleavases (the hammerhead, HDV, hairpin, and X-motif ribozymes, and the 10–23 and 8–17 deoxyribozymes), ligases, and versions of the self-splicing group I ribozyme have all been engineered or selected to be aptazymes.

13.2 Creating Aptazymes via Rational Design and In Vitro Selection Methodologies 13.2.1 Rational Design of Aptazymes

Aptazymes can be created by rational design, selection, or a combination of the two. The first aptazymes that could respond to non-oligonucleotide effectors were created by appending a known ATP-binding sequence in place of stem II of a trans-acting hammerhead ribozyme using a series of connecting stems (Tang and Breaker, 1997). The resulting constructs were assayed for activity in the presence and absence of ATP and several were identified with allosteric activity (examples are shown in Fig. 13.1). Aptazymes that were responsive to the small molecules theophylline (Tang and Breaker, 1997) and flavin mononucleotide (FMN) (Araki et al., 1998) were created by similar design-and-screen methods, but showed only modest activation (a greater rate in the presence than in

Fig. 13.1 Aptazyme design. Appending an ATP-binding aptamer (bold) to a hammerhead ribozyme via different connecting stems (boxed) yielded aptazymes which were either activated or inhibited by ATP. The cleavage site on the substrate (italics) is denoted by an arrow.

13.2 Creating Aptazymes via Rational Design and

the absence of the effector molecule) or inhibition. While selection methods will prove to be more powerful for imbuing ribozymes with allosteric properties, rational design methods continue to be an important means of generating initial aptazyme constructs, and a number of rational designs will be discussed in later sections. Nonetheless, the throughput of rational design methods is limited (although it is likely to be greatly expanded by computational design). Additionally, common sense suggests that it is likely easier to design constructs in which ligand-binding perturbs an active structure than to engineer ligand-dependent repair of an inactive structure. This is unfortunate, as a catalyst which can be turned on in the presence of its effector is generally more desirable than one that can be turned off. In vitro selection techniques have been able to solve both of these limitations. Briefly, in vitro selection is a cyclic process whereby a large number of individual molecules are partitioned based on fitness for a particular task. Selected molecules can be isolated and amplified, and the cycle repeated until only highly functional variants remain. This process allows a large number of potential catalysts to be simultaneously interrogated until only a handful of sequences (ideally the best sequences) remain in the pool to be assayed in detail. Selection schemes can also be designed such that activation (rather than inhibition) is required for survival. 13.2.2 In Vitro Aptazyme Selection

Different types of random sequence pools can be used to initiate aptazyme selections. One type of pool is a modular design in which a known aptamer sequence is connected to a known ribozyme motif by a random sequence stem (Fig. 13.2a).

Fig. 13.2 Pools for the selection of ligase aptazymes. The letter N signifies a randomized position which contains any nucleotide. (a) The FMNn8 pool consisted of the FNM aptamer (bold) connected to the L1 ligase by either three or four nucleotides in each strand

(boxed); this pool has a complexity of 102 400 members. (b) The L1-N50 pool contained a minimized L1 ligase with fifty random nucleotides in place of Stem B; this pool has a complexity of Z1.27 q 1030 members.

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This configuration is similar to the designed aptazymes described above, except that a “communication module” between aptamer and ribozymes will be selected instead of designed (Soukup and Breaker, 1999). Because communication module pools have relatively low complexity, each possible stem variant is expected to be represented in the initial pool. For example, the pool shown in Fig. 13.2a has 102 400 possible sequence combinations. A selection for FMN-dependent aptazymes (Robertson and Ellington, 2000) was initiated with 100 pmol of this pool; this amount corresponds to Z6 q 1013 molecules and thus every possible combination was likely to have been present. A second type of pool contains a purely random sequence in place of one or more structurally or functionally important parts of a ribozyme. Usually such an appended random pool has a much higher complexity than a communication module pool, and therefore can potentially be used to identify novel allosteric sites and mechanisms; for example, allosteric sites for an effector can be selected even when there is no known aptamer for that effector. An example of this type of pool is shown in Fig. 13.2b. Stem C of a minimized L1 ligase ribozyme was replaced with a region containing 50 random nucleotides. Of course, the relatively high complexity of an appended random pool means that only a small fraction of the possible sequence space will actually be sampled. The pool shown in Fig. 13.2b could contain approximately 1.27 q 1030 possible sequences; if each of these combinations was actually present the pool would weigh nearly 70 000 000 kg. Instead, selections from this particular pool have been initiated with on the order of 1015 molecules (Robertson and Ellington, 2001; Robertson et al., 2004), or approximately 55 mg of RNA. Researchers sometimes think that the incomplete coverage of sequence space afforded by longer pools may somehow be disadvantageous during selection experiments, compared with shorter, less complex pools that can be fully sampled. However, this prejudice has no basis in fact. The longer pool already contains all of the sequences that would be present in a shorter pool, and some fraction of longer motifs as well. That is, a longer pool may be incomplete, but it is nonetheless more complex than a shorter pool. The selection scheme used to acquire aptazymes is basically the same regardless of which type of pool is used. For aptazymes that are to be activated by their effectors, each round consists of both a negative and a positive selection step in which sequences that are active in first the absence and then the presence of an effector are eliminated from or retained in the pool, respectively. The scheme depicted in Fig. 13.3 is for an aptazyme ligase which should catalyze the cis addition of a substrate RNA to its own 5l end in the presence of an effector. A round begins with a negative selection step in which the aptazyme pool is incubated with the oligonucleotide substrate in the absence of effector. The substrate oligonucleotide (along with any ligated ribozymes) is then captured and removed from the population. For the positive selection step, effector molecules are then added to the reaction mixture, along with additional substrate oligonucleotides. The substrate and ligated ribozymes are captured, washed to remove any unligated RNA, and amplified by reverse transcription, polymerase chain reaction (PCR), and transcription. The resultant, more highly activated pool is used as the

13.3 Effector Activation

Fig. 13.3 Selection scheme for an effectoractivated aptazyme. Clockwise from the top: negative selection (right) removes species which are active in the absence of effector,

while positive selection (left) isolates species which become active upon the addition of effector, as indicated by the (–) and (+) incubations, respectively.

input for the subsequent round of selection and amplification. The stringency of the selection is increased as rounds progress by increasing the time allowed for ligation in the absence of the effector and decreasing reaction time in its presence. Once the pool demonstrates a desired level of activation (or no longer shows improvement in activation), individual aptazymes are cloned, sequenced, and assayed for activity and activation. A similar selection scheme can be concocted for aptazymes that are inhibited by an effector, except that the negative selection step consists of identifying variants that fail to react in the presence of the effector, but are then active once the effector is removed.


As mentioned above, aptazymes have been engineered or selected to utilize a variety of effectors. The specificity of target recognition by aptazymes is comparable to that of aptamers, allowing discrimination between even closely related mole-

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cules. For example, the specificity of an anti-theophylline aptamer has been imparted to a theophylline-activated hammerhead aptazyme derived from the aptamer (Soukup et al., 2000). The aptamer bound theophylline 6.3-fold tighter than it bound 3-methylxanthine (a molecule which differs from theophylline by the absence of the 1-methyl group). However, the aptazyme was activated 18-fold better by theophylline than by 3-methylxanthine; a 2.8-fold increase in specificity over the aptamer alone. The increased discrimination shown by aptazymes is likely due to the fact that the energy of ligand binding to the aptamer domain must be further transduced into conformational changes in the appended ribozymes. This increased discrimination may ultimately be limited by background reaction rates. For instance, while the aptamer demonstrated a 10 900-fold specificity for theophylline over caffeine, the aptazyme was unable to equal this, since its overall activation was only 2300-fold. However, the most remarkable kinetic property exhibited by aptazymes is not the diversity of effectors or the specificity with which they can be recognized, but rather the levels of effector dependence that have been achieved. In general, the activation parameters exhibited by in vitro selected aptazymes dwarf those of known allosteric protein enzymes. For example, aptazymes that were selected to be activated by small organic molecule effectors or cations have shown activity levels that are three to five orders of magnitude faster in the presence of saturating effector than in the absence of effector, while many known allosteric protein regulators are generally activated by only tens- to hundreds-of-fold. Each of the different design and selection methods cited above has resulted in generating aptazymes with significant activation parameters. A high affinity antitheophylline aptamer is known to undergo a significant conformational change upon binding its target, and this makes it a particularly good candidate for modular aptazyme design. A rationally designed L1 ligase was created in which a functionally unnecessary portion of stem C was replaced with a theophylline-binding domain (Robertson and Ellington, 2000). The resulting aptazyme (Fig. 13.4a) was activated by 1625-fold. Similarly, a hammerhead ribozyme pool was created in which the anti-theophylline aptamer replaced stem II of a cis-acting hammerhead ribozyme, and the two domains were joined by a library of 10 random residues (five randomized positions on each of two opposing strands) (Soukup et al., 2000). The selection for theophylline dependence yielded individual aptazymes that were activated by up to 4700-fold (Fig. 13.4b). Finally, a pool was created in which stem II of a hammerhead ribozyme was replaced with 25 completely random nucleotides. Rather than selecting for theophylline activation, selections were carried out with a series of nucleotide 3l,5l cyclic-monophosphates (Koizumi et al., 1999). A range of aptazymes were generated, one of which was activated greater than 5000-fold by cGMP (Fig. 13.4c). Aptazymes have been selected for dependence on proteins and peptides, as well as small organics. A random sequence pool derived from the L1 ligase (L1-N50, shown in Fig. 13.2b) was used as a starting point for the selection of proteindependent aptazyme ligases (Robertson and Ellington, 2001). Even though this pool does not contain any extant protein-binding sequence or structural motifs,


Fig. 13.4 Small-molecule-activated aptazymes. (a,b) The structure of the theophylline effector is shown along with the secondary structures of (a) a L1 ligase aptazyme (L1-THEOd1) and (b) a hammerhead-based aptazyme (cm+theo2). Ligand binding domains are in bold and the selected joining

region is boxed. (c) 3l5l-cyclic GMP is shown with a cGMP-dependent hammerhead aptazyme (cGMP-1). The selected activation domain is shown in bold. Hammerhead ribozyme cleavage sites are denoted by an arrow.

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13.4 Aptazyme Structural and Functional Diversity m Fig. 13.5 Protein-activated aptazymes. (a) The secondary structure of a Cyt18-dependent ligase (L1-cyt7-2) is shown with the selected activation domain bolded. (b) The activation domain of a Rev ARM peptide-dependent ligase (L1-Rev 8-4) is shown; the remainder of the ribozyme corresponds to the ligase sequence shown in (a).

(c) A ligation assay for L1-Rev 8-4 demonstrates the formation of product within minutes in the presence of the Rev peptide, while ligation in the absence of the peptide produced little product within hours. (d) The rate of L1Rev 8-4 ligation is orders of magnitude greater in the presence of either the Rev peptide or protein.

the power of the selection method is such that it seemed likely that aptazymes could be identified that would both bind protein ligands and undergo protein-dependent conformational changes. In fact, after nine rounds of selection and amplification the L1-N50 pool yielded aptazymes with some of the greatest activation parameters yet observed. Selected aptazyme ligases were up to 3100-fold activated in the presence of hen egg white lysozyme, while a selection that targeted a tyrosyl tRNA synthetase from Neurospora mitochondria (Cyt18) yielded an aptazyme that was activated by 94 000-fold (Fig. 13.5a). Because no known protein-binding motif was initially incorporated into the design of the aptazyme pool, it was unknown whether selected aptazymes would be specific for their target proteins. However, the only cross-reactivity that was observed was for a lysozyme dependent clone, lys11-2, which was cross-activated by turkey lysozyme (98% similar to chicken) but not by human lysozyme (70% similar). The L1-N50 pool has also been used to select for peptide-dependent aptazymes (Robertson et al., 2004). In these selections, the 17-amino-acid arginine-rich motif (ARM) peptide from the HIV-1 Rev protein was used as a target. One of the selected aptazymes (Fig. 13.5b) was activated 18 000-fold by the presence of its ARM peptide effector and was also shown to be activated several thousand fold by the protein from which the peptide epitope was derived (Fig. 13.5c,d). The specificity of the selected aptazyme was confirmed by assaying it against a number of other ARM peptides. The aptazyme could in general recognize the wild-type Rev peptide better than those with single amino acid substitutions, and the only other cross-reactivity that was observed was modest activation by the HIV Tat ARM. Reselection of the aptazyme from a doped sequence pool yielded aptazymes that were even more specific, in that they were able to better discriminate against the Tat peptide. In addition, activation parameters were further improved; the most active reselected Rev ARM-dependent clone was activated by 38 000-fold, a value which could be further increased by additional incubation time prior to initiation of the reaction.

13.4 Aptazyme Structural and Functional Diversity

As described above, many aptazymes have been derived from either the hammerhead ribozyme (a naturally occurring RNA cleavase) or the L1 ligase ribozyme (discovered through in vitro selection) using either rational design or selection

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methods. However, a number of additional natural and unnatural ribozyme motifs have also been adapted to function as aptazymes. Group I introns are self-splicing RNAs that utilize an exogenous guanosine cofactor to catalyze three successive transesterfication reactions, ultimately resulting in the removal of the ribozyme sequence from within an mRNA and concomitant ligation of the mRNA exons. In the wild-type ribozyme, the terminal tetraloop of the P5 stem docks with the P6 stem during ribozyme folding. Atsumi et al. (2001) have designed a derivative of the Tetrahymena group I self-splicing intron in which a portion of the P5 and P6 stems have been replaced with known protein-binding RNA motifs, bacteriophage l box B and the HIV RBE, which bind to the ARMs of the lN and Rev proteins, respectively (Atsumi et al., 2001). A chimeric peptide that adjoined the two ARM peptides brought the P5 and P6 stems into close proximity and enabled folding of the designed aptazyme. The peptide effector activated the aptazyme by up to 8-fold. Aptazyme activation was then further optimized via selection of the peptide component. A peptide library was expressed in cells that also contained the aptazyme inserted into the b-galactosidase gene. Cells that were able to grow on lactose were selected, yielding a peptide with a selected linker region that in turn promoted an 11-fold increase in the extent of aptazyme splicing. The hepatitis delta virus (HDV) is a circular RNA that undergoes rolling circle replication to yield RNA concatamers. The concatamers are processed by an embedded self-cleaving ribozyme, the HDV ribozyme. Kertsburg and Soukup (2002) have utilized the HDV ribozyme as a platform for the in vitro selection of aptazymes activated by theophylline (Kertsburg and Soukup, 2002). The selection was initiated with a pool in which the P4 stem of the ribozyme was replaced with a random stem of either 9 or 10 nucleotides adjoined to a minimal anti-theophylline aptamer. The selection yielded aptazymes that were activated by up to 115-fold in the presence of theophylline. The selected communication modules could actually be swapped between different aptamers and ribozyme platforms, resulting in new aptazymes. Both the anti-FMN and anti-ATP aptamers, in conjunction with the selected communication module, could activate the HDV ribozyme. Likewise, the X-motif (an in vitro selected self-cleaving ribozyme) and group I intron ribozymes could both be activated by appending the selected theophylline-dependent domain. The hairpin ribozyme, another RNA cleavase of viral origin, has also been adapted by the Famulok lab to function in an effector-dependent manner. A large internal loop of the hairpin ribozyme was designed to be complementary to an anti-thrombin DNA aptamer (Hartig et al., 2002). The addition of this aptamer led to the formation of a long double-stranded region and caused misfolding of the ribozyme, yielding low catalytic activity. In the presence of thrombin, the native quadruplex structure of the aptamer was stabilized relative to the extended double-stranded structure, releasing the ribozyme from structural constraints and thereby allowing it to efficiently cleave substrate RNAs. This schema was particularly interesting because of its modular nature. Because the ribozyme and aptamer domains were on separate nucleic acid strands, individual aptamer or ribo-

13.4 Aptazyme Structural and Functional Diversity

zyme domains could be readily swapped with other aptamers or ribozymes in order to generate aptazymes that responded to new effectors or that had new catalytic activities. Indeed, the authors demonstrated that the same system could be used to generate thrombin-activated hammerhead aptazymes. Ribozyme catalysis is not the province of RNA alone. A number of DNA aptamers and catalysts (deoxyribozymes) have been called into being by in vitro selection. Since DNA has been shown to be capable of both ligand binding and catalysis, it stands to reason that it should also be possible to create deoxyaptazymes. Potential advantages of deoxyaptazymes relative to aptazymes are the increased stability of DNA to biological and environmental degradation, and the greater ease (and reduced expense) of DNA synthesis. The first deoxyribozyme adapted to function as an aptazyme was a DNA ligase selected by Levy and Ellington (2002b). The deoxyribozyme was initially shown to catalyze the ligation of DNA strands with 3l phosphorothioates to strands containing a 5l iodine moiety, forming a bridging phosphorothioate (Fig. 13.6a). This chemistry was initially popularized by Eric Kool and co-workers (Xu and Kool, 1997) as a means of chemically (rather than enzymatically) ligating DNA, but the selected deoxyribozyme can still modestly (10-fold) enhance the rate of reaction. The deoxyribozyme can act either in cis (the catalyst itself terminates in a 5l iodine) or in trans. A known anti-ATP DNA aptamer was appended to the deoxyribozyme, and deoxyaptazymes were engineered by both rational design and selection methods. In rationally designed constructs stem B of a minimized ligase was replaced with a series of short stems topped by the anti-ATP aptamer (Levy and Ellington, 2002a). These designed deoxyaptazymes displayed up to 250-fold activation by ATP when substrates were presented in trans (55-fold in cis). As with other aptazyme selections, a pool was then created that contained either three or four random nucleotides on either side of the aptamer domain, and ATP-dependent deoxyaptazymes were selected. The best selected deoxyaptazyme (shown in Fig. 13.6b) was activated by 400- and 460-fold for the trans and cis ligation reactions, respectively. Deoxyaptazyme cleavases as well as ligases have been engineered. Starting with two deoxyribozyme cleavases (10–23 and 8–17) that were originally selected in vitro by the Joyce lab, the Sen lab has developed general methods for engineering trans-cleaving deoxyaptazymes (Wang and Sen, 2001; Wang et al., 2002a,b). The two enzymes catalyze cleavage adjacent to bulged G or A residues, respectively, in RNA strands base-paired to the deoxyribozymes. The anti-ATP DNA aptamer was inserted into and interrupted the hybridizing arms of the deoxyribozymes (Fig. 13.6c,d). In the presence of saturating adenosine, the best designs catalyzed 30-fold faster cleavage of RNA linkages. One advantage of this design strategy is that it is quite modular, and any of a variety of aptamers can potentially be positioned so as to influence the free energies of base pairing and hybridization to substrates.

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Fig. 13.6 ATP-dependent deoxyaptazymes. (a) The reaction catalyzed by the Levy deoxyribozyme ligase. (b) A trans-acting, ATP-dependent deoxyaptazyme ligase is shown. The ATPbinding domain (bold) and the selected joining region (boxed) are indicated, and the substrates are shown in italics with the ligation

junction denoted by an asterisk. (c,d) Designs for a two-piece, ATP-dependent 10-23 (c) and a one-piece, ATP-dependent 8-17 (d) cleavase deoxyaptazymes are shown with the ATP-binding domain in bold and the RNA substrate in italics. The cleavage site is denoted by an arrow.

13.5 Uses of Aptazymes in Biology and Biotechnology

13.5 Uses of Aptazymes in Biology and Biotechnology 13.5.1 Aptazymes as Biosensors

Traditionally, biosensors depend on the capture of an analyte by either a protein (antibody) or nucleic acid (aptamer) receptor. Binding events can potentially be directly reported by label-free techniques such as surface plasmon resonance (SPR) or quartz crystal microbalance (QCM) measurements. Alternatively, sensors often utilize labeled target molecules or secondary capture agents (in the case of a sandwich-type assay) which themselves contain some form of label or reporter molecule. Aptazymes may have a number of advantages over biopolymer receptors, especially in those instances where a given binding event is transient and potentially rare, such as at analyte concentrations significantly below the affinity of the receptor. While analytes must generally remain associated with a receptor, once analyte-mediated catalysis has occurred with an aptazyme, the change in covalent bond state will persist and can be detected even if the analyte is washed away. For research purposes, the products of aptazyme activity can be separated on polyacrylamide gels and visualized using autoradiography or nucleic acid stains. However, these methods are not particularly useful for medical, industrial, or military biosensors. Nonetheless, the fact that the products of an aptazyme reaction generally have masses and charges that differ significantly from the original substrates facilitates detection via label-free techniques, such as those described above. In addition, the ability to chemically synthesize nucleic acids enables the facile incorporation of a variety of specific reporters, such as fluorophores, that can further enhance detection. Hammerhead aptazymes have been shown to act upon fluorescently-labeled substrates (Fig. 13.7), and to thereby transduce analyte recognition into optical signals. Most schemes depend on the incorporation of two optically active molecules: either a fluorophore/quencher pair or a fluorescence resonance energy transfer (FRET) pair. Upon cleavage, one label is liberated from its proximal partner, resulting in a change in fluorescence intensity, wavelength or both. Such “signaling aptazymes” have been utilized to tackle specific sensing problems in biology. For example, the Famulok lab has used an aptazyme-based screen to identify small molecules which bind to the Rev protein and which might therefore serve as drugs for the inhibition of HIV-1 (Hartig et al., 2002). These researchers appended an anti-Rev aptamer to a hammerhead ribozyme in place of stem II and generated an aptazyme that was 36fold inhibited by the presence of Rev. The addition of a small molecule that could compete with the aptazyme for binding to Rev resulted in reactivation of the aptazyme, which in turn cleaved apart a fluorophore and quencher pair on a substrate RNA. The anti-Rev aptamer was also appended to the 5l end of the ribozyme, such that it folded into an inactive hairpin conformation that also prevented access to the substrate oligonucleotide. In the presence of Rev, the native conformation of the

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Fig. 13.7 Fluorescent signaling aptazymes. Cleavage and subsequent dissociation of products liberates a fluorophore from a proximal quencher, causing an increase in fluorescent emission (a and c) or liberates an acceptor fluorophore from its donor, resulting in a loss of FRET signal (b).

anti-Rev aptamer was stabilized, allowing the substrate to bind and thus allowing cleavage of the optically active substrate. Aptazyme activity could be monitored in real time. One of three Rev-binding drug candidates identified by these screens was shown to be specific enough to reduce HIV-1 replication in cultured cells without significant cellular toxicity. Similar signaling aptazyme schemes have been used to report other biological phenomena. Aptazymes have been used to report protein modification states (Vaish et al., 2002). Stem II of the hammerhead ribozyme was again replaced with aptamers, but this time with aptamers specific for either the unmodified or doubly-phosphorylated form of the MAP kinase ERK2. However, the aptazymes were also designed such that one of the substrate-binding arms interacted with the new chimeric stem II. Protein binding stabilized the aptamer, displaced the substrate-binding arm, and allowed access to substrate. The two designed aptazymes showed activation levels Z230-fold and 50-fold for the modified and unmodified proteins, respectively, and each aptazyme was not activated significantly by the non-cognate form of the protein. These aptazymes were able to detect the presence of their protein effectors in the low nanomolar range, and a combination of these two aptazymes was shown to accurately report the relative level of protein phosphorylation. The authors also demonstrated ERK2 detection in a mammalian cell lysate. Aptazyme-based assays can also be used to measure protein activity rather than just detect protein structure. A beautiful demonstration of this was carried out by researchers at the company Archemix, in which an ADP-activated hammerhead aptazyme was selected from a pool in which a known anti-ADP aptamer was con-

13.5 Uses of Aptazymes in Biology and Biotechnology

nected to the core ribozyme via a random stem (Srinivasan et al., 2004). Protein kinases transfer the gamma phosphate of an ATP molecule to their target protein, releasing ADP; thus the detection of ADP can be used as an indicator of protein activity. The selected aptazyme could detect ADP to concentrations as low as 1–5 mmol/L and was utilized in a coupled phosphorylation/aptazyme cleavage assay to report the in vitro activity of phopshorylated ERK2 on a synthetic peptide substrate. Fluorescent molecules are not the only modification that can allow optical detection of ribozyme activity. For example, thiol-modified DNA and gold nanoparticles have recently been used to create a colorimetric aptazyme assay (Liu and Lu, 2004). DNA-functionalized nanoparticles were aggregated by complementary oligonucleotide strands containing a substrate for the adenosine-dependent 8–17 aptazyme. In an aggregated state, these nanoparticles appear blue. However, aptazyme cleavage of the hybridizing oligonucleotide frees gold particles from the aggregate, resulting in a return of the normally observed red color. Photometric measurements of the aggregated and free particle states have been used to quantitate adenosine concentrations in the 0.1–1 mmol/L range. Aptazymes which have been created to date are all based on RNA or DNA molecules that can rearrange phosphodiester bonds, such as cleavases, ligases, and self-splicers. However, a significant number of other chemical reactions can now be catalyzed by ribozymes and deoxyribozymes. For example, in vitro selections have yielded Diels-Alderase and peptidyltransferase ribozymes, to name a few. The adaptation of ribozymes with novel chemistries into aptazymes may allow them to be used in a greater variety of detection schemes. One ribozyme that would be particularly amenable for use as a biosensor is a redox active ribozyme selected by the Suga lab (Tsukiji et al., 2003). This ribozyme, called “ribox02,” has been shown to catalyze the NAD+-assisted oxidation of benzyl alcohol. An appropriately engineered redox aptazyme could be introduced into an electrochemical sensor in much the same way that horseradish peroxidase or glucose oxidase are now routinely used in enzyme-coupled electrodes. An additional drawback to using only known RNA or DNA enzymes for the analysis of biological samples is their relative fragility in the presence of nucleases and other environmental insults. However, modified RNAs, which may contain phosphorothioate linkages, or residues in which the 2l-hydroxyl is replaced with fluorine or a hydroxymethyl group are much more stable to degradation. A large number of aptamers have now been selected that contain modified nucleotides, and their functional lifetimes in a variety of harsh environments have been demonstrated to be markedly longer than their non-modified counterparts; for example, modified anti-vascular endothelial growth factor (VEGF) aptamers were shown to be stable for days in sera (Green et al., 1995). Concurrently, polymerases that can more efficiently incorporate modified nucleotides have been engineered (Chelliserrykattil and Ellington, 2004). In addition to aptamers, nuclease-resistant ribozymes have been designed or selected. A logical next step will be the creation of modified aptazymes that can function in biological samples such as cell lysates, blood, or urine.

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13.5.2 Aptazymes as Molecular Logic Gates

In 1994 Len Adleman demonstrated that DNA oligonucleotides could be designed to carry out molecular computations, and in fact solved a partial, directed Hamiltonian path problem (Adleman, 1994). Most DNA computations are based on “hybridization logic” in which programmed sequences assemble into answers. In contrast, the ability of aptazymes to transduce a molecular input (the effector) into a distinctly different output makes them more akin to electronic parts (like transistors) that can “read” a variety of input information, carry out a transformation, and then “write” to another part or to an output. More importantly, the ability of aptazymes to carry out reactions of biological interest (for example, mRNA cleavage) makes them perfect tools for translating research into nucleic acid computation into more practical applications. Aptazymes are particularly well-suited to act as Boolean logic gates. Many of the aptazymes already presented herein are activated or inhibited by a given effector molecule; these can be thought of as YES or NOT gates, respectively. Furthermore, all of the L1-ligase based aptazymes function as AND gates, since they retain the parental ribozyme’s dependence on the oligonucleotide 18.90a as well as their engineered or selected dependencies. A dual-effector (AND gate) hammerhead that was dependent on the presence of both theophylline and FMN for efficient self-cleavage has also been reported by the Breaker lab (Jose et al., 2001). The simplicity of Watson–Crick base-pairing facilitates the engineering of oligonucleotide-activated or oligonucleotide-inhibited aptazyme logic gates. Stojanovic and Stefanovic have created several such gates based on selected RNA-cleaving deoxyribozymes, including XOR, ANDNOT, and iAANDiBANDNOTiC (input A and input B and not input C) gates (Stojanovic et al., 2002; Stojanovic and Stefanovic, 2003a). These authors have joined 23 such logic gates that operate on fluorescent substrates into a finely programmed hierarchy that operates as a molecular automaton (Stojanovic and Stefanovic, 2003b). When judiciously placed in a physical 3 q 3 matrix these logic gates encode an algorithm that allows the automaton to play tic-tac-toe with a human player in such a way that the automaton will either win or play to a draw. 13.5.3 Aptazyme Arrays

Aptazyme biosensors and logic gates can also be immobilized into array formats. The multiplication of a single ribozyme motif, such as the hammerhead cleavase or L1 ligase, into a series of different immobilized aptazymes can help to standardize assays that will report the presence and concentration of numerous analytes in parallel. For example, a number of small molecule-activated hammerheads that were responsive to cobalt ions, cAMP, cCMP, cGMP, theophylline, and FMN were immobilized on a gold surface via a terminal phosphorothioate moiety (Seetharaman et al., 2001). Interestingly, this array also contained the aptazyme logic gate

13.5 Uses of Aptazymes in Biology and Biotechnology Fig. 13.8 Aptazyme ligase array. (a) A variety of effectors can be detected. Ribozymes are labeled along rows, and effectors along columns; (–) and (+) signs indicate the absence of and presence of all analytes, respectively. (b) Response curves for arrayed small-molecule (FMN), peptide (Rev), and proteindependent (lysozyme) aptazymes.

that was activated only in the presence of both theophylline and FMN. The hammerhead aptazyme array could not only specifically indicate the presence of the expected cognate analytes, but was robust enough to be used to phenotype three strains of E. coli based on their ability to accumulate cAMP in growth media. However, the assay format relied on the recovery of a hammerhead aptazyme cleavage product, and proved too variable for accurate effector quantitation. An aptazyme array based on the L1 ligase has also been generated and was capable of not only detecting, but quantitating a diverse range of analytes (Hesselberth et al., 2003). The array included L1 ligase variants that were responsive to an oligonucleotide effector, the small molecules ATP, FMN, and theophylline, and the Rev peptide and the protein lysozyme. This array relied on the capture and subsequent visualization of ligated aptazymes (Fig. 13.8). Any unligated RNAs were removed via a washing step, enabling higher discrimination relative

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to background and better limits of detection (into the low nanomolar range) than were possible with the hammerhead aptazyme array. 13.5.4 Aptazyme Use In Vivo

Ribozymes are often used in vivo, usually to destroy a particular mRNA and knock down the expression of its corresponding protein. In this context, aptazymes could provide an additional level of control over when and how mRNA degradation occurred. For example, Mulligan and his co-workers have inserted a cis-acting hammerhead ribozyme into the mRNA of b-galactosidase, such that cleavage resulted in low protein expression (Yen et al., 2004). Induction of protein expression was then brought about by inhibiting the hammerhead either with a complementary oligonucleotide, or with a small-molecule antibiotic that was known to inhibit the ribozyme. Amazingly, this system was shown to work in both cultured cells and live mice. Although the ribozyme was not an engineered aptazyme per se, these results demonstrate the possibilities inherent in aptazyme-mediated genetic control. Alternatively, it may be possible to regulate the induction (as well as destruction) of mRNA and protein expression. For example, the Ellington lab has used the group I self-splicing intron found within the bacteriophage T4 thymidylate synthase (td) gene as a starting point for engineering aptazymes (Thompson et al., 2002). Aptazymes were generated by appending an anti-theophylline aptamer in place of stem structures within the intron, and the resultant constructs were assayed for theophylline-dependent activation in vitro. Assays were also carried out in vivo by inserting the aptazymes in place of the wild-type intron in an interrupted td gene, and then observing growth in an E. coli thymidine auxotroph. Some aptazymes allowed theophylline-dependent rescue of the bacteria from death or slow growth. However, activity in vitro did not guarantee activity in vivo, a disconnect that has previously been observed for both aptamers and ribozymes as well. It should be possible to extend aptazyme functionality in vivo beyond known cleavage or splicing reactions, and to eventually use aptazymes for biomedical applications. For example, trans-splicing group I ribozymes have been used to restore p53 activity in cancer cells by replacing the mutant 3l portions of p53 mRNAs with the wild-type sequence. Trans-splicing aptazymes that could be used to allow a small-molecule pharmaceutical to control when and potentially where, such mRNA repair would take place. In general, by melding aptazyme and gene therapy technologies it may prove possible to better control gene expression and to develop financially viable models for gene therapy that pharmaceutical companies might participate in. Aptazymes could also control gene expression based on the intracellular concentrations of metabolites, mRNAs, or proteins, and could thereby be introduced into novel genetic circuits and signal transduction pathways for the autonomous diagnosis and treatment of diseases.

References

Acknowledgements

The authors wish to gratefully acknowledge support from the National Institute of Health.

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(2002). Protein-dependent ribozymes report molecular interactions in real time. Nat Biotechnol 20, 717–722. Hesselberth, J., Robertson, M. P., Jhaveri, S., Ellington, A. D. (2000). In vitro selection of nucleic acids for diagnostic applications. J Biotechnol 74, 15–25. Hesselberth, J. R., Robertson, M. P., Knudsen, S. M., Ellington, A. D. (2003). Simultaneous detection of diverse analytes with an aptazyme ligase array. Anal Biochem 312, 106– 112. Jose, A. M., Soukup, G. A., Breaker, R. R. (2001). Cooperative binding of effectors by an allosteric ribozyme. Nucleic Acids Res 29, 1631–1637. Kertsburg, A., Soukup, G. A. (2002). A versatile communication module for controlling RNA folding and catalysis. Nucleic Acids Res 30, 4599–4606. Koizumi, M., Soukup, G. A., Kerr, J. N., Breaker, R. R. (1999). Allosteric selection of ribozymes that respond to the second messengers cGMP and cAMP. Nat Struct Biol 6, 1062–1071. Levy, M., Ellington, A. D. (2002a). ATP-dependent allosteric DNA enzymes. Chem Biol 9, 417–426. Levy, M., Ellington, A. D. (2002b). In vitro selection of a deoxyribozyme that can utilize multiple substrates. J Mol Evol 54, 180–190. Liu, J., Lu, Y. (2004). Adenosine-dependent assembly of aptazyme-functionalized gold nanoparticles and its application as a colorimetric biosensor. Anal Chem 76, 1627– 1632. Porta, H., Lizardi, P. M. (1995). An allosteric hammerhead ribozyme. Biotechnology (NY) 13, 161–164. Rimmele, M. (2003). Nucleic acid aptamers as tools and drugs: recent developments. Chembiochem 4, 963–971. Robertson, M. P., Ellington, A. D. (1999). In vitro selection of an allosteric ribozyme that

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Thompson, K. M., Syrett, H. A., Knudsen, S. M., Ellington, A. D. (2002). Group I aptazymes as genetic regulatory switches. BMC Biotechnol 2, 21. Tsukiji, S., Pattnaik, S. B., Suga, H. (2003). An alcohol dehydrogenase ribozyme. Nat Struct Biol 10, 713–717. Vaish, N. K., Dong, F., Andrews, L., Schweppe, R. E., Ahn, N. G., Blatt, L., Seiwert, S. D. (2002). Monitoring post-translational modification of proteins with allosteric ribozymes. Nat Biotechnol 20, 810–815. van der Horst, G., Christian, A., Inoue, T. (1991). Reconstitution of a group I intron self-splicing reaction with an activator RNA. Proc Natl Acad Sci USA 88, 184–188. Wang, D. Y., Sen, D. (2001). A novel mode of regulation of an RNA-cleaving DNAzyme by effectors that bind to both enzyme and substrate. J Mol Biol 310, 723–734. Wang, D. Y., Lai, B. H., Feldman, A. R., Sen, D. (2002a). A general approach for the use of oligonucleotide effectors to regulate the catalysis of RNA-cleaving ribozymes and DNAzymes. Nucleic Acids Res 30, 1735– 1742. Wang, D. Y., Lai, B. H., Sen, D. (2002b). A general strategy for effector-mediated control of RNA-cleaving ribozymes and DNA enzymes. J Mol Biol 318, 33–43. Xu, Y., Kool, E. T. (1997). A novel 5’-iodonucleoside allows efficient nonenzymic ligation of single-stranded and duplex. Tetrahedron Lett 38, 5595–5598. Yen, L., Svendsen, J., Lee, J. S., Gray, J. T., Magnier, M., Baba, T., D’Amato, R. J., Mulligan, R. C. (2004). Exogenous control of mammalian gene expression through modulation of RNA self-cleavage. Nature 431, 471–476.

14.1 Introduction

14 Conversion of Aptamers into Small-Molecule Lead Compounds Andreas Jenne

14.1 Introduction

Many pharmaceutical companies are primarily concerned with developing orally administered small-molecule drugs. This is because small-molecule drugs are inexpensive to produce and can potentially become blockbusters. Initially, compound libraries are screened in cell-based or biochemical assays to identify hits: these are subsequently optimized in pharmaceutical hit-to-lead programs. Classical screening approaches are often supported by in silico methods to aid drug development. The ultimate goal of these combined efforts is to build a strong pipeline of novel lead compounds for preclinical and clinical testing. The development of appropriate screening assays is an integral part of every high-throughput screening (HTS) campaign. Functional assays are usually preferred over affinity-based assays since they are more likely to deliver active “hits.” Generally, such hits provide a suitable starting point for subsequent lead compound development. Biochemical competition assays in which small molecules displace a natural interaction partner of the target are high on the list of screening strategies. The development of such assays, however, is often time-consuming and laborious, for example, when the substrate of an enzyme target is difficult to label or the natural interaction partner has yet to be identified. This review focuses on aptamers as an elegant solution to such problems. The aptamer approach shows bright prospects for generating small-molecule inhibitors with new mechanisms of action for both comprehensively validated targets and genomic targets with unknown interaction partners. Practical examples include inhibition of protein–protein, or protein–RNA interactions as well as enzyme activity.


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14.2 Rational Drug Design

In recent years, structure-based drug design has identified a number of promising development candidates, and contributed to a better understanding of their mode of actions (Vassilev et al., 2004). Usually, X-ray or NMR structures of protein or protein–ligand complexes provide the basis for visualizing small-molecule docking to the active site of the protein target. The availability of several structures with and without ligand is an advantage as in silico models become more reliable. Such meaningful models are the key to success since they reveal potential active or regulatory sites and provide the basis for predicting compound affinity and selectivity (Oprea and Matter, 2004). Structural analyses of aptamer–protein complexes could provide a wealth of data to support rational drug design. To date, only a few structures of aptamer– protein complexes are resolved, but it seems likely that more data will be published as a result of aptamer technologies becoming more widespread. Aptamer–protein co-crystal structures of bacteriophage MS2 coat protein, transcription factor NFkB, and thrombin have provided valuable insights into the molecular recognition mechanisms adopted by aptamers (Padmanabhan et al., 1993; Rowsell et al., 1998; Huang et al., 2003). Comparison of these structures, for example, revealed that aptamers can sometimes cover relatively large surfaces (Jaeger et al., 1998), or alternatively only make small footprints (Rowsell et al., 1998). Typically, aptamer binding involves ionic interactions, hydrogen bonding, and base stacking (Herrmann and Patel, 2000), but other features, for example, phosphate backbone flexibility and shape complementarity, also contribute to aptamer affinity (Horn et al., 2004). In some cases aptamers can fold into intrinsically stable structures, as is best exemplified by the very small 15-mer G-quartet-forming anti-thrombin aptamer (Kankia and Marky, 2001). In other cases relatively large RNA motifs of 70 nucleotides or more are required for high-affinity recognition, obviously because these aptamers must adopt rather complex structures (Sakamoto et al., 2005). Many aptamers are potent inhibitors of protein activity because they tend to bind functional sites on proteins in a manner similar to small-molecule drugs (Bacher and Ellington, 1998). Besides blocking the active site of a protein, aptamers have also been found to interfere with protein activity by allosteric or non-competitive inhibition (Lupold et al., 2002). These features make aptamers highly attractive tools for rational drug design and biochemical drug screening (see next section). Referring to the latter point, structural analyses of aptamer– protein complexes may also contribute to a more in-depth understanding of the mechanisms involved when a protein-bound aptamer is displaced by a small molecule. Presumably, this question will need to be answered on a case-by-case basis since aptamer–protein recognition can adopt various modes and configurations. It is fairly certain, however, that in an unbound state aptamers are rather flexible molecules compared with the more rigid antibodies, for example. Upon binding to their targets, aptamers usually undergo structural changes to adopt a well-defined three-dimensional structure within the framework of the protein’s binding

14.3 Biochemical Screening

pocket (Herrmann and Patel, 2000). When a small molecule interacts with this aptamer-binding site, the aptamer might no longer adopt the active conformation required for tight binding and target recognition. As a consequence, such small molecules can displace the aptamer, even if the small molecule only blocks a single or a few interaction sites.


Biochemical competition assays enable the screening of vast compound libraries under high-throughput conditions. These assays usually require natural or synthetic target interaction partners: if a library compound competes with the interaction partner it suggests that this compound has a potential biological activity. Indeed, many small molecules derived from functional screenings exhibit target inhibition. In many cases it is desirable to obtain hits with low micromolar inhibition constants from the first screening round since these facilitate optimization into more potent lead compounds with fewer side-effects. Medicinal chemistry and in silico methods can be used to specifically enhance the molecule’s potency and ADMET parameters. Nevertheless, following screening and iterative rounds of optimization these lead compounds need to be tested further in appropriate animal models. Only then is it possible to decide if the compound is suitable for testing in humans. The following examples illustrate how aptamers can be effectively integrated into early drug discovery to facilitate the development of new lead compounds. Green and co-workers provided the proof-of-concept for aptamer-based screening in 2001 (Green et al., 2001). The authors employed high-affinity aptamers for two protein targets, platelet-derived growth factor (PDGF) and wheat germ agglutinin (WGA). It was demonstrated previously that these aptamers effectively inhibit target activity in functional assays with affinities in the low nanomolar range (anti-WGA aptamer 11.20, Kd = 5.7 nmol/L; anti-PDGF aptamer 20tb, Kd = 0.4 nmol/L) (Green et al., 1996; Bridonneau et al., 1999; Floege et al., 1999). WGA and PDGF were each screened with a different set of small molecules. Both screenings yielded inhibitors capable of inhibiting target activity in cell culture with low micromolar and in some cases nanomolar IC50 -values. In the case of PDGF suramin, an anti-tumor agent belonging to the naphthalene sulfonic acid class and several of its derivatives were screened for their ability to displace the anti-PDGF aptamer from the PDGF-binding site. This compound class was selected because naphthalene sulfonic acids are known to inhibit the interaction between PDGF and its receptor (Powis et al., 1992). In the case of WGA, a set of oligosaccharides and sialylated derivatives were screened, including Lewis A, X, Y and several multimers of the carbohydrate (GlcNAc)n. Triacetylchitotriose (GlcNAc)3, a molecule binding to the WGA active site, was used during

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SELEX to elute aptamers from the protein and was therefore already known to compete with the anti-WGA aptamer (Bridonneau et al., 1999). In competition experiments radiolabeled aptamers were pre-incubated with varying concentrations of the small molecule to be tested. After adding the target protein, PDGF or WGA, the binding reaction was allowed to reach equilibrium. In both cases an anti-VEGF aptamer served as a negative control. The amount of aptamer/protein complex in the presence of the small-molecule competitor was determined by separating aptamer/protein complexes from the non-bound aptamers by filter binding. These experiments allowed the calculation of Kd-values for the small molecule/protein interactions. Subsequently, active hits were examined further in functional assays for their ability to inhibit target function. Although only 12 compounds were screened for PDGF it was obvious that the anti-PDGF aptamers can be effectively displaced with low molecular weight compounds (Fig. 14.1a). It was striking that the affinities of all tested compounds clearly correlated with their inhibitory potency in a cell-based functional assay (i.e. PDGF-induced [3H]thymidine incorporation in rat smooth muscle cells). Remarkably, the first evidence for a structure–activity relationship became obvious when correlating the Ki-values with the screened structures. Similar results were obtained in the case of WGA where 16 related compounds were screened and analyzed for their ability to inhibit WGA-induced agglutination of sheep erythrocytes. The tested multimers of GlcNAc inhibited WGA-induced agglutination with IC50 -values between 4 and 94 mmol/L (see Fig. 14.1b). As illustrated by double logarithmic plots of Kd versus IC50 -values, inhibition constants again corresponded well with the binding affinities of the screened compounds (Kd = 0.7– 83 mmol/L). Other sugar derivatives such Fuc1-alpha-4-GlcNAc, which is structurally related to (GlcNAc)2, showed significantly lower or no detectable inhibition. Researchers at NascaCell (Munich, Germany) assembled further examples of aptamer-competition screens involving the therapeutic targets thrombin, extracellular regulated kinase 2 (Erk2), G protein-coupled receptor kinase 2 (GRK2), HIV Rev protein, and cytohesin-1, a key regulator of leukocyte adhesion (Burgstaller et al., 2002; Mayer et al., 2003). In proof-of-concept screenings they simulated conditions that are typically applied in pharmaceutical companies. Besides isotopic assays, various common read-outs were established, including fluorescence intensity (FI), fluorescence polarization (FP), and fluorescence lifetime (FLT). In the case of HIV Rev 2500, drug-like small molecules were screened in a 384well format (Graettinger et al., 2003). Compounds were hand picked from commercially available libraries to cover as many individual core structures as possible (M. Blind, personal communication). Rev is an essential viral protein for HIV replication (Yu et al., 2005). After reverse transcription Rev binds to the rev responsive element (RRE) of the virus RNA and facilitates transport of this RNA from the nucleus to the cytoplasm. High-affinity anti-Rev aptamers were selected previously and reported to block HIV replication when expressed under the control of the U6 promoter in HIV cells (Good et al., 1997). For FP assay development the 33-nucleotide anti-Rev aptamer (RBE 79.9; Kd = 5 nmol/L) was 5l-labeled with the fluorescent dye Cy3. Screening was performed at a compound concentration of


Fig. 14.1 Small-molecule hits for the protein targets platelet-derived growth factor (PDGF) and wheat germ agglutinin (WGA) obtained in aptamer competition screenings (Green et al., 2001). (a) PDGF: The most potent hit Evans Blue as well as two further inhibitors are depicted. SPADNS and NTSA are both tri-anions

but show a 50-fold difference in the inhibition of PDGF. (b) WGA: (GlcNAc)6 was found to be the most potent inhibitor. Structurally related compounds (GlcNAc)2 and Fuc1-alpha-4GlcNAc show at least a 60-fold difference in the inhibition of WGA.

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Fig. 14.2 Aptamer conversion assay for Rev screening based on fluorescence polarization (Graettinger et al., 2005a). Displacement of the Cy3-labeled aptamer (black) from the protein active site (gray) by a small molecule hit

(white) results in a decreased fluorescence polarization (FP) signal. Signals from free aptamer (B) and target-bound aptamer (A) can be discriminated with high fidelity (Zl = 0.82).

Fig. 14.3 Small-molecule hits for the HIV Rev protein obtained in aptamer competition screenings (Graettinger et al., 2003). IC50 values were determined by Prof. J. Eberle, University of Munich (Germany) and ViroChem Pharma (Canada).


25 mmol/L with aptamer and protein concentrations of 5 nmol/L and 20 nmol/L, respectively. As illustrated in Fig. 14.2 the Rev-bound aptamer shows a higher FP signal compared with the unbound aptamer. Therefore, displacement of the aptamer by small-molecule hits was expected to result in a decreased FP signal, as was indeed the case for 0.16% of the compounds. In conclusion, screening results were highly reproducible and identified small-molecule hits that specifically bind to Rev. After 4-fold hit validation in vitro the three most potent hits were further analyzed in an HIV-replication assay over a time course of several days post infection. All three hits inhibited HIV replication at micromolar concentrations (Fig. 14.3). Further analyses in vivo, however, were not pursued because unspecific cell toxicity was observed within a concentration range similar to the Ki-values (J. Eberle, G. Dionne, personal communication). In another example, an anti-cytohesin-1 aptamer was used for FI assay development (Graettinger et al., 2003). Cytohesin-1, a GDP/GTP-exchange factor (GEF), mediates leukocyte adhesion upon stimulation of the T-cell receptor (Geiger et al., 2000). As reported previously, anti-cytohesin-1 aptamer M69 (Kd = 16 nmol/L) specifically inhibits GEF activity in vitro and leukocyte adhesion in cell-based assays (Mayer et al., 2001). For assay construction cytohesin-1 was immobilized on microtiter plates and pre-incubated with library compounds at 25 mmol/L amounts. Aptamer M69 was added and stained with an RNA intercalating fluorescent dye. After a washing step small-molecule competitors were identified by the resulting reduction in fluorescence intensity. Again, the diverse library of 2500 small molecules (see above) as well as 2500 natural products were screened. Natural products were included since they are likely to illicit different interaction patterns compared with synthetic small molecules. Screenings with the two libraries yielded several low micromolar hits. About 40% of these hits showed biological activity in the leukocyte adhesion assay, with IC50 -values between 12 and 40 mmol/L. These results clearly demonstrate that aptamer displacement assays can lead to qualified hits that have inhibition profiles closely resembling the aptamer used as the surrogate ligand. The compound with the highest affinity in vitro was a natural product with an IC50 of 0.5 mmol/L. The inhibitory effect on leukocyte adhesion, however, was significantly lower in comparison, which may be attributed to poor cell permeability of the compound. In order to develop a pharmacophore model the small-molecule hits were further analyzed at Tripos (Bude, UK) using the Tripos Benchware software (http://www.tripos.com/data/benchware/ benchware_overview_final.pdf). Five hit clusters were identified and 314 related compounds from the Tripos Lead Quest library were tested in a secondary screening (Graettinger et al., 2005b). In this screening the hit rate increased by a factor of 4.5, providing evidence for a structure/activity relationship with hits derived from the aptamer competition screening (Fig. 14.4). Besides directly measuring aptamer displacement, alternative methods have also proved successful in screening. These most elegant methods combine fluorescence technologies with the intrinsic properties of nucleic acids to both bind to a target molecule and at the same time report its presence, absence, or activity. Several recent publications describe how nucleic acids can be precisely manipu-

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Fig. 14.4 Non-linear mapping (NLM) of Tanimoto similarities of compound actives identified in the primary screen using the anticytohesin-1 aptamer (Graettinger et al., 2005b, M. Mohr). Colorcoding of data points: black to gray for increasing activity.

lated to signal specific recognition of an analyte even at very low concentrations. Such nucleic acids exist in manifold guises and have been termed aptazymes, riboreporters, aptamer beacons, reporter ribozymes, aptamer sensors, or signaling aptamers (Stojanovic et al., 2001; Gold, 2002; Rajendran and Ellington, 2002; Nutiu and Li, 2003; Silverman, 2003; Breaker, 2004; Tan et al., 2005). A shared feature is that they consist of two domains, an aptamer domain and one or more signaling sequences that report aptamer-target recognition (e.g. by a shift or change in fluorescence). Famulok and co-workers, for example, designed fluorescence-based reporter ribozymes that enable screening for inhibitors of proteins, protein–RNA and protein–protein interactions (Hartig et al., 2002). In a feasibility study the authors screened a library of 96 antibiotics for inhibitors of the Rev protein. Three compounds – coumermycin A1, nosiheptide, and patulin – were found to specifically bind to, and inhibit Rev in cellular assays. Monitoring side-products formed during enzyme reactions provides another way to discover small-molecule inhibitors for enzyme targets. Aptamers and their catalytic relatives seem almost predestined for this purpose since they recognize chemical structures with excellent specificities, even when closely related compounds are present in excess amounts (Feigon et al., 1996; Famulok, 1999). In principle, aptamers and aptazymes are applicable to any enzyme reaction that involves the formation of characteristic side-products. The following two examples describe the identification of protein kinase and phosphatase inhibitors. Researchers at Archemix screened for protein kinase inhibitors using an ADPspecific aptazyme (Srinivasan et al., 2004). Protein kinases catalyze the transfer of a phosphate group from a nucleotide triphosphate, usually ATP, to a protein substrate, resulting in the formation of phosphorylated substrate and ADP. Instead of


detecting phosphorylation of the substrate (which usually differs from case to case) the aptamer approach detects ADP, the common side-product of the kinase reaction. This assay format has the advantage that it measures kinase activity without the need for individual substrates, or antibodies for their detection. The aptazyme construct used for kinase screening comprised an ADP-specific aptamer and a ribozyme reporter domain. Signaling of the ribozyme domain was based on dequenching of fluorescence resonance energy transfer (FRET) as has been described previously (Jenne et al., 1999). Upon binding to ADP, the ribozyme domain undergoes self-cleavage and releases a short fluorescein-labeled oligonucleotide, which ultimately results in an increase of fluorescence. Before screening it was established that the fluorescence signal was specifically associated with ADP-binding and was proportional to the ADP concentration, with a lower detection limit of about 1 mmol/L. In a proof-of-concept screen 77 druglike compounds, including the known kinase inhibitor staurosporin, an ATP-mimetic, were tested for Erk inhibition. Screening was performed at 25 nmol/L Erk, 10 mmol/L test compound and 100 mmol/L each of ATP and substrate. When the initial rates of the fluorescence increase were measured and plotted, the aptazyme unambiguously identified the known kinase inhibitor staurosporin, whereas control compounds gave no signal. Furthermore, it was demonstrated that the aptazyme assay was both sensitive and robust enough to be used in HTS. The authors speculate that improvement of the aptazyme recognition properties for ADP will allow screening of nearly every known kinase. Such aptamer optimization for an improved discrimination of the nucleotide phosphorylation status is certainly possible. Sazani and co-workers, for example, recently reported an anti-ATP aptamer that discriminates between ATP and ADP by a factor of 1100 (Kd ATP = 5 mmol/L, Kd AMP = 5.5 mmol/L) (Sazani et al., 2004). Li and co-workers at McMaster University developed aptamer-based screening reagents that may be best described as “structure-switching signaling aptamers” (Nutiu and Li, 2004). This class of aptamers exploits the structural rearrangement of the aptamer upon target binding, monitoring the presence of that target by fluorecence signaling. In a proof-of-concept study Nutiu and Li screened inhibitors of alkaline phosphatase (ALP), an ATP dephosphorylating enzyme (Nutiu et al., 2004). The authors used a structure-switching aptamer that has a higher affinity for adenosine than for its 5l-phosphorylated analogs ATP, ADP, or AMP. In the absence of adenosine, the aptamer hybridizes with two short antisense oligonucleotides, each labeled with a different fluorophore. In this arrangement both fluorophores are positioned close to each other, resulting in the fluorescence signal being quenched by FRET so that only a low background signal is detected. When this system is exposed to adenosine, however, one of the two antisense oligonuclotides is released because the aptamer switches to binding its target, adenosine. Since release of the oligonucleotide abolishes the FRET effect this structure-switching event results in an increase of fluorescence intensity. This means formation of adenosine as a result of enzymatic dephosphorylation of AMP can be conveniently detected by a real-time fluorescence read-out.

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Fig. 14.5 Signaling aptamers monitor enzyme activity of alkaline phosphatase (ALP) in the presence of various small molecules in realtime. The two ALP inhibitors levamisole and

teramisole were reliably identified as indicated by the slower rate of fluorescence intensity increase relative to the uninhibited reaction (Nutiu et al., 2004).

A library of several small molecules was screened in a 96-well format, including the known ALP inhibitor, levamisole and its racemic mixture, tetramisole. Nanomolar amounts of the signaling aptamer were pre-incubated with an excess of 750 mmol/L AMP. The test compounds at 100 mmol/L were then added and the reaction started with 0.01 units of ALP per mL. The increase of fluorescence intensity was monitored and activity of ALP in the presence of the compound was calculated relative to the uninhibited control reaction. As can be seen from Fig. 14.5, the known inhibitors levamisole and tetramisole could be readily detected by the aptamer reagent. In summary, the example of Li and co-workers demonstrates that structure-switching signaling aptamers can be used in HTS to detect small-molecule inhibitors of enzyme reactions. The authors believe that further improvement of their system should extend its scope of applications, for example through reaching lower detection limits by optimizing aptamer specificity for the target versus the reactants and test compounds.

14.4 Summary and Outlook

Although the aptamer approach to drug discovery is still in its infancy, several examples from pharmaceutical and academic research clearly illustrate its considerable potential (Famulok, 2005; Mayer and Jenne, 2004). It has been impressively demonstrated that aptamers antagonizing target function can be readily converted into small-molecule hits with similar inhibitory activities. On the one hand this

14.4 Summary and Outlook Table 14.1 Aptamer advantages for drug discovery Features of aptamers

Advantages for drug discovery

Specific inhibition of target function

Small molecule hits are likely to be inhibitors as well

High-affinity binding to most proteins

Almost unlimited target space including orphan and genomic targets

Binding mode different from natural binding partner

Small molecules potentially exhibit a novel mode of action

Directed against specific epitopes

Targeting of functional domains, active sites, specific conformations, allosteric sites

Selected in vitro

Fast development of screening assays

Production by chemical synthesis

Consistently high batch quality and low cost of reagents when produced in small scale

Site-specific labeling by phosphoramidite chemistry

Integration in all common assay formats

can be achieved by employing aptamers as substitute interaction partners for protein targets in biochemical screening assay. On the other hand, aptamers can serve as sensitive reporters of enzyme inhibition by translating product formation into easily detectable signals. Aptamers offer numerous advantages for drug discovery (see Table 14.1). Being chemicals rather than biologicals, any reporter groups and labels can be introduced into aptamers at specific sites during synthesis. Importantly, labeling usually does not interfere with protein binding, meaning aptamer screening probes retain their high affinity and specificity for their targets. This great flexibility allows aptamers to cover a large spectrum of common read-out formats used in HTS. Above all, aptamer-based screening assays only need short development times, require no labeling of targets or compounds, and are characterized by their excellent reproducibility. Aptamers have the potential to streamline drug discovery by linking it directly with target validation: aptamers that block protein function can be readily converted into small-molecule lead compounds. In principle, aptamer-based drug discovery is applicable to almost any target protein but may be especially advantageous in such cases where other methods have proved too laborious or shown little promise. This applies in particular to those targets considered “difficult” or “non-druggable,” such as protein–protein interactions and orphan receptors. The examples reviewed here are intended to demonstrate how aptamers and aptazymes can be adapted to solving different kinds of problems, in drug screening and rational drug design.

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Acknowledgments

I thank Mira Graettinger and Michael Blind of NascaCell for their assistance and helpful advice in preparing the manuscript, and Matthias Mohr of Tripos for providing Fig. 14.4.

References Bacher, J.M., Ellington, A.D. (1998). Nucleic acid selection as a tool for drug discovery. Drug Discov Today 3, 265–273. Breaker, R.R. (2004) Natural and engineered nucleic acids as tools to explore biology. Nature 432, 838–845. Bridonneau, P., Chang, Y.F., Buvoli, A.V., O’Connell, D., Parma, D. (1999). Site-directed selection of oligonucleotide antagonists by competitive elution. Antisense Nucleic Acid Drug Dev 9, 1–11. Burgstaller, P., Jenne, A., Blind, M. (2002). Aptamers and aptazymes: accelerating small molecule drug discovery. Curr Opin Drug Discov Devel 5, 690–700. Famulok, M. (1999). Oligonucleotide aptamers that recognize small molecules. Curr Opin Struct Biol 9, 324–329. Famulok, M., (2005). Allosteric aptamers and aptazymes as probes for screening approaches. Curr Opin Mol Ther 7, 137–143. Feigon, J., Dieckmann, T., Smith, F.W. (1996). Aptamer structures from A to zeta. Chem Biol 3, 611–617. Floege, J., Ostendorf, T., Janssen, U., Burg, M., Radeke, H.H., Vargeese, C., Gill, S.C., Green, L.S., Janjic, N. (1999). Novel approach to specific growth factor inhibition in vivo: antagonism of platelet-derived growth factor in glomerulonephritis by aptamers. Am J Pathol 154, 169–179. Geiger, C., Nagel, W., Boehm, T., van Kooyk, Y., Figdor, C.G., Kremmer, E., Hogg, N., Zeitlmann, L., Dierks, H., Weber, K.S., Kolanus, W. (2000). Cytohesin-1 regulates beta2 integrin-mediated adhesion through both ARF-GEF function and interaction with LFA-1. EMBO J 19, 2525–2536. Good, P.D., Krikos, A.J., Li, S.X., Bertrand, E., Lee, N.S., Giver, L., Ellington, A., Zaia, J.A., Rossi, J.J., Engelke, D.R. (1997). Expression of small, therapeutic RNAs in human cell nuclei. Gene Ther 4, 45–54.

Gold, L. (2002). RNA as the catalyst for drug screening. Nat Biotechnol 20, 671–672. Graettinger, M., Bandilla, B., Klenzke, K., Rudolph, P., Faulhammer, D., Blind, M. (2003). Accelerating the drug discovery process by the conversion of aptamers into lead compounds. SBS 9th Annual Conference and Exhibition, Portland, USA, Poster Presentation. Graettinger, M., Bandilla, B., Klenzke, K., Rudolph, P., Blind, M. (2005a). Flexible and universally applicable screening assays based on aptamer inhibitors. Screening Europe, Geneva, Switzerland, Poster Presentation. Graettinger, M., Mohr, M., Wendt, B., Bandilla, B., Blank, M., Fessele, S., Blind, M., Kane P., Proske, D. (2005b). Aptamer-based approach for target validation and drug discovery: case study inflammation. ScreenTech, San Diego, USA, Poster Presentation. Green, L.S., Jellinek, D., Jenison, R., Ostman, A., Heldin, C.H., Janjic, N. (1996). Inhibitory DNA ligands to platelet-derived growth factor B-chain. Biochemistry 35, 14413–14424. Green, L.S., Bell, C., Janjic, N. (2001). Aptamers as reagents for high-throughput screening. Biotechniques 30, 1094–1110. Hartig J.S., Najafi-Shoushtari S.H., Grune I., Yan A., Ellington A.D., Famulok M. (2002). Protein-dependent ribozymes report molecular interactions in real time. Nat Biotechnol 20, 717–722. Herrmann, T., Patel D.J. (2000). Adaptive recognition by nucleic acid aptamers. Science 287, 820–825. Horn, W.T., Convery, M.A., Stonehouse, N.J., Adams, C.J., Liljas, L., Phillips, S.E., Stockley, P.G. (2004). The crystal structure of a high affinity RNA stem-loop complexed with the bacteriophage MS2 capsid: further challenges in the modeling of ligand-RNA interactions. RNA 10, 1776–1782.

References Huang, D.B., Vu, D., Cassiday, L.A., Zimmerman, J.M., Maher, L.J. 3rd, Ghosh, G. (2003). Crystal structure of NF-kappaB (p50)2 complexed to a high-affinity RNA aptamer. Proc Natl Acad Sci USA 100, 9268–9273. Jaeger J., Restle T., Steitz T.A. (1998). The structure of HIV-1 reverse transcriptase complexed with an RNA pseudoknot inhibitor. EMBO J 17, 4535–4542. Jenne, A., Gmelin, W., Raffler, N., Famulok, M. (1999). Real-time characterization of ribozymes by fluorescence resonance energy transfer (FRET). Angew Chem Int Ed Engl 38, 1300–1303. Kankia, B.I., Marky, L.A. (2001). Folding of the thrombin aptamer into a G-quadruplex with Sr2+: stability, heat, and hydration. J Am Chem Soc 123, 10799–10804. Lupold, S.E., Hicke, B.J., Lin, Y., Coffey, D.S. (2002). Identification and characterization of nuclease-stabilized RNA molecules that bind human prostate cancer cells via the prostate-specific membrane antigen. Cancer Res 62, 4029–4033. Mayer, G., Jenne A. (2004). Aptamers in research and drug development. BioDrugs 18, 351–359. Mayer, G., Blind, M., Nagel, W., Bhm, T., Knorr, T., Jackson, C.L., Kolanus, W., Famulok, M. (2001). Controlling small guanine-nucleotide-exchange factor function through cytoplasmic RNA intramers. Proc Natl Acad Sci USA 98, 4961–4965. Mayer, G., Graettinger, M., Blind M. (2003). Aptamers: Multifunctional tools for target validation and drug discovery. DrugPlus Int 6–10. Nutiu, R., Li, Y. (2003). Structure-switching signaling aptamers. J Am Chem Soc 125, 4771–4778. Nutiu, R., Li, Y. (2004). Structure-switching signaling aptamers: transducing molecular recognition into fluorescence signaling. Chem Eur J 10, 1868–1876. Nutiu, R., Yu, J.M., Li, Y. (2004). Signaling aptamers for monitoring enzymatic activity and for inhibitor screening. Chembiochem 5, 1139–1144. Oprea, T.I., Matter, H. (2004). Integrating virtual screening in lead discovery. Curr Opin Chem Biol 8, 349–358. Padmanabhan, K., Padmanabhan, K.P., Ferrara, J.D., Sadler, J.E., Tulinsky, A. (1993). The structure of alpha-thrombin inhibited

by a 15-mer single-stranded DNA aptamer. J Biol Chem 268, 17651–17654. Powis, G., Seewald M.J., Melder D., Hoke M., Gratas C., Christensen T.A. (1992). Inhibition of grwoth factor binding, Ca2+ signaling and cell growth by polysulfonated azo dys related to the antitumor agent suramin. Cancer Chemother Pharmacol 31, 223–228. Rajendran, M., Ellington, A.D. (2002). Selecting nucleic acids for biosensor applications. Comb Chem High Throughput Screen 5, 263– 270. Rowsell, S., Stonehouse, N.J., Convery, M.A., Adams, C.J., Ellington, A.D., Hirao, I., Peabody, D.S., Stockley, P.G., Phillips, S.E. (1998). Crystal structures of a series of RNA aptamers complexed to the same protein target. Nat Struct Biol 5, 970–975. Sakamoto, T., Oguro, A., Kawai, G., Ohtsu, T., Nakamura, Y. (2005). NMR structures of double loops of an RNA aptamer against mammalian initiation factor 4A. Nucleic Acids Res 33, 745–754. Sazani, P.L., Larralde, R., Szostak, J.W. (2004). A small aptamer with strong and specific recognition of the triphosphate of ATP. J Am Chem Soc 126, 8370–8371. Silverman, S.K. (2003). Rube Goldberg goes (ribo)nuclear? Molecular switches and sensors made from RNA. RNA 9, 377–383. Srinivasan, J., Cload, S.T., Hamaguchi, N., Kurz, J., Keene, S., Kurz, M., Boomer, R.M., Blanchard, J., Epstein, D., Wilson, C., Diener, J.L. (2004). ADP-specific sensors enable universal assay of protein kinase activity. Chem Biol 11, 499–508. Stojanovic, M.N., de Prada, P., Landry, D.W. (2001). Aptamer-based folding fluorescent sensor for cocaine. J Am Chem Soc 123, 4928–4931. Tan L, Li Y, Drake TJ, Moroz L, Wang K, Li J, Munteanu A, Chaoyong JY, Martinez K, Tan W. (2005). Molecular beacons for bioanalytical applications. Analyst 130, 1002–1005. Vassilev, L.T., Vu, B.T., Graves, B., Carvajal, D., Podlaski, F., Filipovic, Z., Kong, N., Kammlott, U., Lukacs, C., Klein, C., Fotouhi, N., Liu, E.A. (2004). In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 303, 844–848. Yu, X.G., Lichterfeld, M., Addo, M.M., Altfeld, M. (2005). Regulatory and accessory HIV-1 proteins: potential targets for HIV-1 vaccines? Curr Med Chem 12, 741–747.

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15 Aptamers as Ligands for Affinity Chromatography and Capillary Electrophoresis Applications

15 Aptamers as Ligands for Affinity Chromatography and Capillary Electrophoresis Applications Eric Peyrin

15.1 Introduction

Since the first reports of the systematic evolution of ligands by exponential enrichment (SELEX) procedure by three independent laboratories (Ellington and Szostak, 1990; Tuerk and Gold, 1990; Robertson and Joyce, 1990), the development of in vitro selection has allowed the discovery of aptamers against various targets, such as small molecules, including amino acids and nucleosides, proteins, cells, etc. (Jayasena, 1999). The use of aptamers as tools in analytical chemistry is a very promising and exciting field of research due to their ability to bind specifically the target molecules with an affinity equal or superior to those of antibodies. Aptamers present many advantages over antibodies (O’Sullivan, 2002). They can be regenerated within minutes via a denaturation–renaturation step and are, at least for DNA aptamers, relatively stable over time. The in vitro selection can be manipulated to obtain binding and kinetic properties desirable for specific assays. Aptamers can be produced by chemical synthesis with little or no batch-to-batch variation and reporter molecules can be attached to aptamers at precise locations. Furthermore, they are produced through an in vitro process which does not require animals. Taking all these background features into account, various analytical aptamer-based formats have been exploited, including ELISA-type (ELONA) assays (Ito et al., 1998), biosensors (aptasensors) (Potyrailo et al., 1998), aptazymes (Famulok, 2005), flow cytometry (Davis et al., 1996) or separation techniques. The applications of aptamers as specific ligands in affinity chromatography and capillary electrophoresis are addressed in this chapter.


15.2 Aptamers as Ligands in Affinity Liquid Chromatography (and Electrochromatography)

15.2 Aptamers as Ligands in Affinity Liquid Chromatography (and Electrochromatography) 15.2.1 General Principles of Affinity Chromatography

Affinity chromatography is rapidly becoming the separation method of choice in biochemical, biotechnology, and pharmaceutical sciences. It can be defined as a liquid chromatographic technique that makes use of a biological interaction for the separation, analysis, and purification of specific analytes within a sample. The ligand of interest is classically immobilized on a chromatographic support covalently or via a streptavidin–biotin interaction. The simplest operating scheme (direct approach) for affinity chromatography involves the injection of the sample onto the affinity column under conditions in which the target analyte will bind to the immobilized ligand with high affinity. Because of the specificity of the analyte–ligand interaction, undesirable solutes in the sample tend to have little or no affinity for the immobilized ligand. The elution buffer is then applied to dissociate the retained analyte. Alternatively, chromatographic (or flow-injection assays) based on competitive or sandwich approaches have been developed for the determination of trace analytes that do not produce a detectable signal by themselves in direct affinity chromatography. When low-to-moderate affinity ligands are immobilized to the chromatographic support, the chromatographic analysis is performed under isocratic operating conditions, allowing the elution of different analytes in relation to their respective affinities for the stationary phase. Although various ligands have been used in affinity chromatography, the most popular format uses the high-affinity and specificity of antibodies to create efficient immunoaffinity columns. However, there are some constraints that reduce the effectiveness of antibodies. The linkage of antibodies to column often results in couplings that are not uniform, leading to reduced binding capacity and can allow leaching of the antibody from the column. Furthermore, antibodies are relatively large, which limits the ligand density at the chromatographic surface. Finally, the elution conditions can be harsh, requiring extremes of pH, detergents, organic solvents, or chaotropic salts, leading to denaturation of the antibody and possibly the target (protein for example). Expected advantages of aptamers relative to antibodies for affinity chromatography include their smaller size, enabling higher density stationary phases, novel approaches for elution, and the possibility of immobilizing the ligand to a chromatographic surface at a precise location. Moreover, a number of recent reports have shown great interest in the use of immobilized aptamers (DNA or RNA) as affinity ligands in chromatography or electrochromatography. To date, these techniques have been applied to the separation/purification of proteins, separation of small molecules, or chiral resolution (Table 15.1).

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15 Aptamers as Ligands for Affinity Chromatography and Capillary Electrophoresis Applications Table 15.1 Aptamers as ligands in affinity chromatography for analyte capture and separation Targets/Species

Oligonucleotides

Separation systems

Applications

References

L-selectin

DNA

LC

Capture

Romig et al., 1999

Thrombin

DNA

Proteins CEC

Capture

Connor and McGown, in press

HCV RNA polymerase RNA and replicase

LC/chip

Capture

Cho et al., 2004; Chung et al., 2005

Non-target proteins

G-quartet DNA

CEC

Separation

Rehder and McGown, 2001; Rehder-Silinski and McGown, 2003; Dick et al., 2004

Non-target analytes

G-quartet DNA

CEC

Separation

Kotia et al., 2000; Charles and McGown, 2002; Vo and McGown, 2004

Adenosine and analogs

DNA

Nano-LC

Separation Capture

Deng et al., 2001; Deng et al., 2003

CEC

Separation

Clark and Remcho, 2003a,b

Small molecules

Flavin mononucleotide RNA and other molecules Enantiomers Vasopressin

DNA

LC Separation narrowbore column

Michaud et al., 2003

Adenosine

DNA

Micro-LC

Separation

Michaud et al., 2004

Amino acids and derivatives

DNA RNA l-RNA

Micro-LC

Separation

Michaud et al., 2004; Brumbt et al., 2005; Ravelet et al., 2005

LC, liquid chromatography; CEC, capillary electrochromatography.

15.2.2 Separation/Purification of Proteins

Drolet and co-workers (Romig et al., 1999) presented the first work concerning the use of an immobilized DNA aptamer as an affinity stationary phase. An aptamer of 36 nucleotides in length, specific and with high affinity (Kd = 2 nmol/L) for human L-selectin, was applied to the chromatographic purification of recombinant L-selectin–immunoglobulin (Ig) fusion protein from Chinese hamster ovary cellconditioned medium. The 5l-biotinylated anti-L-selectin DNA aptamer was immobilized on a streptavidin sepharose support which was packed in a short column.


Fig. 15.1 Aptamer affinity chromatography of partially purified L-selectin receptor globulin fractions. Approximately 220 mg of total protein loaded on a 1 mL aptameric column at a volumetric flow-rate of 0.75 mL/min. After washing the column with phosphate-buffered saline (PBS), L-selectin receptor globulin is eluted

from the column by a linear EDTA gradient. Because EDTA absorbs weakly at 280 nm, a slight rise in the baseline is observed for these elution profiles. Arrows indicate the initiation of the gradient. Reprinted from Romig et al. (1999) with permission.

After loading onto the aptamer affinity column, the L-selectin–Ig fusion protein was eluted under conditions that do not denature proteins: based on the known divalent cation dependence of the active tertiary structure of the aptamer, a linear EDTA gradient appeared to be very efficient to release the captured protein (Fig. 15.1). Application of the aptamer column as the initial purification step resulted in a 1500-fold purification with an 83% single step recovery, demonstrating that aptamers can be effective as affinity purification reagents. More recently, the use of a thrombin-binding DNA aptamer as a protein capture system in affinity capillary chromatography has been reported (Connor and McGown, in press). The 5l-thiol modified aptamer (15 nucleotides) was covalently attached to the inner surface of a bare fused-silica capillary via an organic linker to serve as stationary phase. After protein capture at 25 hC by incubation overnight, the bound thrombin was released using an elution scheme involving 8 mol/L urea and a capillary temperature of 50 hC. The results showed that the aptamer stationary phase was able to bind approximately three times as much thrombin as the control column (scrambled sequence oligonucleotide), in the presence or absence of human serum albumin. However, due to the low binding capacity of the opentubular capillary, only around 60 pmol of protein can be captured in this system.

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15 Aptamers as Ligands for Affinity Chromatography and Capillary Electrophoresis Applications Fig. 15.2 Schematic view of microaffinity purification process using photolytic elution method. (a) Injection of the protein mixture into the microchip packed with RNA aptamer-modified microbeads. (b) Purification of the target protein. (c) UV irradiation. (d) Analysis of the photolytically eluted protein. Reprinted from Chung et al. (2005) with permission.

Via an elegant elution strategy based on a photolytic method (Fig. 15.2), detection of hepatitis C virus (HCV) RNA polymerase and replicase from a protein mixture using a microbead affinity chromatography on a chip has been achieved (Cho et al., 2004; Chung et al. 2005). The RNA aptamer directed against the protein was coupled to the beads using a 7-fluorenylmethoxycarbonyl (Fmoc) photo-cleavable linker and the aptamer-immobilized beads were loaded and packed in a microfluidic chip composed of a transparent microchannel. A protein sample was injected onto the microchamber and incubated for 5–30 min. After washing, the microchannel was irradiated by UV light at 360 nm and the captured protein was eluted with the pumped flow of mobile phase. In the first approach for the detection of HCV RNA polymerase (Cho et al., 2004), subsequent trypsin treatment was performed and the peptide mixture was concentrated and applied to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) analysis. The detection limit of this system was estimated to be around 10 fmol of HCV RNA polymerase. In the second approach for the analysis of HCV RNA replicase (Chung et al. 2005), the protein was labeled with fluorescein and the eluted target was quantitatively detected via the fluorescence intensity measurement using a confocal microscope. It has been shown that such system was able to detect the HCV RNA replicase from a mixture containing 170 fmol of the target protein. Alternatively, McGown’s group has used the two-plane G-quartet sequence of the antithrombin DNA aptamer and an analogous four-plane G-quartet oligonucleotide of greater stability to separate various non-target proteins in open-tubular capillary electrochromatography (CEC). This approach is quite different from those reviewed previously, as the binding and specificity properties of aptamers are not stricto sensu used to discriminate the proteins. Using the same oligonucleotide covalent immobilization as that reported above (Connor and McGown, in press), the four-plane G-quartet stationary phase has been used for the separation of bovine b-lactoglobulin variants A and B (LgA and LgB), which differ by only two amino acid residues (Rehder and McGown, 2001). In control experiments (i.e. with an oligonucleotide of similar base composition but which did not form the G-quartet structure) no separation was achieved, indicating that


the folded structure plays a role in the protein discrimination. Furthermore, this approach has been extended to the separation of various bovine milk proteins (Rehder-Silinski and McGown, 2003). Both the two-plane and four-plane structures have been tested. It has been shown notably that the four-plane G-quartet displayed good resolution for the caseins while the two-plane G-quartet stationary phase did not allow complete separation of these proteins. As the experiments were carried out in the absence of stabilizing K+, the authors attributed this behavior difference to the loss of the G-quartet structure for the less stable oligonucleotide. Finally, these two stationary phases have been also evaluated in presence of 1 mmol/L of K+ to separate albumins of different species (Dick et al., 2004). It appeared that the two-plane phase behaved differently from the four-plane, with a significant resolution between albumins from different species and among the variants within a single albumin variant. Compared with the rigid four-plane structure, the two-plane structure was expected to offer greater flexibility that could facilitate interactions with proteins. All these studies have shown that there are advantages to using G-quartet DNA stationary phases for separation of proteins that exhibit only weak, non-specific interactions with DNA. 15.2.3 Separation of Small Molecules

McGown’s group has reported the use of DNA aptamers as stationary phases to separate small solutes. Using two-plane G-quartet sequences and open-tubular CEC systems similar to those described above for the separation of proteins, these authors demonstrated that some non-target small analytes such as amino acids and polycyclic aromatic hydrocarbons can also be resolved (Kotia et al., 2000). The influence of the addition of organic solvents in the mobile phase has been tested. It has been shown notably that the increase of acetonitrile to 60% improved the resolution of naphthalene and benzo-perylene. As shown for the protein separation, the G-quartet conformation was found to play a role in the separation of polycyclic aromatic hydrocarbons. In presence of KCl using a hydro-organic mobile phase (30% of methanol), naphthalene and benzo-perylene were partially resolved while no distinct or resolved peaks were observed in the absence of KCl. Both two-plane and four-plane G-quartet stationary phases have also been investigated to separate the isomeric dipeptides Trp-Arg and Arg-Trp (Charles and McGown, 2002). Although temperature studies suggested that resolution was enhanced when the G-quartet structure was partially destabilized, control experiments in which K+ was not included in the mobile phase confirmed that the folded structure nevertheless played a role in the separation (Fig. 15.3). This work has been further extended using three G-quartet-forming DNA oligonucleotides for the analysis of homodipeptides and alanyl dipeptides in CEC (Vo and McGown, 2004). It has been demonstrated notably that the replacement of G by T in the central loop of the two-plane aptamer increased the affinity of DNA

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15 Aptamers as Ligands for Affinity Chromatography and Capillary Electrophoresis Applications Fig. 15.3 Separation of non-target analytes Trp-Arg and Arg-Trp (0.5 mmol/L each) using 25 mmol/LTris, pH 7.2 as mobile phase, 15 kV voltage, 75 mm internal diameter capillaries, 25 hC. (a) Electropherogram using a capillary coated with a two-plane G-quartetforming DNA aptamer (antithrombin aptamer), with 2 mmol/L KCl in the mobile phase. (b) Capillary electrophoresis using a bare capillary, other conditions same as (a). (c) Electropherogram without KCl in the mobile phase, other conditions same as (a). Reprinted from Charles and McGown (2002) with permission.

for the dipeptide Ala-Glu, which would indicate that the site of interaction is near the central loop of the G-quartet. The authors have also proposed that the lower stability of this aptamer variant facilitated interactions with the dipeptide by giving greater flexibility to the structure. This was supported by the results for the homodipeptides, in which the resolution of Trp-Trp was better on the two-plane phase than on the more stable four-plane. Kennedy and co-workers have been the first researchers to report an immobilized aptamer that can selectively retain and separate related target compounds by weak affinity chromatography (Deng et al., 2001). Using the target specificity of the anti-adenosine DNA aptamer, these authors have described the preparation and characterization of an aptamer affinity nano-column for the analysis of adenosine and various derivatives such as NAD, AMP, ATP, and ADP. The 3l-biotinylated DNA aptamer was immobilized to the streptavidin chromatographic surfaces via a streptavidin–biotin bridge. The DNA-modified beads were packed in fused-silica capillaries of internal diameter of 150 or 50 mm. Using frontal analysis, it was found that (1) the aptamer immobilization did not alter the adenosinebinding properties of the oligonucleotide and (2) a greater surface coverage (about three-fold) was obtained relative to that classically obtained for antibodies. The various species were separated in isocratic conditions in relation to their different dissociation constant values.


The influence of the operating parameters such as particle diameter, capillary internal diameter, buffer composition, ionic strength, and mobile phase pH were evaluated to obtain optimal separation. It appeared notably that when Mg2+ was removed from the column, the affinity of all the analytes was no longer observed (except of some residual retention of adenosine), due to probably the loss of the active Mg2+-dependent tertiary structure. This aptamer affinity nano-column was further used to develop an efficient adenosine assay in microdialysis samples (Deng et al., 2003). Using an aqueous mobile phase containing 20 mmol/L of Mg2+, adenosine was strongly retained on the column. Various elution schemes have been tested to optimize the target UV detection. Although the elution by chelation of Mg2+ with EDTA would be a suitable approach, a large background disturbance was observed in the chromatograms due to the large change in EDTA concentration. So, a competitive elution with divalent cations such as Ni2+, which is presumed to complex nitrogen atoms in adenosine involved in binding to the aptamer, was preferred (Fig. 15.4). Up to 6 microL of 1.2 micromol/L adenosine could be injected onto the 150 mm (internal diameter) q 7-cm-long nano-column without loss of adenosine. The detection limit was found to be 120 fmol. This assay has been successfully applied to the determination of the adenosine concentration in microdialysis samples (without required preparation) collected from the somatosensory cortex of chloral anesthetized rats. Finally, an immobilized specific oligonucleotide ligand based on an RNA aptamer has been designed for CEC applications (Clark and Remcho, 2003a). A 5lamino-modified antiflavin mononucleotide (FMN) 35-base RNA aptamer was covalently bound to the inner walls of fused capillaries and used in open-tubular CEC and flow-counterbalanced open-tubular CEC. As the three-dimensional structure of this oligonucleotide is known to be stabilized by the presence of divalent cations, the influence of Mg2+ on the retention of FMN and flavin adenine dinucleotide (FAD) was evaluated. The retention increased for FAD between 0 and 0.5 mmol/L, while the affinity of FMN for the immobilized ligand exhibited a maximum at 0.2 mmol/L. The authors have proposed that the increase to higher

Fig. 15.4 Chromatogram illustrating gradient elution of 1.2 mmol/L adenosine (2 mL injected) from an anti-adenosine DNA aptamer nanocolumn 70 q 0.15 (i.d.) mm. Mobile phase A consists of 20 mmol/LTris, 20 mmol/L NaCl, 20 mmol/L MgCl2 at pH 6.6 and mobile phase B consists of 20 mmol/LTris, 20 mmol/ L NaCl, and 20 mmol/L NiCl2 at pH 3.45. The gradient is 2% B from 0 to 72 s with a linear increase to 90% B from 72 to 180 s. Reprinted from Deng et al. (2003) with permission.

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concentration of divalent cations may shield the phosphate group of FMN so that the interaction between this group and a guanine present in the aptamer was lessened. This aptameric column has been further studied to evaluate its ability to discriminate between the target and other molecules which do not contain the flavin moiety recognized by the aptamer (Clark and Remcho, 2003b). It has been shown notably that FMN and anthracene can be separated in this system. However, the well-known inherent instability of RNA, which is significantly higher than that of DNA due to the ability of the 2l-hydroxyl groups to act as intramolecular nucleophiles in both base- and enzyme-catalyzed hydrolysis, is expected to be the major drawback for use of such RNA stationary phase. Unfortunately, the stability of the immobilized ligand over the time was not evaluated in detail in this study. 15.2.4 Target-specific Chiral Separation

For chiral compounds, the efficient monitoring of the selection procedure has allowed in most cases a very high specificity, exemplified by the ability of the aptamer to bind the target enantioselectively. The chiral discrimination properties of aptamers selected against a target enantiomer have been used by our group to create a new class of target-specific chiral stationary phases. First, the enantiomers of arginine-vasopressin were separated using an immobilized 55-base DNA aptamer known to bind stereopecifically the d-enantiomer of the oligopeptide (Michaud et al., 2003). Immobilization was achieved using the biotin–streptavidn interaction, as previously described by Kennedy and co-workers (Deng et al., 2001, 2003). The influence of various parameters (such as column temperature, eluent pH, and salt concentration) on the l- and d-peptide retention has been investigated in order to provide information about the binding mechanism and then to define the utilization conditions of the aptamer column. Very important apparent enantioselectivity was observed, the non-target enantiomer not being retained by the column. More, it has been shown by thermodynamic analysis that both dehydration at the binding interface, charge–charge interactions, and adaptive conformational transitions contributed to the specific d-peptide– aptamer complex formation. Furthermore, it was established that the aptamer column was stable over an extended period of time. In further work, this approach has been extended to the chiral resolution of small molecules of biological interest (Michaud et al., 2004). The DNA aptamers used were selected against the d-adenosine and l-tyrosinamide enantiomers. An apparent enantioseparation factor of around 3.5 (at 20 hC) was observed for the anti-d-adenosine aptamer chiral stationary phase, while very high enantioselectivity was obtained with the immobilized anti-l-tyrosinamide aptamer. This allowed baseline resolution to be attained even at a relatively high column temperature. The anti-d-adenosine aptameric stationary phase can be used for 2 months without loss of selectivity, while some performance degradation was observed for the anti-l-tyrosinamide column over this period.


Fig. 15.5 (A) Chromatograms for the resolution of arginine using an anti-l-arginine d-RNA chiral stationary phase. Amount of d-, l-arginine injected: (a) 10 ng and (b) 100 ng. Column 370 q 0.76 (i.d.) mm; mobile phase phosphate buffer 25 mmol/L, NaCl 25 mmol/L, MgCl2 5 mmol/L, pH 7.3; column temperature 4 hC; injection volume 100 nL; flow rate 50 mL/min; detection at 208 nm. (B) Chromatogram for the

resolution of arginine using an anti-d-arginine l-RNA chiral stationary phase. Column 370 q 0.76 (i.d.) mm; mobile phase phosphate buffer 25 mmol/L, NaCl 25 mmol/L, MgCl2 5 mmol/L, pH 7.3; column temperature 4 hC; amount of d-, l-arginine injected 50 ng; injection volume 100 nL; flow rate 50 mL/min; detection at 208 nm. Reprinted from Brumbt et al. (2005) with permission.

Most of the aptamers reported in the literature are related to RNA sequences (70% of aptamers are RNAs). The ability of RNA aptamers to bind targets with very high stereoselectively has also been observed (Geiger et al., 1996). Thus, the enantioselective properties and the stability of an anti-l-arginine d-RNA aptamer target-specific chiral stationary phase have been tested (Brumbt et al., 2005). It was found that this immobilized ligand was very quickly degraded by RNases under usual chromatographic utilization and storage. In order to overcome this severe limitation for a practical use, it appeared fundamental to develop a RNA molecule intrinsically resistant to the classical cleaving RNases. A very interesting strategy involving the mirror-image approach has been successfully developed to design biostable l-RNA ligands (Spiegelmers) for potential therapeutic or diagnostic applications (Klussmann et al., 1996). As the structure of nucleases is inherently chiral, the RNases only accept a substrate in the correct chiral configuration (i.e. the “natural” d-oligonucleotide). Thus l-oligonucleotides are expected to be resistant to naturally occurring enzymes. This concept has been successfully

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applied to create a biostable RNA chiral stationary phase. It was demonstrated that a chiral stationary phase based on l-RNA, that is the mirror-image of the “natural” d-RNA aptamer, was stable for an extended period of time (about 1600 column volumes of mobile phase) under usual chromatographic conditions of storage and experiments. In addition, as expected from the principle of chiral inversion (i.e. the mirror-image of the “natural” aptamer recognizes with the same affinity and specificity the mirror image of the target), d-arginine interacted with the l-RNA stationary phase while l-arginine was not significantly retained by the column. This was responsible for the reversed elution order of enantiomers relative to that obtained using the various d-RNA columns (Fig. 15.5). Finally, we recently reported for the first time an aptamer-based chiral stationary phase which was able to resolve racemates of not only the target but also various related compounds (Ravelet et al., 2005). The enantiomers of tyrosine and analogs (11 enantiomeric pairs) were separated using an immobilized anti-tyrosine-specific l-RNA aptamer (Fig. 15.6). It was also found that the immobilized RNA aptamer could be used under hydro-organic mobile phase conditions without alteration of the stationary phase stability (about 3 months of experiments).

Fig. 15.6 Chromatographic resolution of (a) tryptophan, (b) 2-quinolyl-alanine, (c) N-acetyltryptophan, and (d) l-methyl-tryptophan using a anti-d-tyrosine l-RNA aptamer chiral stationary phase. Column 350 q 0.76 (i.d.) mm; mobile phase Tris–HCl buffer 8 mmol/L, NaCl

25 mmol/L, MgCl2 5 mmol/L, pH 7.4; column temperature 10 hC; injected concentration 0.50 mmol/L; injection volume 100 nL; flow rate 15 mL/min; detection at 220 nm. Reprinted from Ravelet et al. (2005) with permission.

15.3 Aptamers as Ligands in Affinity Capillary Electrophoresis

15.3 Aptamers as Ligands in Affinity Capillary Electrophoresis 15.3.1 General Principles of Affinity Capillary Electrophoresis

Capillary electrophoresis (CE) may be carried out at high field strengths and provides highly efficient separation of a wide range of analytes. Affinity CE can be defined as electrophoretic separation where the separation patterns are influenced by molecular binding interactions taking place during the separation process. Whereas many of the affinity techniques have been adapted to CE, one area of great importance is the characterization of affinity interactions and the quantification of analyte concentration. Ligands may interact with the analyte before and/or during electrophoresis. The analysis of preincubated target–ligand mixtures is an approach that is especially useful for quantitative measurements of analytes that form high-affinity complexes with the ligand. Such CE assays are based on separating the analyte–ligand complexes from the free analyte and free ligand by CE. Both competitive and non-competitive (direct) assays can be used. In the competitive assay, the analyte is labeled and competes with the unlabeled solute in the sample for binding to a limited amount of the corresponding ligand. A CE separation of the mixture produces two distinct peaks corresponding to the free labeled analyte and the labeled analyte bound to the ligand, allowing the quantification of the target. In the direct assay, the ligand is labeled and added at a constant concentration to the sample for binding the target. Detection and quantification of the complex peak or free ligand peak is related to the amount of the analyte in solution. Nearly all CE assays rely on laser-induced fluorescence detection because of the sensitivity and selectivity of detection. Various interacting systems have been studied by CE using proteins, antibodies, or double-stranded DNAs as specific ligands. CE assays based on the formation of antigen–antibody complexes are the most frequently reported format. In principle, direct assays possess several advantages over competitive assays, including a larger dynamic range, detection limits that are less dependent upon binding constant between the analyte and the ligand, and the ability to distinguish between cross-reactive species. However, antibodies present some drawbacks for application in such a non-competitive approach. They are electrophoretically heterogeneous and do not migrate as a single sharp peak. Moreover, the fluorescent label may interfere with binding if it is too close to the binding site. On the other hand, aptamers possess several advantages over antibodies that render them especially valuable in direct affinity CE. They are simple to label fluorescently, and they possess predictable electrophoresis properties and a relatively low molecular mass, which simplifies the separation of complex from free aptamer. At the present time, aptamers have been used in affinity CE in a direct approach for the quantification of protein and determination of binding parameters (Table 15.2).

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15 Aptamers as Ligands for Affinity Chromatography and Capillary Electrophoresis Applications Table 15.2 Aptamers as ligands in affinity capillary electrophoresis for protein quantification Proteins

Oligonucleotides

Particular methodologies

References

IgE

DNA

–

German et al., 1998; Buchanan et al., 2003

HIV type 1 reverse transcriptase

DNA

–

Pavski and Le, 2001

Focusing

Wang et al., in press

Thrombin

DNA

–

German et al., 1998; Buchanan et al., 2003

Stabilizing PEG

Huang et al., 2004

NECEEM

Berezovski et al., 2003

Competitive

Huang et al., 2004

Antithrombin III

DNA

PEG, polyethylene glycol; NECEEM, non-equilibrium capillary electrophoresis of the equilibrium mixture.

15.3.2 Affinity Capillary Electrophoresis for Target (Protein) Quantification

Kennedy and co-workers first reported the use of aptamers as ligands in affinity probe CE (German et al., 1998). A DNA aptamer directed against IgE was labeled with fluorescein and used as a specific fluorescent tag for the quantification of IgE using laser-induced fluorescence detection. Aptamer solutions and the target were mixed and incubated for at least 3 min and then injected into the separation capillary hydrodynamically. In order to prevent complex dissociation during the electrophoretic run, the analysis time was reduced to less than 1 min, applying vacuum to the outlet and using a separation distance of just 7 cm. Using fluorescein as internal standard, IgE can be detected with a dynamic linear range of 105 and a detection limit of 46 pmol/L. It should be noted that the dissociation constant can also be easily calculated from the free labeled aptamer peak via this methodology. It was found that the assay was highly specific and can be conducted in complex samples such as human serum without significant interferences (Fig. 15.7). This suggests that such assays could have broad utility, including clinical applications. Another study performed by the same group has evaluated the effects of buffer, electric field, and separation time on the detection of aptamer–ligand complexes (Buchanan et al., 2003). The results showed that the best conditions for the detection of the complexes involved the use of the minimal column length and electric field necessary to achieve separation. A similar CE assay has been reported for the determination of HIV type 1 reverse transcriptase (HIV-1 RT) using specific DNA aptamers labeled with 5l-car-

15.3 Aptamers as Ligands in Affinity Capillary Electrophoresis Fig. 15.7 Determination of IgE in serum using an anti-IgE DNA aptamer by a non-competitive affinity capillary electrophoresis method. Electropherograms obtained for samples prepared in reconstituted human serum. Each sample contains a final concentration of 300 nmol/L of labeled aptamer (A*) and 0, 100, and 400 nmol/L IgE in (a)–(c), respectively. Reprinted from German et al. (1998) with permission.

boxyfluorescein (Pavski and Le., 2001). The assay was capable of quantifying up to 50 nmol/L HIV-1 RT and was not affected by the presence of other reverse transcriptases. Recently, this CE assay has been further improved by this group using a DNA-driven focusing procedure (Wang et al., in press). The key for focusing is to establish electrophoresis conditions under which the mobility of the DNA–protein complex is between those of the electrolytes in the sample and in the running buffer. This has been achieved by using different anions in the sample and in the running buffer, which have different mobilities. The Tris–glycine buffer was used as the running buffer as glycine is characterized by low mobility while acetate of higher electrophoretic mobility was used in the sample. It was shown that the separation efficiency of the aptamer–HIV-1 RT complex reached 5 million theoretical plates/m and the sensitivity for the detection was enhanced by 70–120-fold. CE assays have been also performed using an antithrombin aptamer (German et al., 1998; Buchanan et al., 2003). However, as this thrombin aptamer is a significantly weaker binder than the IgE and HIV-1 RT aptamers (dissociation constant Kd of Z200 nmol/L versus Kd of Z10 nmol/L or 1–2 nmol/L), a significant loss of the complex during the electrophoretic process was observed, allowing a low detection limit (40 nmol/L). In order to overcome this severe limitation for unstable target–aptamer complexes, Tan and co-workers proposed the use of a relatively long capillary so that no interference occurs between the free labeled aptamer and the aptamer–thrombin peaks (Huang et al., 2004). In this case, the aptamer–thrombin complex would almost completely decay during the long analysis time, which was well suited for detection based solely on changes in the free aptamer peak. The increase in thrombin in the sample was then reflected by a large decrease in the free labeled apatmer. As this methodology allowed the linear inactive aptamer conformer peak to be differentiated from the folded G-quadruplex

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15 Aptamers as Ligands for Affinity Chromatography and Capillary Electrophoresis Applications Fig. 15.8 Determination by affinity capillary electrophoresis of thrombin and antithrombin III using an antithrombin DNA aptamer and a PEG-containing sample matrix. Each sample contained a final concentration of 200 nmol/L aptamer and (a) 0, (b) 50, and (c) 200 nmol/L thrombin. In (e)–(g), each sample contained a final concentration of 200 nmol/L aptamer, 200 nmol/L thrombin, and (e) 50, (f) 100 and (g) 200 nmol/L antithrombin III. (d) and (h) are the calibration curves constructed with various concentrations of thrombin and antithrombin III, respectively. The sample matrix consisted of 10 mmol/L Tris–HCl, 15 mmol/L KCl, and 2% PEG at pH 8.4. The electrophoresis buffer was 10 mmol/LTris–HCl and 15 mmol/L KCl at pH 8.4. Reprinted from Huang et al. (2004) with permission.

active conformer peak, the limit of detection was improved four-fold (10 nmol/L versus 40 nmol/L) relative to the previously reported data (German et al., 1998). Another approach reported by the same authors was to stabilize the weak aptamer–thrombin complex via the addition of soluble linear polymer (polyethylene glycol, PEG) to the electrophoretic buffer (Huang et al., 2004). Such polymers are known to promote the stabilizing cage effect, retard the dissociation step, and increase local concentrations of analytes. Using a short coated capillary, the addition of 2% PEG allowed the aptamer–thrombin peak to be detected with low interference of the free aptamer peak (Fig. 15.8). A detection limit of 10 nmol/L was also obtained using the stabilizing effect of PEG. On the basis of competitive experiments, this CE assay approach has been extended to the quantification of antithrombin III (Huang et al., 2004). As shown in Fig. 15.8, the increasing concentration of antithrombin III caused a decrease of the aptamer–thrombin complex associated with an increased peak of free aptamer. This confirmed an earlier report that demonstrated that the binding of antithrombin III to thrombin induced a conformational change in thrombin that rendered the analyte binding to the aptamer unstable (Fredenburgh et al., 2001). Finally, Krylov and co-authors have proposed a new method that allows the use of low-affinity aptamers as affinity probes in quantitative analysis of proteins (Berezovski et al., 2003). This is based on the non-equilibrium capillary electro-

15.3 Aptamers as Ligands in Affinity Capillary Electrophoresis

Fig. 15.9 Electropherograms generated by the non-equilibrium capillary electrophoresis of the equilibrium mixture (NECEEM) of thrombin and antithrombin DNA aptamer. Peak 1 corresponds to the equilibrium fraction of free aptamer (A1 area). Exponential part 2 corresponds to the equilibrium fraction of the complex (A2 area). From the A1 and A2 areas,

the dissociation constant Kd between the protein and the aptamer can be easily determined so that target quantification is obtained. The inset illustrates fitting of experimental data (black line) with the single-exponential function (red line). Reprinted from Berezovski et al. (2003) with permission.

phoresis of the equilibrium mixture (NECEEM) of a protein with its labeled aptamer. Generally, NECEEM generates an electropherogram with three characteristic features: two peaks that correspond to the free aptamer and protein–aptamer complex and an exponential curve, which is ascribed to the complex decaying during separation. The NECEEM method has been applied to the analysis of thrombin using its specific aptamer. The aptamer was indirectly labeled via an additional 16-mer oligonucleotide sequence that was associated to a complementary 15-mer sequence labeled with fluorescein. Using a relative long analysis time, the NECEEM electropherograms of the thrombin–aptamer mixtures showed a first peak corresponding to the free aptamer and an exponential curve associated with the monomolecular decay of the complex; the second peak, which should correspond to the thrombin–aptamer association, was not detected due to the complete dissociation of the complex (Fig. 15.9). From the areas of the first peak (A1) and the exponential part (A2), the dissociation constant was calculated and the unknown concentration of the protein was classically derived from this value. Using this approach, the detection limit of thrombin was found to be 60 nmol/L, comparable to that reported previously for the equilibrium method. Such an NECEEM-based method appears to be of interest because it would be universally applicable to the analysis of proteins, even when the aptamer forms an unstable complex with its target.

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15.4 Concluding Remarks

Although DNA and RNA aptamers have been exploited successfully in the separation, purification, and quantification of analytes using chromatography or capillary electrophoresis techniques, it some major drawbacks still remain that could limit broad practical applications in routine analysis. As reported above, the well-known role of nucleases in RNA aptamer degradation is problematic, notably in chromatography or electrochromatography, where the stationary phases have to be reusable and then stable over time. This has been resolved by applying the mirror-image strategy (Klussmann et al., 1996), with special interest in the chiral separation field (Brumbt et al., 2005). Alternatively, the substitution of the 2l-hydroxyl on the ribose by a more stable functional group such as 2l-O-methyl or 2l-fluoro may protect such RNA aptamer ligands. The stationary phases in high-performance liquid chromatography (HPLC) or CEC are commonly characterized by a limited binding capacity, due possibly to the presence of inactive/different conformers at a given temperature, and slow mass transfer kinetics related, at least in part, to the target binding-dependent conformational changes of the ligand (Michaud et al., 2003; Brumbt et al., 2005). This makes the binding properties of the immobilized aptamer highly concentration dependent, with severe limitations of the efficiency performances and peak shape. This is exemplified in Fig. 15.5A for the separation of the arginine enantiomers on an anti-d-arginine RNA chiral stationary phase. In addition, due to the relatively high cost of the aptamers, the applications have been limited to miniaturized systems in most cases, including chips, capillary electrophoresis, and micro/nano HPLC. These constraints probably preclude, at least at the present time, any applications at the preparative level. Finally, another major problem is related to the SELEX methodology. Although highly efficient, this requires highly sophisticated equipment, expensive reagents, and can be relatively time-consuming. Furthermore, the number of aptamers directed against small molecules of importance such as the drugs is relatively restrained at this time. However, improved methods of selection have been recently developed (Murphy et al., 2003; Mendosa and Bowser, 2005) which could allow rapid generation of new aptamers of great interest for the development of efficient separation analytical tools in the pharmaceutical or biochemical fields.

References

References Berezovski, M., R. Nutiu, Y. Li, S. N. Krylov (2003). Affinity analysis of a protein-aptamer complex using nonequilibrium capillary electrophoresis of equilibrium mixtures. Anal Chem 75, 1382–1386. Brumbt, A., C. Ravelet, C. Grosset, A. Ravel, A. Villet, E. Peyrin (2005). Chiral stationary phase based on a biostable L-RNA aptamer. Anal Chem 77, 1993–1998. Buchanan, D. D., E. E. Jameson, J. Perlette, A. Malik, R. T. Kennedy (2003). Effect of buffer, electric field, and separation time on detection of aptamer-ligand complexes for affinity probe capillary electrophoresis. Electrophoresis 24, 1375–1382. Charles, J. A. M., L. B. McGown (2002). Separation of Trp-Arg- and Arg-Trp using Gquartet-forming DNA oligonucleotides in open-tubular capillary electrochromatography. Electrophoresis 23, 1599–1604. Cho, S., S. Lee, W. Chung, Y. Kim, Y. Lee, B. Kim (2004). Microbead-based affinity chromatography chip using RNA aptamer modified with photocleavable linker. Electrophoresis 25, 3730–3739. Chung, W., M. Kim, S. Cho, S. Park, J. Kim, Y. Kim, B. Kim, Y. Lee (2005). Microaffinity purification of proteins based on photolytic elution: toward an efficient microbead affinity chromatography on a chip. Electrophoresis 26, 694–702. Clark, S. L., V. T. Remcho (2003a). Electrochromatographic retention studies on a flavin-binding RNA aptamer sorbent. Anal Chem 75, 5692–5696. Clark, S. L., V. T. Remcho (2003b). Open tubular liquid chromatographic separations using an aptamer stationary phase. J Sep Sci 26, 1451–1454. Connor, A. C., L. B. McGown. Aptamer stationary phase for protein capture in affinity capillary chromatography. J Chromatogr A (in press). Davis, K. A., B. Abrams, Y. Lin, S. D. Jayasena (1996). Use of a high affinity DNA ligand in flow cytometry. Nucleic Acids Res 24, 702– 706. Deng, Q., I. German, D. Buchanan, R. T. Kennedy (2001). Retention and separation of adenosine and analogues by affinity

chromatography with an aptamer stationary phase. Anal Chem 73, 5415–5421. Deng, Q., C. J. Watson, R. T. Kennedy (2003). Aptamer affinity chromatography for rapid assay of adenosine in microdialysis samples collected in vivo. J Chromatogr A 1005, 123–130. Dick Jr., L. W., B. J. Swinteck, L. B. McGown (2004). Albumins as a model system for investigating separations of closely related proteins on DNA stationary phases in capillary electrochromatography. Anal Chim Acta 519, 197–205. Ellington, A. D., J. W. Szostak (1990). In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822. Famulok, M (2005). Allosteric aptamers and aptazymes as probes for screening approaches. Curr Opin Mol Ther 7, 137–143. Fredenburgh, J. C., A. R. Stafford, J. I. Weitz Dagger (2001). Conformational changes in thrombin when complexed by serpins. J Biol Chem 276, 44828–44834. Geiger A., P. Burgstaller, H. von der Eltz, A Roeder, M. Famulok (1996). RNA aptamers that bind L-arginine with sub-micromolar dissociation constants and high enantioselectivity. Nucleic Acids Res 24, 1029–1036. German, I. D. D. Buchanan, R. T. Kennedy (1998). Aptamers as ligands in affinity probe capillary electrophoresis. Anal Chem 70, 4540–4545. Huang, C., Z. Cao, H. Chang, W. Tan (2004). Protein–protein interaction studies based on molecular aptamers by affinity capillary electrophoresis. Anal Chem 76, 6973–6981. Ito, Y., S. Fujita, N. Kawazoe, Y. Imanishi (1998). Competitive binding assay for thyroxine using in vitro selected oligonucleotides. Anal Chem 70, 3510–3512. Jayasena, S. D. (1999). Aptamers: an emerging class of molecules that rival antibodies in diagnostics. Clin Chem 45, 1628. Klussmann, S., A. Nolte, R. Bald, A. Erdmann, J.P. Furste (1996). Mirror-image RNA that binds D-adenosine. Nat Biotechnol 14, 1112–1115. Kotia, R. B., L. Li, L. B. McGown (2000). Separation of nontarget compounds by DNA aptamers. Anal Chem 72, 827–831.

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15 Aptamers as Ligands for Affinity Chromatography and Capillary Electrophoresis Applications Mendonsa, S. D., M. T. Bowser (2005). In vitro selection of aptamers with affinity for neuropeptide Y using capillary electrophoresis. J Am Chem Soc 127, 9382–9383. Michaud, E., E. Jourdan, A. Villet, A. Ravel, C. Grosset, E. Peyrin (2003). A DNA aptamer as a New Target-Specific Chiral Selector for HPLC. J Am Chem Soc 125, 8672–8679. Michaud, M., E. Jourdan, C. Ravelet, A. Villet, A. Ravel, C. Grosset, E. Peyrin (2004). Immobilized DNA aptamers as target-specific chiral stationary phases for resolution of nucleoside and amino acid derivative enantiomers. Anal Chem 76, 1015–1020. Murphy, M. B., S. T. Fuller, P. M. Richardson, S. A. Doyle (2003). An improved method for in vitro evolution of aptamers and applications in protein detection and purification. Nucleic Acids Res 31, e110. O’Sullivan, C. K. (2002). Aptasensors – the future of biosensing? Anal Bioanal Chem 372, 44–48. Pavski, V., X. C. Le (2001). Detection of human immunodeficiency virus type 1 reverse transcriptase using aptamers as probes in affinity capillary electrophoresis. Anal Chem 73, 6070–6076. Potyrailo, R. A., R. C. Conrad, A. D. Ellington, G. M. Hieftje (1998). Adapting selected nucleic acid ligands (aptamers) to biosensors. Anal Chem 70, 3419–3425. Ravelet, C., Boulkedid Rym, A. Ravel, C. Grosset, A. Villet, J. Fize, E. Peyrin (2005).

A L-RNA aptamer chiral stationary phase for resolution of target and related compounds. J Chromatogr A 1076, 62–70. Rehder, M. A., L. B. Mc Gown (2001). Opentubular capillary electrochromatography of bovine b-lactoglobulin variants A and B using an aptamer stationary phase. Electrophoresis 22, 3759–3764. Rehder-Silinski, M. A., L. B. McGown (2003). Capillary electrochromatographic separation of bovine milk proteins using a Gquartet DNA stationary phase. J Chromatogr A 1008, 233–245. Robertson, D. L., G. F. Joyce (1990). Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA. Nature 344, 467–468. Romig, T. S., C. Bell, D. W. Drolet (1999). Aptamer affinity chromatography: combinatorial chemistry applied to protein purification. J Chromatogr B 731, 275–284. Tuerk, C., L. Gold (1990). Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505–510. Vo, T. U., L. B. McGown (2004). Selectivity of quadruplex DNA stationary phases toward amino acids in homodipeptides and alanyl dipeptides. Electrophoresis 25, 1230–1236. Wang, H., M. Lu, X. C. Le. DNA-driven focusing for protein-DNA binding assays using capillary electrophoresis. Anal Chem (in press).

16.1 In Vivo Imaging: Modalities and Requirements

16 Aptamers for In Vivo Imaging Sandra Borkowski and Ludger M. Dinkelborg

16.1 In Vivo Imaging: Modalities and Requirements 16.1.1 Imaging Modalities

In clinical in vivo diagnostics today, several imaging modalities are used that provide different types of information (Table 16.1). Conventional imaging modalities in radiology such as computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound mainly provide anatomical information about the human body with a high spatial resolution (morphological imaging). Because of their significantly higher sensitivities, single photon emission computer tomography (SPECT), positron emission tomography (PET), or optical imaging (OI) devices allow the generation of pathophysiological information on biological processes at the cellular or even subcellular level, making use of molecular targeting principles (molecular imaging). (Fig. 16.1). PET in particular is a growing technology and new tracers and molecule classes addressing a plethora of interesting preclinical and clinical questions are currently evolving (Rohren et al., 2004). The intrinsic spatial resolution of these technologies is inferior when compared with the above mentioned morphological imaging modalities. However, the recent combi-

Fig. 16.1 Molecular targeting in imaging by single photon emission computer tomography (SPECT), positron emission tomography (PET), and optical approaches. The Aptamer Handbook. Edited by S. Klussmann Copyright c 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-31059-2

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16 Aptamers for In Vivo Imaging

CT image of a lung cancer (non-small cell lung Fig. 16.2 Computed tomography (CT), 18Ffluorodeoxyglucose (18F-FDG) positron emis- cancer) patient with lymph node metastasis. sion tomography (PET) image and fused PET– From von Schulthess (2003), with permission. Table 16.1 Comparison of imaging modalities Modality mode

Information

Spatial resolution

Blood concentration

X-Ray/CT

Anatomical

300 mm

>50 mmol/L

MRI

Anatomical

800 mm

>50 mmol/L

Ultrasound

Anatomical

500 mm

500 nmol/L

SPECT

Functional and molecular

5–10 mm

nmol/L–pmol/L

PET


2–8 mm

nmol/L–pmol/L

Optical


1–5 mm

nmol/L–pmol/L

Modified after von Schulthess (2003).

nation of the high spatial resolution of morphological imaging (CT) and the high sensitivity of molecular imaging (PET and SPECT) in one instrument (PET/CT and SPECT/CT) combines the strength of both procedures synergistically in one device. For example, in a lung cancer patient the pathophysiological information of enhanced metabolism in lymph node metastases with high localized precision can be clearly demonstrated by fusing a 18F-fluorodeoxyglucose (FDG) PET image with a CT image (Fig. 16.2) (von Schulthess, 2003). In anatomical imaging the morphological information gained by the use of imaging technologies such as CT, MRI, and ultrasound is enhanced by the application of contrast agents. The kinetics of these contrast agents is mainly determined by their physicochemical characteristics (size, charge, etc.) and the biodistribution is heavily dependent on the blood supply to the targeted organ (e.g. a tumor). The typical blood concentrations of contrast agents lie within the micromolar range. Molecular imaging approaches need a signal-generating moiety such as an radioisotope or a near infrared dye attached to a targeting agent, allowing the localization of the injected tracer by the dedicated imaging device (PET, SPECT, OI). The injected amounts of molecular imaging agents are far below those of contrast agents. Provided a high specific activity can be achieved, injected amounts below 100 mg are typically sufficient to obtain diagnostically effective images. Therefore, toxic side-effects caused by molecular targeting agents, especially in the SPECT and PET field, are not expected.

16.1 In Vivo Imaging: Modalities and Requirements

In this review we will focus on the use of aptamers for SPECT and PET. We will mainly concentrate on cancer indications, but also touch upon other diseases such as neurological disorders, infection, and inflammation. 16.1.2 Requirements for Imaging

Along with small molecules, peptides, and antibody fragments, aptamers are promising tools in molecular imaging. In research, aptamers have been successfully applied for molecular targeting of nucleotides and proteins. As well as high target specificity and affinity, appropriate chemical stability, rapid pharmacokinetics, and the exclusion of immunogenicity and toxic side-effects are prerequisites for effective imaging agents. In order to achieve an early and high signalto-noise ratio, rapid tissue penetration and a high target binding affinity are the dominant requirements as described already in the concept of “escort” aptamers (Hicke et al., 2000). In contrast to therapeutic interventions, in imaging, direct interference with the disease process such as the activation or inhibition of enzymes or other signaling pathways is not necessary. The accumulation of the radiolabeled targeting agent in the diseased organ or tissue can occur merely by binding and elimination of the unbound material via renal or hepatobiliary excretion. In general, rapid tissue penetration and target accumulation combined with fast excretion is preferred for SPECT and PET applications, which mainly use isotopes with a short half-life, like 99m Tc (t⁄2 = 6 h) for SPECT and 18F (t⁄2 = 2 h) for PET. Therefore, high signal-tonoise ratios at early time points are needed in routine nuclear medicine diagnosis. Important parameters in this regard are high tumor-to-blood ratios, tumor-to-normal tissue ratios, and urinary excretion that occurs more rapidly than hepatobiliary clearance (see Table 16.2 for optimal properties of diagnostic tracers in oncology). In contrast to therapeutic applications, in diagnostic imaging a high concen1

1

Table 16.2 Optimal properties of in vivo targeting agents in radiodiagnostics of cancer

In vitro characteristics Pharmacokinetics in mice

Property

Parameters (examples)

High affinity to the target

Ki or Kd in nanomolar range

High specificity to the target

Specific blocking by i70%

Rapid uptake in target tissue

Maximum tumor uptake within 15 min

Accumulation and retention in the target tissue

Wash-out from tumor I 50% within 1 h

Low non-target tissue retention

Tumor-to-tissue ratios i5

Rapid blood clearance

Tumor-to-blood ratios i10,

High urinary excretion

>70% ID in the urine

345

346

16 Aptamers for In Vivo Imaging Table 16.3 Tumor and blood uptake (in % ID/g) and tumor-toorgan ratios of 99mTc-and 125I-labeled TTA1 (5 h post injection) in comparison to 125I-anti-TN-C-IgG1 (4 h post injection) in U251 tumor-bearing mice (n = 3; e SD) 99m

125

Tumor (% ID/g)

0.45 e 0.46

1.18 e 0.17

9.12 e 0.64

Blood (% ID/g)

0.05 e 0.01

0.46 e 0.14

17.88 e 1.00

T/Spleen

5.40 e 5.71

4.95 e 1.75

2.11 e 0.47

T/Liver

2.36 e 1.79

1.53 e 0.24

1.84 e 0.27

T/Kidneys

2.81 e 2.83

2.10 e 0.05

2.14 e 0.23

T/Lung

7.14 e 7.91

3.52 e 1.37

0.74 e 0.10

Tc-TTA1 (5 h p.i.)

I-TTA1 (5 h p.i.)

125

I-anti-TN-C-IgG1 (4 h p.i.)

T/Blood

11.28 e 10.75

2.69 e 0.48

0.51 e 0.05

T/Muscle

21.76 e 15.88

20.72 e 8.10

20.67 e 7.66

T/Stomach

2.14 e 1.87

0.63 e 0.32

1.29 e 0.64

T/Intestine

0.19 e 0.22

0.42 e 0.14

5.08 e 1.17

TTA1, aptamer targeting human tenascin-C (TN-C).

tration of a radiolabeled aptamer at the target site is less important than a high signal-to-noise ratio which can even be achieved by a low tracer uptake at the diseased organ if the elimination from healthy organs and tissues occurs rapidly. Table 16.3 compares the biodistribution of 99mTc and 125I-labeled TTA1, an aptamer targeting the human matrix protein tenascin-C, with an iodinated fullsize antibody against the same target in mice. Although the antibody exhibits a much higher tumor uptake, the tumor-to-tissue ratios and especially the tumorto-blood ratio is significantly lower than those achieved with the TTA1 aptamer. However, a high number of target molecules and the efficient and rapid delivery of labeled aptamers to these targets are remaining critical factors for generating sufficient signal-to-noise ratios in imaging.

16.2 Aptamers for In Vivo Imaging 16.2.1 Oligonucleotide Properties for In Vivo Applications

Aptamers against several interesting targets, and with promising in vitro characteristics, have been generated making use of the SELEX process. However, only a few aptamers have been reported so far to be successful in in vivo imaging experiments. Since an extrapolation of in vitro data of oligonucleotides and other targeting agents to the in vivo situation is often not possible, studying the biodistribu-

16.2 Aptamers for In Vivo Imaging

tion and imaging characteristics of oligonucleotides after radiolabeling is a valuable method to characterize their in vivo behavior. This relates to both the use of oligonucleotides as tools for drug discovery as well as for development of oligonucleotides themselves as drugs. Two key properties – stability in biological fluids and systemic elimination – determine the bioavailability of aptamers. A main obstacle to the development of this compound class as drugs is still their instability against plasma endoand exonucleases. A rapid degradation of aptamers in the blood prevents target binding, leading to insufficient signal-to-noise ratios for imaging. In this respect, RNA-based aptamers were proven to be more stable than DNA-based aptamers. Post-SELEX modifications of the oligonucleotide backbone (such as phosphodiester, phosphothioate, methyldiester) or the introduction of a 2l-amino, -fluoro or -O-methyl groups on the ribose can improve the stability of oligonucleotides significantly. Appropriate positions within the aptamer sequence for these postSELEX modifications have to be identified because they can significantly influence binding affinity or the in vivo properties of the aptamer (Schmidt et al., 2004). Phosphothioate oligonucleotides exhibit high plasma protein binding and persistent liver and kidney uptake (Tavitian et al., 1998), and their metabolites were found to be excreted into the urine. In contrast 2l-O-methyl RNA or 2l-fluoropyrimidine aptamers are mainly excreted intact via the kidneys. Substitutions of nucleotides or other modifications such as increasing the molecular size by PEGylation also heavily influence the systemic clearance rate. Furthermore, the choice of the radioisotope (see also Table 16.3) as well as its chelate have an important influence on the biodistribution of radiolabeled oligonucleotides (Zhang et al., 2000 and Khnast et al., 2000). The commonly used capping of the 3l end to prevent degradation by 3l exonucleases in the plasma (Dougan et al., 1997) increases the blood stability of aptamers significantly. Other strategies to gain higher in vivo stability resulted in the synthesis of Spiegelmers, locked nucleic acids (LNAs), and peptide nucleic acids (PNAs). Due to their higher stability and rapid body clearance, PNAs are especially interesting for imaging. In addition, they can be designed to cross cell membranes (Mier et al., 2000), opening the possibility that they could be used to image intracellular targets (for review see Tung, 2000). Although stabilization of aptamers by one or more of the aforementioned strategies is a prerequisite for their use in vivo, it has to be considered that higher stability might also lead to a higher background activity leading to a low signalto-noise ratio in imaging. For example, LNA derivatives of the aptamer TTA1 have been investigated for in vivo tumor targeting after labeling with 99mTc (Schmidt et al., 2004). Although the higher plasma stability led to an increased tumor uptake in mice, the observed increased tissue background and slower renal and hepatobiliary excretion resulted in a lower signal-to-noise ratio (Table 16.4). Additionally, 2l-O-methyl and LNA backbone modifications of TTA1 shifted the murine biodistribution towards higher urinary clearance and kidney uptake, whereas the unmodified TTA1 exhibited higher intestinal uptake and more rapid fecal excretion. In conclusion, the balance between the two key properties

347

348

16 Aptamers for In Vivo Imaging Table 16.4 Tumor and blood uptake and tumor-to-organ ratios of 99mTc-labeled TTA1 and its stabilized 2l-O-methyl and locked nucleic acid (LNA) analogs in U251 tumor-bearing mice 1 hour post injection (n = 3; e SD) 99m

99m

Tc-TTA1.1 (2l-O-methyl analog)

99m

0.87 e 0.25

1.28 e 0.06

2.89 e 0.55

Tc-TTA1

Tumor (% ID/g)

Tc-TTA1.2 (LNA analog)

Blood (% ID/g)

0.21 e 0.01

0.24 e 0.04

1.03 e 0.09

T/Spleen

2.09 e 0.718

0.89 e 0.34

0.78 e 0.30

T/Liver

0.78 e 0.20

0.13 e 0.02

0.23 e 0.05

T/Kidneys

1.47 e 0.49

0.06 e 0.01

0.14 e 0.02

T/Lung

4.42 e 1.49

3.69 e 0.82

1.95 e 0.78

T/Blood

4.23 e 1.42

5.38 e 1.02

2.81 e 0.55

T/Muscle

13.91 e 2.82

8.70 e 3.50

8.56 e 4.09

T/Stomach

4.17 e 1.26

1.52 e 0.04

1.14 e 0.13

T/Intestine

0.06 e 0.02

1.16 e 0.28

4.44 e 0.78

of aptamers – stability, allowing for a sufficient targeting and body clearance, and low background activity in non-targeted organs and tissues – must be optimized for every individual aptamer. Rapid renal clearance can result in very high signalto-noise ratios early after administration, provided a specific target retention can be achieved. It has been observed in animals that aptamers have a tendency to non-specifically stick to serum proteins or cells and that they are retained in non-target tissues such as excretory organs after systemic administration. The high negative charge of the aptamer’s backbone seems to be responsible for this phenomenon, which increases the background activity during imaging. Since efficient delivery of aptamers to their target is another main obstacle of their in vivo use, several attempts have been made to improve perfusion across endothelial cell layers and enhance cellular uptake and internalization. Vectors such as cationic lipids, polyamines, or synthetic vehicles have been used to overcome the highly charged backbone of most oligonucleotides, which prevents them from binding to intracellular targets. However, successful vector applications for aptamers in imaging have not been reported. 16.2.2 Comparison of Different Classes of Targeting Agents

With approximately 10–15 kDa, aptamers have a molecular weight between peptides and antibody fragments. Compared with antibodies and antibody fragments (scFv, Fab) aptamers have several characteristics that are advantageous for imaging:

16.2 Aptamers for In Vivo Imaging x

x

x

x x

Because of their smaller size, aptamers can diffuse more rapidly into tissues and organs, leading to faster targeting (and imaging) when compared with antibodies. The lower molecular weight of aptamers can result in a shorter circulation time and faster body clearance, leading to a low background noise during imaging. The fast body clearance decreases the radiation dose (total body dose) to the patient. Aptamers do not induce immune responses. Because they are fully synthetic, the production of aptamers is less expensive.

Peptides are much smaller than aptamers and have been used for targeting cell surface proteins such as G-protein-coupled receptors. Together with small molecules they represent useful tools, especially in the PET imaging field where rapid pharmacokinetics are needed due to the short half-lives of most PET isotopes. Because of the fact that high-throughput screening of aptamer libraries is feasible by use of the SELEX process, their high affinity and specificity as well as their automated synthesis, aptamers combine the advantages of antibodies and peptides for in vivo imaging (Hicke and Stephens, 2000). Based on their molecular weight they still seem to be small enough and attractive for use in PET imaging. Although oligonucleotides seem to be excellent tools for rational drug design, their systemic therapeutic use has rarely been transferred to in vivo applications. Reasons for this situation are their inherent fragility, low bioavailability, and tendency to non-specific interactions (Younes et al., 2002). Opposite to the limitations in systemic therapeutic use, the general suitability of oligonucleotides has been proven in imaging and a growing use especially of radiolabeled aptamers in imaging is foreseen. 16.2.3 Aptamer Targets for Imaging

Medical needs in targeted imaging arise from the localization and staging of a disease, patient selection for an individualized treatment, and early therapy monitoring. Due to the negative charge of the oligonucleotide backbone, aptamers lack the ability to efficiently cross cell membranes. This inherent characteristic limits their in vivo application to extracellular targets. Therefore, aptamers for imaging applications are best suited for targeting of spatially confined extracellular disease targets. Vascular targets seem to be well suited and are often selected in indications like thrombosis, restenosis/intimal hyperplasia and angiogenesis (White et al., 2000). Also with therapy as the goal, both DNA and RNA aptamers have been raised against thrombin (Bock et al., 1992), platelet-derived growth factor (PDGF) (Leppnen et al., 2000), and vascular endothelial growth factor (VEGF) (Ruckman et al., 1998).

349

350


Making use of the SELEX process, RNA aptamers were generated binding to human L-selectin for use in imaging of inflammation (Ringquist and Parma, 1998). In vitro tests demonstrated that these aptamers preferentially recognize intracellular L-selectin with a high specificity and sensitivity. However, in vivo application have not been reported yet. Besides cardiovascular diseases, several questions in oncology (e.g. determining the aggressiveness of a tumor, predicting the best therapeutic intervention, and the early identification of those patients responding to a given treatment) can be addressed with molecular (targeted) imaging. 18F-Fluorodeoxyglucose (FDG) is a prominent tracer for PET imaging in oncological indications. Tumors are diagnosed with 18F-FDG, making use of the well-known “Warburg effect” whereby tumors have a higher glucose consumption than other tissues. However, because FDG uptake is also enhanced in inflammation, more specific tracers that are able to differentiate between tumor growth and inflammation are desired. Therefore, there is a clear need for alternative tracers from different molecular classes to overcome these limitations of FDG in tumor diagnosis and also to allow for the combination of imaging and therapy approaches with the same compound. Targets that are overexpressed in a variety of tumors such as the extracellular matrix protein tenascin-C (TN-C) are well suited for radiolabeled aptamers. The post-SELEX modified RNA aptamer TTA1, generated against human TN-C, has been successfully applied in tumor imaging in mice using SPECT (Hicke et al., in press). Another example for oncological indications are stabilized RNA aptamers that have been generated by SELEX against the extracellular domain of prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer (Lupoldt et al., 2002). Although high in vitro binding affinities in the nanomolar range have been reported, no in vivo applications have been carried out so far. Several examples of in vivo applications of oligonucleotides have been reported for both antisense DNAs and RNAs. A 99mTc-labeled antisense oligonucleotide (20-mer) was raised against CAPL, a cancer-related gene (Hjelstuen et al., 1998). Biodistribution experiments in normal mice showed a higher stability and a slower blood and organ clearance when the 3l end was capped by the MAG3 -aminohexyl chelator. Another 18-mer antisense DNA was synthesized against RIa, a subunit of protein kinase A (PKA) (Zhang et al., 2000). The biodistribution of this antisense oligonucleotide in mice was compared after labeling via HYNIC, MAG3, or DTPA chelators with 99mTc. The oligonucleotide labeled via the HYNIC approach led to significantly higher liver, spleen, and kidney uptake, as well as a high intestinal uptake compared with the MAG3 -labeled oligonucleotide (Table 16.5). It has also been demonstrated that an increased sequence length of antisense oligonucleotides not only reduces the achieved labeling yields and specific activities but also strongly influences their pharmacokinetics (Wu et al., 2000). It has to be stated that the lack of well-controlled and convincing results questions the usefulness of antisense approaches for imaging applications (for review see Duatti, 2004). Compared with antisense oligonucleotides, the use of globular extracellular binding aptamers are much more promising for in vivo imaging.

16.3 Labeling of Aptamers Table 16.5 Biodistribution (% ID/g, mean e SD) in normal mice 4 h following the injection of 99mTc labeled to DNA via three different chelators HYNIC, MAG3, or DTPA (n = 5) Organ

HYNIC

MAG3

DTPA

Liver

24.0 e 4.5

3.3 e 0.7

14.0 e 2.5

Heart

0.84 e 0.06

0.37 e 0.09

0.74 e 0.16

Kidney

30.1 e 3.0

2.5 e 0.2

6.0 e 1.0

Lung

1.5 e 0.3

0.51 e 0.07

1.0 e 0.30

Spleen

9.4 e 1.4

1.4 e 0.14

6.1 e 1.6

Stomach

0.46 e 0.08

1.2 e 1.4

0.49 e 0.04

Small intestine

1.5 e 0.3

1.1 e 0.8

0.52 e 0.09

Large intestine

1.9 e 0.4

22.2 e 9.4

0.70 e 0.06

Muscle

0.17 e 0.09

0.13 e 0.01

0.12 e 0.00

Blood

0.21 e 0.02

0.65 e 0.22

1.8 e 0.6

From Zhang et al. (2000).

16.3 Labeling of Aptamers

A general overview of labeling methods for different compound classes with both single photon emitters for SPECT and positron emitters for PET has been extensively reviewed elsewhere (Younes et al., 2002). Without influencing their binding affinities, radiolabeling of aptamers is pursued either via site-specific conjugation of a bifunctional chelator or by attaching a radiolabeled precursor. In order to establish a stable position for the radioisotope, often the 3l end of the aptamer is chosen for chelator conjugation or, if the 5l end is radiolabeled, the 3l end is simultaneously capped, preventing exonuclease activity. Radiolabeling conditions such as high temperatures, the use of organic solvents, and pH ranges between 4 and 9 can be applied, because these conditions do not harm the aptamer structure, stability or specificity. 16.3.1 SPECT Isotopes

For SPECT the isotope of choice is technetium-99m (99mTc) because of its ideal physical properties (pure gamma-emitting decay, imaging equipment-matched energy, short half-life of 6 h). The generator-based radioisotope production technology is convenient and cost-saving. Established chelators allowing for 99mTc labeling are HYNIC, MAG2 (N3S systems), or MAG3, which have already been used successfully for labeling of antisense oligonucleotides and RNA-aptamers

351

110 min

F

68

18

13.2 h 4.2 h

67.3 h 68 min 12.7 h

I I

In Ga 64 Cu

111

124

123

6.0 h

99m

Tc

Half-life

Isotope

PET

SPECT PET PET

SPECT PET

SPECT

Modality

S

N Tc

O

COO-

CH2

N

N

O

O Gl y

O

R

N

N

Gly

Tc

O

N

H

Sn(n-Bu)3

R

X

18

N N CO2H

N CO2H

CO2H

HOOC

HOOC

N N

N N

COOH

COOH

(18F) FBBA

N H

O Br

F two-step labeling of e.g. fluorobenzylbromoacetamide

F

18

HO2C

HO2C

Br

18

F

O

O

(18F)SFB

O N O

or succinimidofluorobenzoate

N H

O

or bromoacetamide

Cys

O

Labeling by metal complexation of e.g. DPTA or DOTA

O

HN

O

H

S

N

O

or peptidergic chelator

lodination of e.g. stannyl phosphoramidite

O

e.g. MAG3

Labeling/Chelator/Precursor

Table 16.6 Common SPECT and PET isotopes with physical half-lives and labeling methods

352


16.3 Labeling of Aptamers

(Table 16.6). High conjugation yields and high specific activities can be achieved in a reproducible and robust manner. Typical 99mTc-radiolabeling efficiencies reported for MAG3 and HYNIC antisense-DNA oligonucleotides were 40% and 60%, respectively with a specific activity of Z85 MBq/nmol (Zhang et al., 2000). MAG3 was also used for 3l end labeling of a 20-mer antisense oligonucleotide aminohexyl derivative. Tc-99m labeling yielded 32–35% with i90% purity and a specific activity of 14.9 GBq/mg (Hjelstuen et al., 1998). A MAG2 -amide RNA aptamer could be labeled with 99mTc with a 95% radiochemical yield and a specific activity of 37 MBq/nmol (Hilger et al., 1998). Comparable results (>95% purity, 40–80% radiochemical yield, specific activity 14.8–29.6 MBq/mg) were achieved by labeling the stabilized tenascin-C targeting RNA aptamer TTA1 and its 2l-Omethyl and LNA analogs via a MAG2 chelator (Schmidt et al., 2004). Indium-111 labeling is pursued either via attaching bifunctional DTPA or DOTA chelators to the aptamer. Iodine-123 or iodine-125 labeling of aptamers can be achieved by indirect iodination. Methods for radioiodination of stannyl-oligonucleotides (DNA aptamers against human thrombin) reported by Dougan et al. (1997) using chloramine-T, iodogen, and iodobeads resulted reproducibly in yields i90%. Specific activities of 37–74 GBq/nmol for 125I and i185 GBq/nmol for 123I were achieved with the same oligonucleotides (Dougan et al., 2003). The yields and specific activities achieved with the aforementioned labeling methods are sufficient to make use of radiolabeled antisense oligonucleotides or aptamers in preclinical and clinical SPECT imaging. 16.3.2 PET Isotopes

Examples of commonly used PET isotopes are 18F, 11C, 15O, 13N, and 68Ga. In contrast to attaching a bifunctional chelator, 18F-labeling of oligonucleotides is usually performed in two steps making use of non-carrier added methods. The first step is the radiolabeling of a synthon or a precursor designed for high reactivity and stable incorporation of halogens. In the following step the halogenated synthon or precursor is conjugated regio-selectively to the oligonucleotide (Tavitian, 2003). With regard to the short half-life of PET emitters, rapid and efficient coupling reactions are needed, taking into account the necessary purification steps. A general method of radiohalogenation using the same synthon is described for 123I, 125I, 76Br, and 18F-labeling of model oligonucleotides (9- and 18-mers) with a phosphodiester backbone (Khnast et al., 2000). The synthon halogenbenzyl-bromoacetamide was successfully labeled and conjugated to these oligonucleotides with yields of 7–13% (decay corrected). For 18F a specific activity of 111–195 MBq/nmol could be achieved. Radioiodination making use of 125I gave a yield of 35% (decay corrected) and a specific activity of 37 MBq/nmol. Biodistribution studies demonstrated that dependent on whether the oligonucleotides have been labeled with 125I or 18F, different metabolites can be observed in vivo. The group of Langstrm (Hedberg and Langstm, 1998) reported another twostep 18F-labeling method by nucleophilic substitution using succinimido-fluoro-

353

354


Fig. 16.3 Microwave-accelerated 68Ga labeling of DOTA oligonucleotides. From Velikyan et al. (2004).

benzoate as an electrophilic reagent. This approach has been successfully applied to proteins and seems to be also useful for oligonucleotides. Depending on the oligonucleotide concentration used, 18- and 20-mers with a 5l-hexylamino linker were labeled with 18F with yields between 7 and 28%. These reported yields and specific activities for 18F-labeling methods are sufficient for clinical PET imaging. The positron emitters 68Ga and 64Cu can be attached to oligonucleotides via conjugation of bifunctional chelators such as DOTA or DO3A. For example, DOTA was conjugated site-specifically to oligonucleotides (17-mers) with a hexylamino linker at the 3l or 5l end and labeled with 68Ga by microwave acceleration (Velikyan et al., 2004). The radiochemical yields ranged between 30 and 53% (decay corrected) and specific activities of 0.1–1.5 MBq/nmol were achieved independent of chemical modifications of the oligonucleotides. The use of microwaves reduced the labeling time significantly, which is crucial for short-lived PET isotopes (Fig. 16.3). An advantage of metal isotopes such as 68Ga or 64Cu over halogens is that ion complexation reactions have a simpler chemistry and allow for the production of cold kits that can provide products having high specific activities. The disadvantage of chelator approaches is related to their high molecular weight which, when they are attached to the oligonucleotides, often heavily influences the in vivo pharmacokinetics and can also disturb binding affinities, especially when applied to small oligomers.

16.4 Oligonucleotides in SPECT and PET Imaging 16.4.1 Non-targeting Aptamers

Non-invasive imaging is a convenient method to test the in vivo characteristics of aptamers and antisense oligonucleotides intended to be used for therapy or diagnostics. In this regard a lot of in vivo imaging studies have been carried out using non-targeting labeled oligonucleotides to examine general biodistribution characteristics and pharmacokinetics. For example an 18-mer RNA oligonucleotide against murine erythroleukemia virus RNA was 3l end labeled with 18F via fluorobenzyl-bromoacetamide and injected into healthy baboons for PET imaging (Tavi-

16.4 Oligonucleotides in SPECT and PET Imaging

tian et al., 1998). The influence of modifications in the sugar–phosphate backbone of the antisense oligonucleotides on pharmacokinetics was investigated. The phosphodiester (PO) was used as control, and 2l-O-methyl RNA (OM) and phosphothioate RNA (PS) were used as modifications. PO showed initial liver and kidney uptake but cleared rapidly out of these excretory organs as well as of the heart and brain. One hour after injection, all of the radioactivity was found in the bladder. The OM modification led to a rapid liver washout but cleared more slowly from heart and brain. High and increasing kidney uptake was also observed. In contrast, PS showed highest and persistent liver uptake and also increasing kidney uptake but rapid clearance from the heart and brain. It was demonstrated that the 18F-labeling procedure of the synthon had no influence on the in vivo behavior of the three oligonucleotides. The control PO differed markedly from the two backbone modifications (OM and PS), exhibiting by far the lowest plasma stability. In contrast, the 2l-O-methyl modification looked most promising in terms of stability and low liver uptake. The same group also investigated the biodistribution of non-targeted DNA and RNA Spiegelmers, which were labeled with 18F using fluorobenzyl-bromoacetamide as a prosthetic group, in baboons (Tavitian, 2003; Boisgard et al., 2005). The Spiegelmers showed high in vivo plasma stability for up to 2 h (Table 16.7), no unspecific binding or retention, and mainly excretion of the intact Spiegelmer via the urine, which is regarded as promising for imaging applications (Fig. 16.4). It remains to be investigated whether the high stability of Spiegelmers might in-

Fig. 16.4 Dynamic positron emission tomography (PET) imaging of 18F labeled l-DNA and l-RNA Spiegelmers in male baboons up to 120 min post injection. From Boisgard et al. (2005).

355

356

16 Aptamers for In Vivo Imaging Table 16.7 Metabolism of 18F-labeled RNA and DNA Spiegelmers in plasma and urine of baboons (% of radioactivity recovered as intact tracer) [18F]-L-RNA

[18F]-L-DNA

Plasma

100a (170 min)

100a (180 min)

Urine

68b

75b

a

b

From Boisgard et al. (2005). Plasma samples were collected after tracer injection at the times indicated in parentheses. Urine samples were collected 360 min after tracer injection.

crease background noise in imaging when they have a targeting function. So far, targeting Spiegelmers have not been tested for in vivo imaging. Knowledge about non-specific binding of backbone modifications in vivo with respect to normal organs is important for imaging dependent on the localization of the target. For example a high kidney or intestinal uptake would be detrimental for imaging of abdominal targets. 16.4.2 Antisense Oligonucleotides

A few in vivo imaging studies using radiolabeled antisense oligonucleotides targeting mRNA of the c-myc oncogene, CAPL, or rat chromogranin A have been reported. Although fast progress was expected in antisense imaging of especially oncogenes, the contradictory results questioned the feasibility of antisense imaging in general (Urbain, 1999). However, a promising imaging application was recently reported of an antisense oligonucleotide (15-mer) against the c-myc oncogene which was labeled with 99mTc via a 5l end-conjugated MAG3 chelator and used for SPECT imaging of atherosclerosis in rabbits. The oligonucleotide could visualize the abdominal atherosclerotic aorta as soon as 2 h post injection and achieved a target-to-background ratio of 4.2 after 4 h post injection. The significant accumulation in the aortic plaques of hyperlipidemic rabbits was shown to be specific by blocking with 400 mg/kg cold antisense oligonucleotide (Fig. 16.5) (Qin et al., 2005). Successful in vivo application for PET imaging of tumors was also recently reported using antisense PNAs (18-mers) against unr RNA (upstream of N-ras) overexpressed in MCF-7 breast tumors. These PNAs were modified by lysines at the C-terminus, making them able to penetrate cell membranes. They were labeled with 64Cu via a bispecific DOTA chelate and used for PET imaging in mice by microPET (Sun et al., 2005). The PNAs showed in general high kidney and persistent liver uptake followed by tumor, blood, and muscle uptake (Fig. 16.6). Accumulation in all other organs was low. MCF-7 tumors could be clearly imaged

16.4 Oligonucleotides in SPECT and PET Imaging

Fig. 16.5 (a) In vivo SPECT imaging of an atherosclerotic rabbit with 99mTc-labeled c-myc antisense oligonucleotide. White arrows indicate the atheroscleoritc abdominal aorta at 4 h post injection. (b) Ex vivo imaging of an

atherosclerotic rabbit aorta. Diffuse tracer accumulation is seen at sites of atherosclerotic plaques along the aortic artery 4 h post injection of 99mTc-labeled c-myc antisense oligonucleotide. From Qin et al. (2005).

by MicroPET as early as 1 h post injection (Fig. 16.7) and tumor-to-blood ratios of up to 6 were found 24 h post injection. These are the first reports of successful applications of antisense oligonucleotides in imaging. Although absolute tumor uptake of the PNAs was low, adequate images could be generated by microPET, demonstrating the usefulness of this imaging modality and technology.

357

358


Fig. 16.6 (a) Biodistribution data of 64CuDOTA-Y-PNA50-K4 conjugates in selected organs of normal mice. Data are presented as %ID/organ e SD. (b) Time–activity curves of 64Cu-DOTA-Y-PNA-K4 conjugates in MCF-7

tumor-bearing mice. Data are obtained from averaging multiple microPET image slices in the selected organs and tumor and presented as mean standardized uptake value (SUV) e SD. From Sun et al. (2005).

16.4 Oligonucleotides in SPECT and PET Imaging Fig. 16.7 MicroPET coronal image of 64 Cu-DOTA-Y-PNA50-K4 in an MCF-7 tumorbearing mouse at 1 h post injection. From Sun et al. (2005).

16.4.3 Targeting Aptamers

Only limited information about aptamer imaging can be found in the literature. For example DNA aptamers (15–29-mers) against human a-thrombin were tested for their thrombus-imaging potential. Although in vitro data looked promising, no specific uptake of the radioiodinated (123I) aptamer was observed during imaging of a rabbit jugular vein model in comparison with ovalbumin (Dougan et al., 2003). Despite the 3l end-capping with biotin the aptamers cleared rapidly in vivo. Another annealed DNA aptamer against neutrophil elastase was labeled with 99mTc and used for SPECT imaging of inflammation in a passive Arthus reaction model in rats (Charlton et al., 1997). Compared with a control IgG, the aptamer achieved a higher target-to-background ratio at an earlier point in time. However, a nonsense control aptamer also showed accumulation at the inflammation site, probably due to unspecific binding. TTA1 is an aptamer targeting the matrix protein tenascin C, which is currently characterized for tumor imaging in a variety of animal models. This RNA aptamer (39-mer) was found by SELEX to bind with high affinity to human tenascin-C, an extracellular matrix protein overexpressed during tumorigenesis (Hicke et al., 2001). TTA1 was stabilized against in vivo degradation by incorporation of 2l-fluoropyrimidines and 2l-O-methyl-purines and a 3l end-blocking by a thymidine cap. Labeling was carried out with 99mTc via a MAG2 chelator and with 68 Ga for PET imaging via DO3A. Biodistribution experiments of 99mTc-TTA1 showed rapid tumor uptake and fast excretion, resulting in favorable tumor-to-tissue ratios for imaging (Tables 16.3 and 16.4) in mice bearing subcutanous human glioblastoma (U251). High intestinal uptake with rapid excretion is reflected by the 99mTc-TTA1 image in mice visualizing a subcutanous U251 tumor in the

359

360


Fig. 16.8 (a) SPECT with digital picture and (b) PET images of TTA1 labeled with 99mTc and 68Ga respectively in U251 tumor-bearing mice.

neck (Fig. 16.8a). However, since TTA1 is specific to human tenascin-C and exhibits a 40 fold lower affinity to murine tenascin-C (Hicke et al., 2001), tumor uptake in mice is lower than it would be expected for the human situation. 99mTcTTA1 is currently in clinical trials and preliminary results demonstrate clear images of lung and breast tumors, including metastases. In contrast to 99mTc, preclinical imaging of 68Ga-DO3A-TTA1 showed high kidney and bladder accumulation in mice due to urinary excretion (Fig. 16.8b), which interferes with visualization of the U251 tumor in the flank. Nevertheless, the results of 68Ga-TTA1 in tumor-bearing mice look promising for the use of this aptamer in oncological PET imaging.

References

16.5 Outlook

Due to its high spatial resolution and the development of dedicated scanners, PET/CT has become a rapidly growing field among the imaging modalities. PET, especially if combined with CT, has gained significant importance, in particular in the oncological field. In addition to small molecules and peptides, aptamers are a promising technology in the PET field. There is continuous and increasing interest in using oligonucleotides for PET imaging, although convincing in vivo applications of this molecular class are still missing. With the existence of targeted aptamers such as TTA1, the potential of this class of molecules will further be studied in different in vivo models.

References Bock LC, Griffin LC, Latham JA, Vermaas EH, Toole JJ (1992). Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature 355, 564–566. Boisgard R, Khnast B, Vonhoff S et al. (2005). In vivo biodistribution and pharmacokinetics of 18F-labelled Spiegelmers: a new class of oligonucleotidic radiopharmaceuticals. Eur J Nucl Med Mol Imaging 32, 470–477. Charlton J, Sennello J, Smith D (1997). In vivo imaging of inflammation using an aptamer inhibitor of human neutrophil elastase. Chem Biol 4, 809–816. Dougan H, Hobbs JB, Weitz JI, Lyster DM (1997). Synthesis and radioindination of a stannyl oligodesoxyribonucleotide. Nucleic Acids Res 25, 2897–2901. Dougan H, Weitz JI, Stafford AR et al. (2003). Evaluation of DNA aptamers directed to thrombin as potential thrombus imaging agents. Nucl Med Biol 30, 61–72. Duatti A (2004). In vivo imaging of oligonucleotides with nuclear tomography. Curr Drug Targets 5, 753–760. Hedberg E, Langstrm B (1998). 18F-labelling of oligonucleotides using succinimido 4-[18F]fluorobenzoate. Acta Chem Scand 52, 1034–1039. Hicke BJ, Stephens AW (2000). Escort aptamers: a delivery service for diagnosis and therapy. J Clin Invest 106, 923–928. Hicke BJ, Marion C, Chang YF et al. (2001). Tenascin-C aptamers are generated using

tumor cells and purified protein. J Biol Chem 276, 48644–48654. Hicke BJ, Stephens AW, Gould T et al. Tumor targeting by an aptamer. J Nucl Med in press. Hilger CS, Willi MC, Wolters M, Pieken W (1998). Synthesis of Tc-99m-labeled, modified RNA. Tetrahedron Lett 39, 9403–9406. Hjelstuen OK, Tonnesen HH, Bremer PO, Verbruggen AM (1998). 3l- 99mTc-labeling and biodistribution of a CAPL antisense oligonucleotide. Nucl Med Biol 25, 651–657. Khnast B, Doll F, Terrazzino S et al. (2000). General method to label antisense oligonucleotides with radioactive halogens for pharmacological and imaging studies. Bioconj Chem 11, 627–636. Leppnen O, Janjic N, Carlsson MA, Pietras K, Levin M, Vargeese C, Green LS, Bergqvist D, Ostman A, Heldin CH (2000). Intimal hyperplasia recurs after removal of PDGFAB and -BB inhibition in the rat carotid artery injury model. Arterioscler Thromb Vasc Biol 20, E89–E95. Lupoldt SE, Hicke BJ, Lin Y, Coffey DS (2002). Identification and characterization of nuclease-stabilized RNA molecules that bind human prostate cancer cells via the prostate-specific membrane antigen. Cancer Res 62, 4029–4033. Mier W, Eritja R, Mohammed A et al. (2000). Preparation and evaluation of tumor targeting peptide-oligonucleotide conjugates. Bioconj Chem 11, 855–860.

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16 Aptamers for In Vivo Imaging Qin G, Zhang Y, Cao W et al. (2005). Molecular imaging of atherosclerotic plaques with technecium-99m-labelled antisense oligonucleotides. Eur J Nucl Med Mol Imaging 32, 6–14. Ringquist S, Parma D (1998). Anti-L-selectin oligonucleotide ligands recognize CD62Lpositive leukocytes: binding affinity and specificity of univalent and bivalent ligands. Cytometry 33, 394–405. Rohren EM, Turkington TG, Coleman RE (2004). Clinical applications of PET in oncology. Radiology 231, 305–332. Ruckman J, Green LS, Beeson J, Waugh S, Gillette WL, Henninger DD, ClaessonWelsh L, Janjic N (1998). 2’-Fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165). Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain. J Biol Chem 273, 20556–20567. Schmidt KS, Borkowski S, Kurreck J et al. (2004). Application of locked nucleic acids to improve aptamer in vivo stability and targeting function. Nucl Acids Res 32, 5757–5765. Sun X, Fang H, Li X et al. (2005). MicroPET imaging of MCF-7 tumors in mice via unr mRNA-targeted peptide nucleic acid. Bioconj Chem 16, 294–305. Tavitian B (2003). In vivo imaging with oligonucleotides for diagnosis and drug development. Gut 52 (suppl IV), iv40–iv47.

Tavitian B, Terrazzino S, Khnast B et al. (1998). In vivo imaging of oligonucleotides with positron emission tomography. Nature Med 4, 467–471. Tung CH (2000). Preparation and applications of peptide-oligonucleotide conjugates. Bioconj Chem 11, 605–618. Urbain JL (1999). Oncogenes, cancer and imaging. J Nucl Med 40, 498–504. Velikyan I, Lendvai G, Vlil M et al. (2004). Microwave accelerated 68Ga-labelling of oligonucleotides. J Label Comp Radiopharm 47, 79–89. von Schulthess GK, ed. (2003). Clinical Molecular Anatomic Imaging: PET, PET/CT and SPECT/CT. Philadelphia: Lippincott Williams & Wilkins. White RR, Sullenger BA, Rusconi CP (2000). Developing aptamers into therapeutics. J Clin Invest 106, 929–934. Wu F, Yngve U, Hedberg E, Honda M, Lu L, Eriksson B, Watanabe Y, Bergstrom M, Langstrom B (2000). Distribution of 76Brlabeled antisense oligonucleotides of different length determined ex vivo in rats. Eur J Pharm Sci 10, 179–186. Younes CK, Boisgard R, Tavitian B (2002). Labelled oligonucleotides as radiopharmaceuticals: pitfalls, problems and perspectives. Curr Pharm Des 8, 1451–1466. Zhang YM, Liu N, Zhu ZH, Rusckowski M, Hantowich DJ (2000). Influence of different chelators (HYNIC, MAG3 and DTPA) on tumor cell accumulation and mouse biodistribution of technecium-99m labeled to antisense DNA. Eur J Nucl Med 27, 1700–1707.

17.2 Aptamer Targets

17 Properties of Therapeutic Aptamers Sharon T. Cload, Thomas G. McCauley, Anthony D. Keefe, Judith M. Healy, and Charles Wilson

17.1 Introduction

Since the invention of the SELEX process and the isolation of the first protein-specific aptamers, parallels to the functional properties of monoclonal antibodies have been drawn. While antibodies have demonstrated utility in a number of settings, their development as therapeutic agents has been long and difficult and it is only in the last 5 years that the field has matured to the stage where one can point to a number of unqualified clinical successes. In many respects, the opportunities for aptamers as therapeutics continue to parallel those for antibodies although the technical challenges have been somewhat different. 2004 was a milestone year for the field with the US Food and Drug Administration (FDA) approval of pegaptanib (Macugenä). In this chapter, we review the properties of aptamers as druglike molecules, highlighting their functional capabilities as target-specific binding agents and our current understanding of how aptamers behave in vivo with respect to distribution, metabolism, and tolerability. The antibody experience provides important lessons for the development of aptamer therapeutics and points the way for future applications which will particularly favor progress with aptamers.


The collected experience of both academic and industry research suggests that aptamers are capable of binding to targets from virtually any protein class (Sullenger and Gilboa, 2002). While early efforts focused largely on nucleic acid-binding targets (e.g. Tuerk and Gold, 1990; Tuerk et al., 1992; Tuerk and MacDougal-Waugh, 1993) and heparin-binding targets (e.g. Bock et al., 1992; Green et al., 1995; Jellinek et al., 1994, 1995; Pagratis et al., 1997; Ruckman et al., 1998), subsequent applications have included a wide variety of proteins, including selectins (O’Connell The Aptamer Handbook. Edited by S. Klussmann Copyright c 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-31059-2

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17 Properties of Therapeutic Aptamers Table 17.1 Example aptamer targets and affinities Protein class

Protein

Affinity

Reference

Cytokines, chemokines, hormones and growth factors VEGF

130 pmol/L

Ruckman et al., 1998

PDGF-BB

100 pmol/L

Green et al., 1996

bFGF

350 pmol/L


KGF

3 pmol/L

Pagratis et al., 1997

NGF

200 nmol/L

Binkley et al., 1995

TGFb1

200 pmol/L


TGFb2

1 nmol/L


Ang2

3.1 nmol/L

White et al., 2003

Vasopressin

0.9 mmol/L

Williams et al., 1997

Oncostatin M

7 nmol/L

Rhodes et al., 2000

MCP-1

180 pmol/L

Rhodes et al., 2001

Neuropeptide Y

370 nmol/L

Proske et al., 2002

Angiogenin

ND

Nobile et al., 1998

hTSA

2.5 nmol/L

Lin et al., 1996

Adhesion molecules, receptors, and other cell surface proteins L-Selectin

60 pmol/L

P-Selectin

29 pmol/L

Watson et al., 2000 Jenison et al., 1998

LFA-1 (CD18)

500 nmol/L– 1 mmol/L

Blind et al., 1999

PSMA

2 nmol/La

Lupold et al., 2002

HER3

45 nmol/L

Chen et al., 2003

CD4

ND

Kraus et al., 1998

CTLA-4

Z50 nmol/L

Santulli-Marotto et al., 2003

Tenascin-C

5 nmol/L

Hicke et al., 2001

Pigpen

ND

Blank et al., 2001

Trypanosoma brucei VSD protein

160 pmol/L

Lorger et al., 2003

Trypanosoma brucei (flagellar pocket protein)

60 nmol/L

Homann et al., 1999

Trypanosoma cruzi cell surface receptor

Z100 nmol/L

Ulrich et al., 2002

17.2 Aptamer Targets Table 17.1 Example aptamer targets and affinities (continued) Protein class

Protein

Affinity

Reference

Antibodies IgE

9 nmol/L


mAb198 (anti-AChR antibody)

6 nmol/L

Lee and Sullenger, 1997

mAb G6–9 (anti-DNA antibody)

2 nmol/L

Kim et al., 2003

mAb 20 (anti-insulin receptor antibody)

30 nmol/L

Lee and Sullenger, 1996

HIV-RT

25 pmol/L

Kensch et al., 2000; Tuerk et al., 1992

HIV Rev

1 nmol/L

Tuerk and MacDougalWaugh, 1993

HIV Tat

ND

Tuerk and MacDougalWaugh, 1993

HIV gp120

5 nmol/L

Khati et al., 2003

HIV integrase

10 nmol/L

Allen et al., 1995

HCV NS5B

1.5 nmol/L

Biroccio et al., 2002

Thrombin

0.5 nmol/L– 200 nmol/L

Bock et al., 1992; Tasset et al., 1997

Factor VIIa

11 nmol/L


Viral proteins

Coagulation cascade components

Factor IXa

600 pmol/L


Activated protein C

110 nmol/L

Gal et al., 1998

Raf-1 RBD

150 nmol/L

Kimoto et al., 2002

Cytohesin 1 (Sec7 domain)

16 nmol/L

Mayer et al., 2001

ERK2

1.3 nmol/L

Seiwert et al., 2000

Complement C5

2 nmol/L

Biesecker et al., 1999

Amyloid beta 4 (Ab4)

29 nmol/L

Rhie et al., 2003

Human neutrophil elastasae (HNE)

10 nmol/L

Smith et al., 1995

Substance P

180 nmol/L

Nieuwlandt et al., 1995

Signaling mediators

Miscellaneous

a

Ki for NAALADase inhibition, not an explicit Kd measurement. ND, not determined.

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17 Properties of Therapeutic Aptamers

et al., 1996; Jenison et al., 1998; Watson et al., 2000), antibodies (Kim et al., 2003; Lee and Sullenger, 1996, 1997; Wiegand et al., 1996), peptide hormones (Lin et al., 1996), cell surface receptors (Ulrich et al., 2002; Chen et al., 2003), tumor markers (Hicke et al., 2001; Lupold et al., 2002; Daniels et al., 2003), and viral coat proteins (Khati et al., 2003), (for examples, see Table 17.1). In the following three sections, we highlight examples from the scientific literature of aptamer selections against proteins expressed at cell surfaces, against proteins expressed intracellularly, and against extracellular protein targets. 17.2.1 Cell Surface Targets

Aptamers to proteins on the surface of cells or virus particles have a variety of potential therapeutic uses. Aptamers to cell surface targets may inhibit cell–cell adhesion, viral–cell adhesion and viral entry or either agonize or antagonize receptor signaling. Additionally, aptamers targeted to cell surface antigens may be utilized as delivery agents for radionuclides or cytotoxins, which can be appended during chemical synthesis. The tumor-to-blood ratio for such therapeutics is driven by both the rate of tumor uptake and clearance for the molecules. The size of aptamers, coupled with the tunability of their half-life in plasma (e.g. Watson et al., 2000) may facilitate both rapid tumor uptake and clearance, demonstrating a potential advantage over antibodies for this application (Hicke and Stephens, 2000). Like antibodies and in contrast to small molecules, aptamers are efficient inhibitors of protein–protein interactions, as they bind with high affinity and form interactions across a broad surface on their target proteins (Green et al., 1996; Jaeger et al., 1998; Huang et al., 2003). Several embodiments of the SELEX method have been used to generate aptamers to cell surface targets. Selection may be carried out against a cell type of interest, with the aim of generating a cell type-specific diagnostic (Hicke et al., 2001; Daniels et al., 2002, 2003), or payload delivery reagents (Hicke et al., 2001; Homann and Goringer, 2001). This broad approach has the advantage that no prior knowledge of the target protein is required. For example, to identify a diagnostic reagent for tumor versus normal vasculature, Blank and co-workers conducted a selection using YPEN-1 rat glioblastoma (tumor) cells as the target (Blank et al., 2001). An aptamer that binds specifically to YPEN-1 cells but not to control N9 microglial cells was generated and then used to isolate and identify the target protein, Pigpen (Blank et al., 2001). With the aim of developing a novel therapeutic for African trypanosomiasis (African sleeping sickness), Homann and co-workers selected an aptamer to an unidentified protein in the flagellar pocket of the trypanosome surface and showed that the aptamer was efficiently endocytosed when conjugated to a mock payload, biotin (Homann and Goringer, 1999, 2001). Enrichment for aptamers to specific targets may be difficult when SELEX is conducted against whole cells, given the vast number of proteins expressed on the surface of a given cell type, many of which may have weak non-specific affinity for nucleic acids (Vant-Hull et al., 1998). However, to improve the odds of se-


lecting an aptamer to a given cell surface protein, pools can be exposed to the target protein in purified form and to cells expressing the target on their surfaces in alternating SELEX rounds. Such toggling may drive specific enrichment for aptamers to bioactive protein expressed on cells (Hicke et al., 2001; Lorger et al., 2003). This strategy was used effectively to generate aptamers to tenascin-C (TC), a marker on the surface of tumor stromal cells (Hicke et al., 2001). The most widely used method of generating aptamers to cell surface targets has been to conduct SELEX against the purified extracellular domain of a target protein (Jenison et al., 1998; Kraus et al., 1998; Lupold et al., 2002; O’Connell et al., 1996; Santulli-Marotto et al., 2003; Watson et al., 2000), such as HER3 (Chen et al., 2003) or gp120 (Khati et al., 2003). L-selectin aptamers that were selected against recombinant protein bind L-selectin on the surface of leukocytes and neutrophils, and inhibit neutrophil–neutrophil adhesion in vitro and lymphocyte trafficking in vivo (O’Connell et al., 1996; Ringquist and Parma, 1998; Watson et al., 2000). Similarly, aptamers selected using recombinant P-selectin inhibit platelet–neutrophil adhesion in vitro (Jenison et al., 1998). An aptamer selected against the extracellular domain of prostate-specific membrane antigen (PSMA), which is upregulated on the surface of prostate cancer cells, specifically binds to the surface of PSMA-expressing LNCaP cells and may be useful as a vehicle for toxin or radionuclide delivery (Lupold et al., 2002). In another interesting application, Santulli-Marotto and co-workers generated an aptamer to cytotoxic T-cell antigen 4 (CTLA-4), a receptor expressed on the surface of activated T-cells that attenuates T-cell response to antigen. This aptamer, Del60, enhanced T-cell proliferation in vitro and a tetravalent aptamer construct enhanced antitumor immunity in melanoma tumor-bearing mice (Santulli-Marotto et al., 2003). A family of aptamers to the extracellular domain of human epidermal growth factor receptor-3 (HER3), a receptor tyrosine kinase that is activated by heregulin, has been characterized. Interestingly, one aptamer, A30, is a potent inhibitor of HER3 signaling while others, A6 and A18, may modestly potentiate heregulin-dependent signaling (Chen et al., 2003). Viral surface proteins can also be targeted by aptamers. HIV-1 surface envelope glycoprotein 120 (gp120) is a critical mediator of virus entry into host cells. An aptamer to recombinant gp120 from a clinically relevant clinical isolate of HIV-1 (R5) neutralized HIV-1 infectivity in PBMCs with an IC50 of 5 nmol/L (Khati et al., 2003). 17.2.2 Intracellular Targets

Aptamers that target a variety of intracellular proteins have been generated (Tuerk et al., 1992; Allen et al., 1995; Kumar et al., 1997; Thomas et al., 1997; Blind et al., 1999; Lebruska and Maher, 1999; Shi et al., 1999; Anwar et al., 2000; Kensch et al., 2000; Mayer et al., 2001; Biroccio et al., 2002; Kimoto et al., 2002), reviewed in (Mayer et al., 2001; Burke and Nickens, 2002). Unlike antibodies, aptamers can fold properly and retain activity in the intracellular environment. An aptamer to the HIV-1 regulatory protein Rev inhibited export of unspliced reporter

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mRNAs to the cytoplasm (Symensma et al., 1996). Similarly, an aptamer to yeast RNA polymerase II expressed under the control of the polymerase III promoter reduced cell viability under conditions of reduced polymerase II expression (Good et al., 1997). Because of their high specificity and action at the protein rather than gene or transcript level, aptamers are a unique tool for assessing target function. Aptamers have been used to help elucidate intracellular contributions to cell– cell adhesion mediated by intercellular adhesion molecule-1 (ICAM-1) and LFA-1 (CD11a/CD18). Blind and co-workers generated an aptamer to a cytoplasmic domain of the integrin LFA-1 (CD18) (Blind et al., 1999). LFA-1 is thought to be activated via an intracellular signaling mechanism after stimulation of leukocytes through T-cell or cytokine receptor activation. Intracellular expression of the aptamer in T-lymphocytes reduced adhesion to immobilized ICAM-1. In a similar study, Mayer et al. (2001) showed that an aptamer to the Sec7 domain of cytohesin-1, a guanine nucleotide exchange factor thought to be involved in the activation of LFA-1, inhibited Sec7 activity in Jurkat cells and concomitantly inhibited cell adhesion to ICAM-1. In the first example of expression of a regulatory aptamer in a multicellular organism, Shi et al. (1999) expressed a multivalent aptamer to B52, a member of the SR protein family, in Drosophila melanogaster. Regulated expression of the aptamer was able to modulate the phenotypic effects that result from overexpression of B52, including loss of salivary glands. Therapeutic use of aptamers that target intracellular proteins will require the development of suitable delivery systems or chemical conjugation strategies to enhance cellular uptake of exogenously added aptamers or, alternatively, engineered aptamer expression inside cells (Ehsan et al., 2001; Famulok et al., 2001). 17.2.3 Extracellular Targets

The majority of aptamers with potential therapeutic utility selected to date target extracellular proteins. Extracellular therapeutic targets, such as growth and coagulation factors of the vasculature, have the advantage of ready access to aptamer intervention without need for enabling aptamer access to cells or tissue spaces. Broad therapeutic areas are represented among aptamers directed against extracellular targets, including angiogenesis/oncology (Green et al., 1995, 1996; Nobile et al., 1998; Ruckman et al., 1998; Lupold et al., 2002; Chen et al., 2003; White et al., 2003), inflammation (Wiegand et al., 1996; Rhodes et al., 2000, 2001), anticoagulation and thrombosis (Bock et al., 1992; Gal et al., 1998; Rusconi et al., 2004; Rusconi et al., 2000, 2002; Tasset et al., 1997), and autoimmune disease (Tsai and Keene, 1993; Doudna et al., 1995; Lee and Sullenger, 1997; Kim et al., 2003). The aptamer drug Macugen (pegaptanib) (Ruckman et al., 1998; Eyetech Study Group, 2002, 2003), which is approved for treatment of age-related macular degeneration (AMD), targets vascular endothelial growth factor (VEGF), a key mediator of angiogenesis, by inhibiting VEGF binding to the receptor tyrosine kinases KDR and Flt-1 (Ruckman et al., 1998).


VEGF blockade for treatment of colorectal cancer has been clinically validated with a monoclonal antibody (Ferrara, 2004), and one can envision a similar application for anti-VEGF aptamers such as pegaptanib. Basic fibroblast growth factor (bFGF), like VEGF, is a mediator of angiogenesis that acts through FGF receptor1 (FGFR-1), a receptor tyrosine kinase expressed on the endothelial cell surface. A high-affinity anti-bFGF aptamer inhibited receptor binding, bFGF-dependent endothelial cell migration and proliferation (Jellinek et al., 1995). Angiopoietin-2 (Ang-2) is an angiogenic factor that potentiates the pro-angiogenic activity of VEGF and bFGF on endothelial cells by acting as a Tie2 receptor tyrosine kinase antagonist (Lobov et al., 2002). White and co-workers showed that an anti-Ang-2 aptamer modulates Tie2 activity in vitro and, more significantly, inhibited bFGFinduced corneal angiogenesis in a rat model (White et al., 2003). The first aptamer of potential therapeutic value was selected against thrombin (Bock et al., 1992) soon after the advent of SELEX (Tuerk and Gold, 1990). Subsequently, inhibitors for several other components of the coagulation cascade have been generated (Tasset et al., 1997; Gal et al., 1998; Rusconi et al., 2000, 2002; Jeter et al., 2004). Complex formation between factor VIIa (FVIIa) and tissue factor (TF) in response to vascular injury initiates the extrinsic arm of the coagulation cascade. Rusconi et al. (2002) showed that an aptamer highly specific for FVIIa inhibited coagulation in vitro, most likely by blocking complex formation between FVIIa and TF. Coagulation factor IXa (FIXa) is a component of the intrinsic clotting cascade that activates factor X. Rusconi and co-workers developed an aptamer (9.3t) to FIXa that suppresses factor X activation and is a potent anticoagulant in vitro (Rusconi et al., 2002). Moreover, the activity of 9.3t can be reversed by a complementary oligonucleotide antidote both in vitro and in vivo (Rusconi et al., 2004). Notable extracellular proteins that play a role in inflammation include cytokines, chemokines, and antibodies. Wiegand et al. (1996) selected aptamers to IgE, a mediator of local inflammation which can cause allergies, atopic dermatitis, and asthma when overexpressed in atopic individuals (Sutton and Gould, 1993). Allergen-specific IgE binds to its high-affinity receptor, FceR1, on the surface of mast cells and basophils, which in turn activates the cells and results in degranulation and release of proinflammatory mediators. IgE-directed aptamers block IgE binding to FceR1, and inhibit degranulation in vitro. In another application, Rhodes et al. (2000) sought an inhibitor of oncostatin M (OSM), a potent proinflammatory cytokine that may be a key mediator of rheumatoid arthritis (PlaterZyberk et al., 2001). The authors showed that an aptamer to OSM specifically inhibited OSM receptor binding and receptor activation in vitro. The same group developed an aptamer against MCP-1, a b/CC chemokine whose levels are elevated in serum from patients with asthma and rheumatoid arthritis (Rhodes et al., 2001). Mouse MCP-1 was used as the bait for SELEX to enable testing of the resulting aptamers in murine disease models. The resulting aptamers were shown to be potent inhibitors of mouse MCP-1-dependent chemotaxis in vitro. Several studies have probed the potential of aptamers for the treatment of antibody-mediated autoimmune disease (Tsai and Keene, 1993; Doudna et al., 1995;

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Lee and Sullenger, 1996; Lee and Sullenger, 1997; Hwang et al., 2003; Kim et al., 2003). In each case, aptamers that recognize and block the activity of specific autoantibodies were generated. Extreme insulin resistance type B is mediated by autoantibodies to a main antigenic epitope on the surface of the insulin receptor (Zhang and Roth, 1991). Antibody binding to the receptor induces internalization, or downmodulation, of the receptor (Roth et al., 1983). A mouse monoclonal antibody, mAb20, which recognizes a primary antigenic epitope on the human insulin receptor, was successfully used as a SELEX target (Doudna et al., 1995; Lee and Sullenger, 1996). The resulting aptamers blocked mAb20 binding to the insulin receptor, and prevented antibody-mediated, but not insulin-mediated receptor internalization (Lee and Sullenger, 1996). Interestingly, one aptamer cross-reacted with patient autoimmune serum and was able to block receptor downregulation induced by patient serum in vitro. Myasthenia gravis (MG) is a neuromuscular disorder that results from an antibody-mediated autoimmune response to the nicotinic acetylcholine receptor (AChR) (Drachman, 1994). Hwang and co-workers selected an aptamer to a rat anti-AChR antibody, mAb198, which is cross- reactive with the human receptor and is also able to induce experimental autoimmune myasthenia gravis (EAMG) in rats (Hwang et al., 2003). The aptamer, tMG, blocked mAb198-dependent AChR downregulation on the surface of TE671 cells, and was also cross-reactive with serum from a MG patient. A polyethylene glycol (PEG)-conjugated (i.e. PEGylated) version of the aptamer, PEG-tMG, improved the clinical score in rats in the mAb198-induced EAMG model. Rats treated with PEG-tMG had higher muscle AChR content than either untreated rats, or rats treated with unPEGylated or scrambled versions of PEG-tMG. Although the results of these and other studies are intriguing, the utility of this treatment modality may actually be limited by the heterogeneity of autoimmune antibodies present in patient sera and the exquisite specificity of aptamers.

17.3 Aptamer Binding Characteristics 17.3.1 Aptamer Affinity

Aptamers bind their targets with affinities comparable to those observed for monoclonal antibodies and scFvs. As the examples in Table 17.1 indicate, aptamer:target equilibrium dissociation constants (Kd-values) range from low picomolar to mid-nanomolar, with the majority falling below 10 nmol/L, although the affinities for peptide targets are generally weaker than those for proteins (e.g. Nieuwlandt et al., 1995; Williams et al., 1997; Blind et al., 1999; Proske et al., 2002). There is no obvious correlation between the affinity of an aptamer and the class of protein it targets; very high-affinity aptamers have been generated to nucleic acid-binding proteins as well as proteins with no specific or non-specif-

17.3 Aptamer Binding Characteristics

ic nucleic acid-binding properties. In addition, simple metrics such as the isoelectric point of a protein are not necessarily adequate predictors of a target’s “aptogenicity.” For example, two of the tightest binding aptamers reported in Table 17.1 bind L-selectin and P-selectin, both of which have predicted isoelectric points lower than pH 5.5, and therefore are likely negatively charged proteins at physiological pH (Jenison et al., 1998; Watson et al., 2000; Warkentin and Greinacher, 2003). Computational modeling of variables that may contribute to high aptamer affinity suggests that the aptogenicity of a protein may be correlated with the presence of concentrated regions of positively charged amino acids in the sequence (Charles Wilson, unpublished results). The high affinities observed for aptamers likely derive from contacts between aptamer and target protein over an extended surface of both molecules (Tasset et al., 1997; Jaeger et al., 1998; Huang et al., 2003). The ability of aptamers to form well-folded stable secondary and tertiary structures also contributes to their binding affinity. Modifications at the 2l-hydroxyl position known to stabilize duplex regions by promoting a 3l-endo sugar pucker can have a marked affect on aptamer affinity (Green et al., 1995). Interestingly, two aptamers with high reported affinities are thought to form tightly compacted pseudo-knot structures (Pagratis et al., 1997; Jaeger et al., 1998; Kensch et al., 2000). For several reasons, one must proceed with caution when considering whether the affinities reported in Table 17.1 are representative of what will be observed for aptamers developed for therapeutic use. First, the methods used to measure the Kd-values vary, each has its own limitations, and sometimes the values obtained differ substantially depending on the method used (e.g. Kensch et al., 2000; Tuerk et al., 1992). More significantly, many of the aptamers described in Table 17.1 are 80–100 nt oligonucleotides hits from SELEX that have not been further optimized. The affinity of an aptamer may sometimes be improved by truncation down to the core target binding element (e.g. White et al., 2001), presumably by reducing the conformational heterogeneity accessible to the full-length sequence. Significant affinity enhancements can also be achieved by a process called biased (or doped) selection, in which the aptamer sequence (parent sequence) is used as the basis for a new starting pool and higher affinity variants of the parent molecule are generated using SELEX. Biesecker and co-workers improved the affinity of an anti-complement C5 aptamer 10-fold through biased reselection (Biesecker et al., 1999). Similarly, the binding potency of an aptamer to substance P was improved more than 30-fold using this method (Nieuwlandt et al., 1995). PostSELEX, changes to the chemical composition of the aptamer that stabilize the aptamer structure or add additional points of contact between the aptamer and protein may also improve affinity (Eaton, 1997; Eaton et al., 1997; Green et al., 1995). For example, Green et al. (1995) found that replacement of the 2l-hydroxyl moieties with helix-stabilizing 2l-O-methyl groups at a majority of the purine positions in an anti-VEGF aptamer afforded a 17-fold increase in affinity. Given these considerations, one could envision that the affinity of many aptamers carried forward through therapeutic development will be on the high end of the spectrum of affinities represented in Table 17.1.

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17.3.2 Aptamer Specificity

The specificity of a therapeutic agent is critical as it will drive both the potency of the molecule and its side effect profile. Aptamers are highly discriminating binders; the factors that contribute to their potent affinities bolster highly specific binding as well (Eaton et al., 1995). Generally, aptamers selected against a member of a family of proteins are specific for the target protein versus other family members. For example, an aptamer (14F3lT) to keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, discriminates by over 10000-fold between KGF and other FGF family members, all of which, notably, are heparin-binding proteins (Pagratis et al., 1997). In fact, aptamers against heparin-binding members of the growth factor family are without exception highly specific, as illustrated by the examples in Table 17.2. Aptamers to VEGF, PDGF-AB, KGF, and bFGF each bind their targets with high affinity and discriminate against other proteins in that set (Green et al., 1995; Jellinek et al., 1995; Pagratis et al., 1997). Similarly, aptamers to heparin-binding members of the coagulation cascade are remarkably specific for their targets versus other members of the coagulation cascade (Bock et al., 1992; Rusconi et al., 2000, 2002). This degree of discrimination is not limited to aptamers that target heparinbinding proteins. An anti-L-selectin aptamer exhibits approximately 8000- to 15000-fold and 200- to 500-fold specificity versus P-selectin and E-selectin, respectively (O’Connell et al., 1996) and a P-selectin aptamer is similarly discriminating (Jenison et al., 1998). As discussed above, specific binding is a general requirement of therapeutic aptamers. While most aptamers bind their targets specifically, discrimination between homologous targets, for example, proteins that share a subdomain, can be programmed into the aptamer through a subtractive SELEX process. Using this procedure, the undesired aptamer target is incubated with the random pool and nucleic acid:protein complexes are then partitioned and discarded. The non-binding pool members are then incubated with the desired target and binders are isolated and amplified. The pool is subjected to iterative rounds of Table 17.2 Example specificities of aptamers to heparinbinding proteins Aptamer

VEGF Kd (nmol/L)

bFGF Kd (nmol/L)

KGF Kd (nmol/L)

PDGF-AB Thrombin Kd (nmol/L) Kd (nmol/L)

Reference

VEGF (NX-213)

0.14

286

NA

91

3060

Green et al., 1995

bFGF (m21a)

426

0.35

450

140

>10 mmol/L


KGF (14F3lT)

N.A.

10

0.0008

50

>10 mmol/L



this process, until it is enriched with molecules that discriminate between the desired and undesired targets. White et al. (2003) used this technique to generate an aptamer that discriminates between the Tie2 receptor tyrosine kinase ligands angiopoietin-2 and angiopoietin-1, which share 60% sequence identity. When aptamers that block specific antibody:antigen binding were sought for applications in autoimmunity, pooled polyclonal antibodies were included in a subtractive steps to eliminate aptamers that target the Fc domain (Lee and Sullenger, 1996; Kim et al., 2003). Demonstration of efficacy in an animal disease model is a key step in the development of therapeutic agents. Aptamer cross-reactivity between the human target and the homologous protein from the model species will therefore be a key feature of many candidate aptamers. For example, cross-reactivity between the human and canine homologs of thrombin enabled assessment of ARC183 as an anticoagulant in a canine model of coronary artery bypass graft (CABG) surgery (DeAnda et al., 1994). In cases where the target sequence identity is not well conserved between species, the exquisite specificity of aptamers may pose a challenge. ADR58 binds with 7 nmol/L affinity to human OSM but shows no detectable binding to murine OSM (43% identical), limiting its evaluation in murine models of rheumatoid arthritis (Rhodes et al., 2000). In such cases, aptamers to the human and relevant non-human target may be developed in parallel, with the second aptamer acting as a surrogate for the anti-human aptamer in animal models. Alternatively, xenogenic models may be used where applicable. Finally, in some cases SELEX may be used to build species cross-reactivity into a candidate aptamer. For example, White et al. (2001) used porcine and human thrombin as the target protein in alternating rounds of SELEX and identified cross-reactive aptamers. This was not necessarily a stringent test of the method, since broadly cross-reactive anti-thrombin aptamers have been selected without using this technique (Bock et al., 1992). In an earlier example, Janjic and Gold (2004) demonstrated the utility of sequential SELEX with homologous targets in their efforts to identify aptamers that specifically recognize VEGF receptor 2, encoded by the KDR (human) and flk-1 (mouse) genes. Five rounds of SELEX against the extracellular domain of the KDR protein yielded individual clones which bound with nanomolar affinity to the human but not the mouse protein. Starting with the KDR-enriched pool, five additional rounds of SELEX were carried out using flk-1 as a target. A different set of sequences were isolated as a result and many individual clones in this second effort successfully bound to both human and mouse proteins. This “cross-SELEX” approach is likely to be successful when used with targets which have low overall homology but that contain highly conserved functional regions or subdomains (e.g. Lorger et al., 2003). 17.3.3 Aptamer Binding Kinetics

The kinetic parameters for complex formation have been determined for a few aptamer–target pairs. In most cases, the dissociation constant (koff) is measured

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by pre-forming the aptamer:protein complex using radiolabeled aptamer, followed by addition of a large excess of unlabeled aptamer and then monitoring the decay of the labeled aptamer:protein complex by dot blot. The association rate (kon) is calculated using the relationship kon = koff/Kd. As illustrated in Table 17.3, the half-lives of aptamer:protein complexes are on the order of minutes rather than hours, with typical kon-values of 106 –107 L/mol.s and off rates from approximately 10 –4 –10 –3 s–1. The fast on rates suggest there may be very little structural reorganization required to enable complex formation on the part of the aptamer or protein, however, there are too few structures available for aptamer:protein complexes to draw broad conclusions (Jaeger et al., 1998; Huang et al., 2003). In the cases studied to date, the kinetics of aptamer binding to cell surface targets are comparable to what is observed for soluble proteins. For example, Watson and co-workers determined koff and kon values for aptamer 1b binding to both soluble recombinant L-selectin and L-selectin expressed on the surface of lymphocytes and found the values to be remarkably similar (Watson et al., 2000). Jenison and co-workers showed that an anti-P-selectin aptamer (PF377) binds with reduced affinity to P-selectin expressed on the surface of activated platelets versus recombinant protein (Jenison et al., 1998). The affinity reduction was impacted by a moderate decrease in the association rate and an increase in the dissociation rate. The dissociation of the aptamer from the surface of platelets (1.2 q 10 –3 s–1) is approximately 1000-fold slower than the rate of P-selectin association with neutrophils, which allows the aptamer to efficiently block platelet–neutrophil association. It is worth noting that the method used for SELEX may drive selection of aptamers with relatively fast off rates. Typically, pools and proteins are incubated for 15–30 min, followed by partitioning of the bound complex. The complexes are

Table 17.3 Example kinetic parameters of aptamers Aptamer

Target

koff (s–1)

kon (L/mol.s)

Kd t1/2 (pmol/L) (min)

T (hC)

Reference

m21a

bFGF

1.96 q 10 –3

5.6 q 106 a

350

5.9

37

Green et al., 1995

36t

PDGF-BB/AB

3.0 q 10 –3

3.1 q 107 a

100

3.8

37

Green et al., 1996

1b

L-Selectin

3.2 q 10

1.1 q 10

300

36

23

Watson et al., 2000

1b

L-Selectin

3.8 q 10 –4

6.3 q 106 a

60

31

37

Watson et al., 2000

PF377s1

P-Selectin

1.9 q 10 –4

1.4 q 107

14

61

23


6a

b

–4

6a

PF377s1

P-Selectin

1.2 q 10

4.8 q 10

250

9.6

23


t2-OMe

VEGF

4.2 q 10 –3

3.2 q 107 a

130

2.8

37

Ruckman et al., 1998

NX-213

VEGF

1.4 q 10 –3

1.0 q 107 a

140

8.2

37

Green et al., 1995

a b c

c

–3

Calculated, based on measured koff and Kd values. Values for binding to L-selectin on the surface of lymphocytes. Values for P-selectin on the surface of platelets.


washed briefly and binders are isolated. If high-affinity aptamers with slower off rates were desired, one could imagine incubating pool and protein for a longer period, partitioning, and then invoking extended washing periods. Davis and Szostak (2002) used such an approach to generate an aptamer to GTP with a koff of 4.5 q 10 –5 s–1 and a Kd of 25 nmol/L, 2 orders of magnitude more potent than other GTP binders that had been obtained using standard SELEX conditions. 17.3.4 Binding versus Function

SELEX is an affinity-driven process and its application against a target will generate molecules that bind typically with high affinity and specificity. In some instances, target binding may be sufficient for therapeutic applications. For example, anti-PSMA aptamers may find use as payload delivery molecules by virtue of binding selectively to the surface of PSMA-expressing tumor cells (Hicke and Stephens, 2000; Lupold et al., 2002). In other cases, inhibition of target function is a requirement. As is the case with antibodies, there is no a priori reason that an aptamer will inhibit the activity of its target. Nonetheless, many of the aptamers selected to date have been functional inhibitors. It is not surprising that aptamers to nucleic acid-binding proteins such as HIV reverse transcriptase are inhibitors, since one might predict that the surface used by the target to recognize its natural substrate would likely also exhibit the features required to drive aptamer selection. The small size of many cytokines and growth factors may cause virtually any aptamer binding site to partially overlap with the receptor binding site, allowing high-affinity aptamers to inhibit receptor binding. With larger proteins that may contain multiple subdomains, generating potent inhibitors may present a challenge. On the one hand, the target may present several “aptogenic” sites on its surface. For example, selection against the extracellular domain of HER3, an 82 kDa protein, yielded a panel of aptamers that apparently bound to several different sites on the receptor. HER3 signaling was affected differently by the different aptamers; some binders had no effect, others were antagonists, and a third set showed possible agonist activity (Chen et al., 2003). In other cases, a large protein may have few aptogenic sites, and they may not be proximal to functional domains. SELEX against the extracellular domain of ICAM-1, a 55 kDa protein with five immunoglobulin-like domains presented on the surface of cells, yielded only one aptamer sequence, which had no inhibitory affect on ICAM-1-dependent Jurkat cell adhesion (Archemix Corp). The structural properties of the target itself may allow for enrichment of functional binders through the SELEX process. As an example, L-selectin binding to its ligand, sialyl Lewis X, is known to require the presence of calcium. This property was exploited during selection for inhibitory aptamers (O’Connel et al., 1996; Watson et al., 2000) by combining the SELEX pools with target in the presence of 1 mmol/L CaCl2, and then eluting with the calcium chelator EDTA. As expected, aptamers resulting from SELEX bind to a Ca2+-dependent L-selectin substructure

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and are potent inhibitors of sialyl Lewis X binding (O’Connell et al., 1996; Watson et al., 2000). In a second interesting case, when Wiegand and co-workers sought aptamers that could bind to the constant region of IgE and block IgE binding to FceR1, they used polyclonal IgE in the selection (Wiegand et al., 1996). In this target mixture, the concentration of the Fc region is relatively high since the same sequence is present on each target molecule. In contrast, the large number of different variable regions (Fv) present within the mixture means that each individual target sequence is present at relatively low concentration. As might be expected, SELEX yielded predominantly molecules that specifically recognize the Fc domain. Going forward, it is envisioned that prudent uses of the SELEX process such as these will guide the selection of inhibitors. For example, if the functional subdomain of a target can be expressed independently, it can be used as a target for SELEX. To ensure selected aptamers bind to the native target, the full-length protein may be included in the SELEX experiment in alternating rounds (White et al., 2001).

17.4 Chemical Modification of Aptamers

The first aptamers were discovered within combinatorial libraries of naturally occurring nucleic acids, RNA and DNA (Ellington and Szostak, 1990; Tuerk and Gold, 1990). These compositions are quickly degraded in vivo by endogenous nucleases, and consequently are too short-lived for many applications. Subsequent efforts have correspondingly been invested to test a range of modifications that can not only significantly improve the nuclease resistance of aptamers but which can also confer significant functional improvements. Considerable stabilization can be achieved by the addition of capping structures at both the 5l and 3l ends of an oligonucleotide and by the incorporation of modifications to both the ribose and phosphate backbone components (Jellinek et al., 1995; Kubik et al., 1997; Lin et al., 1996; Pagratis et al., 1997). Backbone modifications can, in principle, be engineered into aptamers by either direct incorporation into the random sequence pools used to carry out the SELEX process or, alternatively, by postSELEX experimentation. Invariably, the introduction of backbone modifications into an aptamer alters the functional properties and it is rare that an unstabilized aptamer can be globally substituted with a given chemistry without interfering with its proper folding and/or target binding. 17.4.1 2l-Modifications

An early example of the discovery of aptamers within libraries of 2l-modified oligonucleotides (Pagratis et al., 1997) describes the isolation of anti-human keratinocyte growth factor (hKGF) aptamers from 2l-ribopurine 2l-fluoropyrimidine


(rRfY) and 2l-ribopurine 2l-aminopyrimidine (rRaY) random sequence pools. While both SELEX efforts were successful in yielding enrichment for target binding, the rRfY composition appears to be superior based on side-by-side comparison of the properties of the resulting hKGF aptamers. rRfY aptamers had Kd-values in the 0.3–3 pmol/L range and corresponding Ki-values in a cellular assay as low as 34 pmol/L. In contrast, the best rRaY aptamers had approximate Kd-values of 400 pmol/L and Ki-values near 10 nmol/L. The nuclease resistance of these compositions was also investigated by incubation of a fixed aptamer sequence in 90% serum, followed by characterization of the breakdown products by polyacrylamide gel electrophoresis (PAGE). These studies indicated that the rRaY composition was somewhat more stable than the rRfY composition with half-lives, respectively, of 170 h and 90 h. Unmodified RNA (rRrY) by comparison had a half-life in this study of less than 10 s. Another early report (Kubik et al., 1997) presented the discovery of aptamers to human inteferon-g (hIFN-g) within libraries of 2l-fluoropyrimidine (rRfY), 2l-aminopyrimidine (rRaY), and mixed 2l-fluorocytidine-2l-aminouridine (rRfCaU) compositions. All compositions gave aptamers with Kd-values of Z100 nmol/L or lower. The discovery of aptamers within libraries of these compositions is now commonplace, facilitated more recently by the use of mutant T7 RNA polymerases (e.g. Y639F) which give higher yields for many modified transcripts (Huang et al., 1997; Padilla and Sousa, 1999). This polymerase was also recently used to generate 2’-O-methyl-containing transcripts, and subsequently libraries of these transcripts have been used to discover 2’-hydroxy purine, 2’-O-methyl pyrimidine (rRmY) and fully 2’-O-methyl aptamers (Burmeister et al. 2005). 2’-O-methyl substituted nucleotides may have fewer toxicity concerns than other 2’-modified nucleotides because they occur naturally at relatively high abundance in the context of ribosomal RNA (Smith and Steitz 1997). The reported MNA (“methoxyribonucleic acid”) aptamer was highly nuclease-resistant with a 40 kDa polyethylene glycol conjugate showing a blood clearance half-life in the mouse of about one day. Allometric scaling would predict a human halflife for the same aptamer approaching one week (Mordenti, 1985). Post-SELEX optimization of aptamers has been carried out both to allow the introduction of modifications that cannot be introduced enzymatically through transcription and to allow site-specific modifications using chemistries which, if introduced globally, would comprise target binding. In this process, one or a small number of modifications are incorporated into an aptamer using solid-phase chemical synthesis and the resulting derivative subsequently tested for target binding and/or inhibition. Combining data from multiple sets of syntheses enables the design of composite, highly substituted (and therefore stabilized) aptamers. Most commonly, 2l-ribopurines have been replaced with the corresponding 2l-O-methyl nucleotide. Relevant examples include the anti-VEGF pegaptanib (Macugenä) (Green et al., 1995; Ruckman et al., 1998), the PDGF aptamer NX1975 (Green et al., 1996), and the L-selectin aptamer 1d40 (O’Connell et al., 1996). In the case of pegaptanib, all but two nucleotides in the 27-mer could be replaced without compromising activity.

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378


2l-Modifications may also be incorporated post-SELEX to introduce additional functionality to the aptamer. Examples of this approach include a pyrene-derivatized HIV-1 trans-activating response element (TAR) (Blount and Tor, 2003). TAR is a naturally occurring RNA “aptamer” that binds to the HIV-1 protein Tat. The pyrene in this functionalized aptamer is a fluorophore, and its fluorescence intensity is increased by as much as five-fold by the interaction of the aptamer with natural aminoglycosides that are known to inhibit the Tat–TAR interaction. Some 2l-ribose modifications have been shown to confer significant thermodynamic stability to duplexes relative to their naturally occurring counterparts (DNA, RNA), as evidenced by increased melting temperature. In some cases, stabilization of the folded structure of an aptamer through the introduction of these modifications translates directly into improved target affinity. Locked nucleic acid (LNA) nucleotides represent one of the most stabilizing modifications tested to date and with demonstrated capacity to improve aptamer function (Darfeuille et al., 2004). In LNA nucleotides the sugar is made bicyclic by covalently bridging the 2l-oxygen and the 4l-carbon with a methylene group (Kumar et al., 1998). The corresponding reduction in conformational mobility causes an increase in the strength of Watson–Crick base pairing of up to 8 hC per base pair (Schmidt et al., 2004). In addition to increased thermodynamic stability, LNAs are intrinsically resistant to degradation by serum nucleases. 17.4.2 Capping the 3l End

It is known that 3lp5l-directed exonucleases play a critical role in defining the intrinsic stability of oligonucleotides in serum. The introduction of either nucleotide or non-nucleotide caps that block these exonucleases can correspondingly yield dramatic stabilizing effects. One of the most commonly used approaches involves the attachment of an inverted thymidine (3l-idT) to the 3l end of an oligonucleotide to create a 3l–3l linkage. Molecules modified in this way effectively display an additional 5l end and no 3l end and are thus highly resistant to modification by exonucleases that require recognition of the 3l end for activity (Agrawal et al., 1988; Goodchild et al., 1988; Stein et al., 1988). Addition of a 3l-idT to an otherwise unstabilized DNA aptamer confers a serum half-life of approximately 1 h, which can be significantly improved upon by subsequent additional backbone modifications (Floege et al., 1999). Non-nucleotide modifications can have similar effects on aptamer stability. In one example involving ARC183, an antithrombin aptamer composed entirely of DNA, the incorporation of biotin at the 3l-terminus was shown to significantly increase the stability of the aptamer in serum (but not, however, in vivo) (Dougan et al., 2000). Incubating the biotinylated aptamer with streptavidin significantly improves the in vivo half-life, an effect likely resulting from a reduction in renal clearance as a consequence of the increased size of the streptavidin–aptamer complex, rather than through changes in nuclease sensitivity.


17.4.3 Capping the 5l End

To a lesser extent, 5lp3l-directed exonucleases contribute to the degradation of oligonucleotides and the addition of nuclease-resistant caps can improve serum and in vivo stability. Such caps are typically introduced as modified phosphoramidites in the last step in conventional solid-phase synthesis or by post-synthesis coupling. A common synthetic strategy involves the use of a 5l-amino modifier phosphoramidite which adds an alkylamine to a 5l-terminal phosphate and which can serve as a reactive nucleophile for subsequent derivatization. While the alkylamine itself blocks exonucleolytic attack, modification via the 5l-amine makes it possible to further control the in vivo properties of the oligonucleotide conjugate, altering both distribution into tissues and the rate of renal elimination. One of the most common 5l-modifications reported in the scientific literature is with PEG, which is discussed separately below. Other 5l-oligonucleotide substitutions include cholesterol (Manoharan, 2002; Manoharan et al., 1994), fatty acids (Shea et al., 1990), polycations (Vinogradov et al., 1996), and proteins (Jones et al., 1994). For example, conjugation with cholesterol has been reported to increase the circulation half-life of therapeutic nucleic acids, most likely through association with plasma lipoproteins, and to promote hepatic uptake (de Smidt et al., 1991). Recently, delivery peptides apparently able to carry large polar macromolecules, including oligonucleotides, across cellular membranes have also been explored as a means to augment in vivo the range of applications of aptamers (Boomer et al., 2005; Healy et al., 2004). Examples of these modifiers include a 13-amino-acid fragment (Tat) of the HIV Tat protein (Vives et al., 1997), a 16-amino-acid sequence derived from the third helix of the Drosophila antennapedia (Ant) homeotic protein (Pietersz et al., 2001), and short, positively charged cell-permeating peptides composed of polyarginine (Arg7) (Rothbard et al., 2002). 17.4.4 Phosphate Substitutions

Both phosphorothioate and phosphorodithioate modifications have been explored in the context of aptamers. These substitutions introduce sulfur(s) in place of non-bridging phosphodiester oxygen(s) and thereby confer nuclease resistance, lipophilicity, and polarizability to oligonucleotides that contain them. While phosphorothioate oligonucleotides can be generated by either transcription or solidphase chemical synthesis, phosphorodithioate NTPs are not utilized by any polymerases tested to date and thus phosphorodithioate-containing libraries have been prepared by solid-phase chemical synthesis. King et al. (2002) used phosphorothioate SELEX to discover phosphorothioate-containing aptamers (“thioaptamers”) to NF-IL6, NFkB, p65, and p50 proteins. They successfully isolated an aptamer with 800 pmol/L affinity binding to p50, and using an electrophoretic gel-shift approach, attributed enhanced affinities and specificities for this aptamer

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to the presence of phosphorothioate modifications. Tam et al. (1999) discovered an 18-mer fully-phosphorothioate G-rich aptamer that inhibits the expression of CD28. By reducing the number of phosphorothioate linkages the authors showed that only some of these residues are required for nuclease resistance, and that the functional properties of these phosphorothioate linkages are responsible for enhancing the bioactivity of this aptamer. Extending upon the work of King and co-workers, Yang et al. (2002, 2003) have developed in vitro screening methods for generating phosphorodithioate-containing aptamers and have described the application of these methods to isolate antiNFkB aptamers. The inability to directly transcribe sequence pools containing this modification forced these authors to develop a split synthesis method in which each solid-phase support bead contained a single phosphorothioate-containing sequence. Direct, post-screening sequencing of beads with the highest affinity binding made it possible to identify functional aptamers. However, relatively limited data are currently available concerning the characteristics of molecules discovered using this approach. A handful of additional types of phosphate modifications may ultimately be shown to have utility in the context of aptamers. Methylphosphonate linkages have also been considered for oligonucleotides administered for imaging and therapeutic purposes (Younes et al., 2002) and in order to confer nuclease resistance (Agrawal et al., 1997). P-borano linkages can be incorporated into transcripts using T7 RNA polymerase and 5l-(a-P-borano) GTP or 5l-(a-P-borano) UTP, and these modifications have been incorporated into aptamers to ATP using SELEX (Lato et al., 2002). These authors suggested that because 10B can capture thermal neutrons and thereby become radioactive, 10B-containing aptamers could find utility as activatable radiotherapeutics that are specifically delivered to tumor cells. 17.4.5 Base Modifications

A wide variety of modified bases have been suggested as potentially transcribable by the polymerases typically used to carry out the SELEX process (Eaton, 1997; Eaton et al., 1997). Relatively few of these have been incorporated into transcripts, and an even smaller number have been directly used in the selection of aptamers. An early example of this approach (Latham et al., 1994) used 5-(1-pentynyl)-2ldeoxyuridine in place of thymidine in a DNA library, and a family of modification-dependent substituted aptamers was then isolated from this library. Subsequent studies have incorporated functionalities that nucleic acids lack, such as primary amine and thiol residues linked through the pyrimidine 5-position. In one study a 5-amino analog of uridine was incorporated into a library of transcripts from which an ATP-binding aptamer was isolated (Vaish et al., 2003). Binding to ATP was shown to be modification-dependent and multiple substituted uridines were shown to be involved. In other studies a 5-amino analog of dT was used to select modified DNA aptamers to aspartame (Saitoh et al.,


2002) and sialyllactose (Masud et al., 2004) and a 5-thiol analog of dU has been similarly incorporated into DNA using Pwo DNA polymerase (Held and Benner, 2002). 17.4.6 Polyethylene Glycol

Aptamers intended for therapeutic applications that are reported in the scientific literature are often 5’-substituted with PEG with molecular weights in the 20–40 kDa range. PEG is inexpensive, non-immunogenic, inert, and is already utilized in approved therapeutics. It is attached to aptamers because the resultant significant increase in mass decreases renal filtration rates and thereby increases pharmacokinetic half-lives by 10-fold or more (e.g. Ostendorf et al., 2001; Eyetech Study Group, 2003). 17.4.7 Lipid Tags

Lipid groups have been appended to aptamers in an effort to alter their distribution profile in vivo, to extend their pharmacokinetic half-life and to aid their entry into cells. In one example in which a lipid-modified oligonucleotide is preferentially taken up into cells in vivo, a cholesterol-functionalized antisense phosphorothioate oligonucleotide was formulated with low-density lipoprotein and administered to rats intravenously. The oligonucleotide was taken up into Kupffer cells within the liver 6–14 times more extensively than unfunctionalized oligonucleotides (Bijsterbosch et al., 2000, 2001, 2002). Other studies have shown that unsubstituted oligonucleotides, including phosphodiester, phosphorothioate, and methyl phosphonates do not readily cross model cell membranes (Akhtar et al., 1991). Pharmacokinetic half-life extension can result because the aptamer distributes out of the blood and becomes associated with hydrophobic phases within the body, or by co-administration with liposomes which increases the aptamer’s effective molecular weight and thereby reduces the rate of renal clearance. In one such study the plasma residence time of the liposome-anchored aptamer was greatly increased while high-affinity binding to the target (VEGF) was maintained (Willis et al., 1998). The functionalization of oligonucleotides with cholesterol influences pharmacokinetics by increasing cell uptake and serum protein binding. In one study a 3l-cholesteryl phosphorothioate antisense oligonucleotide complementary to ICAM-1 mRNA was dosed to rats intravenously (Bijsterbosch et al., 2000). Hepatic uptake was two-fold higher than for the equivalent unconjugated oligonucleotide with greater than 70% of the conjugated oligonucleotide detectable in the liver, intracellular concentrations within Kupffer cells reaching 75 mmol/L for a 1 mg/kg dose and ICAM-1 expression was also reduced to lower levels with the conjugated aptamer.

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Fatty acids also enhance the transport of oligonucleotides across the intestinal mucosa without being covalently attached. In one such study sodium caprate was used to deliver antisense oligonucleotide to pigs in this fashion (Raoof et al., 2002). In another study in which the delivery of antisense phosphorothioate oligonucleotides to human retinal pigment epithelial cells in vitro was studied without covalent attachment to the delivery agent, the authors concluded that lipid carriers with membrane-active components and small complex sizes are required for efficient delivery (Jaaskelainen et al., 2000). 17.4.8 Peptide Tags

Peptides and proteins are the archetypal functional biopolymers. Consequently they have been attached to aptamers for a variety of purposes including transport across epithelia and cell membranes and pharmacokinetic half-life extension. In an effort to increase the ability of oligonucleotides to enter cells various naturally occurring and artificial peptide sequences have been utilized as tags. Naturally occurring cell-penetrating peptides that have been used include the Tat and antennapedia peptides (Astriab-Fisher et al., 2002; Moulton et al., 2003), fusogenic peptides (Bongartz et al., 1994; Jeong et al., 2003). Non-natural peptides used in this fashion include highly positively charged peptides such as polyarginine and polylysine (Stevenson and Iversen, 1989) and histidyl oligolysines (Pichon et al., 2000) and peptides discovered using genetic selection techniques such as phage display (Shadidi and Sioud, 2003). Peptides have also been utilized to directly increase the activity of aptamers. In one example of this approach (Lin et al., 1995) a DNA aptamer was selected against human neutrophil elastase (hNE) and while this aptamer has a Kd of 17 nmol/L, it did not effectively inhibit elastase activity. Attachment of this aptamer to a tetrapeptide known to weakly inhibit hNE increased the inhibitory activity of the peptide by almost five orders of magnitude to give a Ki of 28 nmol/L.

17.5 Routes of Administration of Aptamers 17.5.1 Parenteral Administration

In contrast to small-molecule drugs, aptamers have molecular weights in the 5–10 kDa range, and these molecular weights can be as high as 50 kDa for aptamers functionalized with PEG, such as Macugen (Eyetech Study Group, 2003). It is generally difficult to deliver drugs of this size across biological barriers such as pulmonary, gastrointestinal, and skin epithelia. When it is desired to deliver an aptamer systemically, the routes of choice are gererally intravenous, intramuscular, intraperitoneal, and subcutaneous administration. The aptamer that is furth-

17.6 Opportunities for Alternative Aptamer Formulations

est advanced towards FDA approval and is delivered systemically is ARC183. This is a 15-mer DNA aptamer that binds to and inhibits thrombin and thereby exerts an anticoagulant effect (Bock et al., 1992). It recently completed in Phase I clinical trials for anticoagulation during CABG surgery. ARC183 is administered continuously intravenously, has a 2 min half-life in cynomolgus monkeys (Griffin et al., 1993) and has been dosed at 0.5 mg/kg/min in dogs during a CABG surgery pilot study (DeAnda et al., 1994). During this study the activated clotting time was observed to rise from 2 min to more than 25 min. The short half-life of ARC183 means that no antidote is required at the conclusion of surgery as is the case with heparin, the current anticoagulant of choice for this indication, which is inactivated with protamine. Alternative routes for systemic dosing have been compared for the anti-VEGF therapeutic aptamer Macugen. The following bioavailabilities were reported for the Sprague–Dawley rat: intraperitoneal 71%, intramuscular 57%, subcutaneous 2%. Subcutaneous dosing to the monkey, however, gave a bioavailability of 78% and a time to peak concentration of 8–12 h (Tucker et al., 1999). 17.5.2 Comparison to Biologics

Biologics are active pharmaceutical ingredients that are produced in living organisms. They include vaccines, blood products, and therapeutic proteins. In contrast, non-biologic therapeutics are produced by chemical processes. Therapeutic oligonucleotides, including aptamers, are chemically synthesized and so do not fall into the category of biologics. The chemical synthesis of oligonucleotides has been shown to readily scale from the microgram to the kilogram scale and beyond. In contrast to this the scale-up of the synthesis of therapeutic proteins, such as antibodies, in bioreactors is problematic. This is because increasing the scale can fundamentally alter the therapeutic properties (e.g. extent of glycosylation) of what is being produced. As a consequence, the market sizes of several therapeutic antibodies are limited by production rather than demand. Building increased production facilities for therapeutic oligonucleotides is, compared to therapeutic proteins, relatively inexpensive.


The vast majority of aptamer studies have relied upon parenteral administration with simple formulations. Work with antisense and other therapeutic oligonucleotides, however, points to the feasibility of a number of alternative formulations that may provide significant advantages, both increasing patient acceptance and improving the therapeutic index.

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17.6.1 Depot

An increasing number of drugs are being approved for delivery via depots, which are able to release drugs in situ over a desired time-course and thereby decrease dosing frequency and promote patient compliance. Several controlled-release studies of oligonucleotides are reported in the literature. In one such example the anti-VEGF aptamer Macugen was formulated with poly(lactic-co-glycolic) acid (PLGA) to yield a controlled-release matrix that was between 2 and 3.5% PEGylated aptamer by mass (0.4–0.6% oligonucleotide by mass) (Carrasquillo et al., 2003). This particulate solid released aptamer over a three-week period in in vitro studies. Additional evidence was presented for the release of the aptamer in vivo using a transcleral route into the eyes of Dutch-belted rabbits. Higher loading levels of PLGA are achievable for oligonucleotides that are not PEGylated. For example Putney et al. (1999) reported loading levels of up to 10% oligonucleotide by mass in the presence of cations such as polyamines or zinc. In this study controlled-release was sustained over 9 days in vitro when the oligonucleotide was formulated in the presence of a 10-fold excess of zinc acetate by mass. In vivo efficacy data in a murine melanoma xenograft model was also presented. PLGA is generally regarded as safe, it is used as a controlled-release matrix in over ten FDA-approved products, and additionally has been used as suture silk for decades. Under physiological conditions, PLGA gradually degrades to non-toxic monomers. 17.6.2 Topical

Although the topical delivery of aptamers has not been reported, there are several examples from the antisense field. Mehta and co-workers (2000) have investigated the local topical delivery of a 20-mer antisense phosphorothioate oligonucleotide that targets the transcript for ICAM-1. This oligonucleotide is being investigated as a potential treatment for psoriasis. In this study the oligonucleotide was formulated into a cream in which the oligonucleotide comprised 2% by mass and this was applied to human cadaver skin and compared with systemic intravenous dosing to SCID mice with human cadaver skin transplants. Additional experiments were performed with hairless mice without transplants. Histological and scintillation-counting approaches demonstrated that concentrations of oligonucleotide in the dermis were 3 mmol/L and in the epidermis 230 mmol/L after 24 h when topically dosed at 0.5 mg/cm2 in a 2 mg/mL (300 mmol/L) cream. These concentrations were 4300- and 160-fold less than were observed in a similar experiment in which the oligonucleotide was dosed by IV at 2 mg/kg. Functional effects of the oligonucleotide were also observed. The concentration of ICAM-1 mRNA was reduced by approximately two-thirds and a reduction in the increase in concentration of ICAM-1 protein was also observed in induction experiments. A scrambled control oligonucleotide did not induce these effects.


The systemic delivery of oligonucleotides via topical administration has also been investigated. A study in which 25 000 combinations of various penetration enhancers were screened for their ability to synergistically increase the permeability of human skin was recently reported (Karande et al., 2004). The authors initially screened for combinations that increased skin conductance. These were then further screened for non-irritance, their ability to sustain a high flux of drug, and ultimately, bioavailability in hairless rats. The very best combinations were able to increase the permeation of a 15 kDa oligonucleotide by approximately 100-fold. Iontophoreisis has also been investigated as a way of increasing the amounts of antisense oligonucleotide delivered to the epidermis and dermis. Sakamoto and co-workers (2004) used iontophoreisis to deliver about 30% of the applied dose of an anti-interleukin-10 (anti-IL-10) antisense oligonucleotide to the epidermis and dermis of mice in a human atopic dermatitis model. IL-10 mRNA and protein levels were observed to decrease, and no effect was observed upon IL-4 mRNA or protein levels. Substituted antisense oligonucleotides have been visualized within the skin in multiple studies. For example, 5-propyne-modified antisense oligonucleotides were observed to penetrate the stratum corneum of psoriatic human skin and become localized in the nuclei of large parakeratotic cells and smaller basal and suprabasal keratinocytes (White et al., 2002). 17.6.3 Oral

There are no reports in the scientific literature of the oral delivery of aptamers. But the delivery of antisense oligonucleotides has been explored by the oral route. One such study reported the oral administration of an 18-mer fully phosphorothioate partially 2l-O-methylated oligodeoxynucleotide that is complementary to the RIa subunit of cAMP-dependent kinase (PKA) (Wang et al., 1999). In this study the oligonucleotide was dosed to SCID mice with LS174T (human colon cancer) xenografts at 10 mg/kg both orally (gavage) and intraperitoneally. Similar significant antitumor activity was found using either delivery route. A scrambled-sequence control oligonucleotide showed no antitumor effect. The oral bioavailability of this oligonucleotide was reported to be 48% at 48 h with a maximum plasma concentration 6.8 h after oral (gavage) dosing. The absorption half-life into plasma was 3.8 h and the elimination half-life from the plasma was 8 h. The absorption half-life into the tumor was 3.8 h while the elimination halflife was 61 h, significantly extended when compared to the plasma. Significant antitumor effects were also observed in SCID mouse xenograft models in which A549 (human lung cancer) and MDA-MB-468 (human breast cancer) tumors had been transplanted. This oligonucleotide was the subject of a Phase I clinical trial (Chen et al., 2000). Similarly high oral bioavailabilities have been reported for other highly stabilized 2l-O-methyl antisense oligonucleotides (Agrawal and Iyer, 1995). Phosphorothioate antisense oligodeoxynucleotides without 2l-O-

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methyl groups do not appear to be sufficiently stable to be detectable in the blood after oral dosing (Nicklin et al., 1998). An alternative approach to delivery via the gastrointestinal tract has been reported by Gonzlez Ferreiro et al. (2002). In this study a variety of fully phosphorothioate oligodeoxynucleotides were formulated into polyalginate/polylysine microparticles and these were administered intrajejeunally in the presence of permeation enhancers. Intrajejeunal administration is a development surrogate for oral administration for compounds not considered stable enough to survive the acidic conditions of the stomach. These microparticles averaged approximately 100 mm in diameter and could be loaded up to 35% by mass. Bioavailabilities of up to 28% were observed in the rat when oligonucleotides were dosed at 40 mg/kg, with blood concentration maxima of around 0.5–1 h. Permeation enhancers included sodium ursodeoxychelate, sodium chenodeoxycholate, sodium caprate, and sodium laurate which were dosed at 200 mg/kg. In the absence of enhancers, no oligonucleotide could be observed in the blood. A second orally dosed antisense oligonucleotide, Monarsen, also known as EN101 (Brenner et al., 2003), has successfully passed through Phase I clinical trials (Sussman, 2003). This is a partially 2l-O-methylated 18-mer that is complementary to an alternative splice-variant of acetylcholinesterase that causes myasthenia gravis. Fifteen out of 16 patients showed improved muscle strength when given 500 mg/kg once-daily orally. 17.6.4 Pulmonary

The feasibility of pulmonary delivery of oligonucleotide therapeutics has been demonstrated with a handful of antisense studies. In one such report, the pulmonary route was used to systemically deliver a 20-mer fully phosphorothioate antisense oligodeoxynucleotide that was complementary to the 3l-untranslated region of human protein kinase C-a mRNA (Nicklin et al., 1998). Intratracheal dosing of male Wistar rats at 6 mg/kg gave bioavailabilities of 40% as determined by scintillation counting of tritiated oligonucletide in blood and tissue samples over a 6 h period. The maximum concentration of oligonucleotide in the blood occurred at 3–4 h after administration. Kidney and liver also showed significant accumulation. Lower doses gave lower bioavailabilities. Additionally “respirable antisense oligonucleotides” (RASONs) are being developed for local delivery to the lung (Ali et al., 2001; Sandrasagra et al., 2002). These studies describe EPI-2010, which is a 21-mer fully phosphorothioate oligodeoxynucleotide complementary to the adenosine A1 receptor, and targets its initiation codon. This oligonucleotide was labeled with 35S, aerosolized and 5 mg was administered to normal New Zealand rabbits using an endotracheal tube. Approximately 1.4% of the administered oligonucleotide was delivered to the lung. This was then eliminated with a half-life of 30 h. Fifteen per cent of the delivered dose was detected in the blood, 6.9% of the delivered dose was detected in the heart, liver and kidneys combined. Over the ensuing 72 h 60% of the total admi-

17.7 Aptamer Pharmacokinetics and Biodistribution

nistered dose was eliminated in the urine, and 7% in the feces. The therapeutic concentration of this oligonucleotide is calculated to fall between 10 and 100 mg/ kg (Nyce and Metzger, 1997; Ali et al., 2001), and so the doses reported to the lung in this study fall within the therapeutic range. Pulmonary delivery by intratracheal dosing of fully phosphorothioate oligodeoxynucleotides has also been shown in rats (Danahay et al., 1999) and mice (Templin et al., 2000). In these studies dose-dependent retention in the lung was demonstrated, with oligonucleotide being detectable in the alveoli. 17.6.5 Ocular

It may not be a coincidence that the only FDA-approved antisense drug (VitraveneTM) and the first approved aptamer drug (MacugenTM) are both administered to the eye by intravitreal injection. The eye is a relatively small and independent compartment. As a consequence systemic toxicity and cost-of-goods issues are favorable when compared with most other dosing strategies. Macugen (pegaptanib) is a 5l-40 kDa-polyethylene glycol-terminated, 3l-inverted dT-terminated 27-mer in which all of the pyrimidines are 2l-fluoro and all of the purines are 2l-O-methyl except for two 2l-hydroxy adenosines. Macugen successfully completed Phase III clinical trials and Phase II clinical trials are reported in the literature (Eyetech Study Group, 2003). Macugen binds to VEGF, which is implicated in the overproliferation of vasculature that accompanies age-related macular degeneration. The Phase II data report that patients received multiple intravitreal injections, that these were well-tolerated and resulted in stabilized or improved vision for 87.5% of the patients, with 25% experiencing a 3-line or greater improvement in their ability to read an ophthalmic test chart. Pharmacokinetic evaluation in Rhesus monkeys showed that for dosing of up to 2 mg/eye, Macugen was cleared intact from the eye with a half-life of 94 h, and that aptamer recovered from the vitreous humor after 28 days was fully active (Drolet et al., 2000). VitraveneTM (fomivirsen) is a 21-mer fully phosphorothioate antisense oligodeoxynucleotide that is complementary to the mRNA of a major immediateearly region protein of cytomegalovirus and is used to treat cytomegalovirus retinitis, especially in AIDS patients. It is dosed bi-weekly by intravitreal injection at 330 mg/eye and is cleared from the vitreous with a half-life of 55 h (Geary et al., 2002).


As stated by Agrawal and Zhang, “understanding the pharmacokinetics of an oligonucleotide provides the basis for its effective use as a therapeutic agent, as well as indicating which organs may be sites of toxicity” (Agrawal and Zhang, 1997). Assessment of the drug and metabolite concentrations in blood and tissues

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guides the preclinical development of a therapeutic aptamer to define the optimal route of administration, dosing frequency, and dosing level. During the transition from preclinical to clinical development, knowledge of the pharmacokinetic characteristics of a therapeutic aptamer is critical to the design of safety pharmacology and toxicology studies (Modi, 2002), and dosing levels must be set so as to provide the appropriate multiples of anticipated clinical doses. Finally, the discovery and development of aptamer therapeutics is an iterative process coordinated between in vivo pharmacology and medicinal chemistry teams to optimize pharmacokinetic and biodistribution properties for particular applications. Once pharmacokinetic and biodistribution properties have been optimized, the next phase in the preclinical development of a therapeutic aptamer is the correlation of pharmacokinetic measurements (i.e. plasma concentration of aptamer as a function of time) with the observed pharmacodynamic measure(s) used to assay the bioactivity of the compound in vivo as well as in vitro. The exploratory pharmacokinetic assessment of a typical therapeutic aptamer begins with a singledose or dose-ranging study after systemic administration (e.g. IV, IP, SC) in a rodent model. Subsequent pharmacokinetic characterization of therapeutic aptamers often involves the performance of an analogous study in non-human primates, in order to refine the pharmacokinetic parameters and dosing estimates derived from the rodent models. In the following sections, we outline general observations likely to apply to many different aptamers that have been learned through repeated studies. 17.7.1 Key Pharmacokinetic and Biodistribution Parameters

It is important for all oligonucleotide-based therapeutics, including aptamers, that their pharmacokinetic properties be tailored to match the desired pharmaceutical application. While aptamers directed against extracellular targets do not have to solve the problem of intracellular delivery (as do antisense and RNAi-based therapeutics), they must be capable of distributing to designated target organs and tissues and persisting in the body unmodified (i.e. be resistant to plasma nucleases and other metabolic enzymes) for a period consistent with the desired dosing regimen. The ideal set of pharmacokinetic properties for a therapeutic aptamer is not universally defined, but depends entirely on the specific indication and intended use, as well as any constraints associated with the anticipated route of administration, patient population, potential interactions with coadministered therapeutic compounds, etc. Similarly, there is no single figure of merit that fully describes the overall pharmacokinetic character of a therapeutic aptamer. The most commonly reported pharmacokinetic parameters include the maximum observed plasma concentration, Cmax; area under the concentration–time curve, AUC; terminal half-life, t1/2 (b); terminal clearance, Cl; and volume of distribution at steady state, Vss. In evaluating and ranking pharmacokinetic parameters derived from compartmental modeling or non-compartmental analysis (NCA) of plasma concentration versus time data, it is important to note which


parameters are primary, and which are secondarily derived or dependent on primary parameters. For NCA analysis, primary parameters are those derived directly from the concentration–time data, as the AUC is by direct integration, while secondary parameters are those such as clearance (Cl = dose/AUC). Among the various pharmacokinetic parameters typically cited, the magnitude of the terminal half-life is often taken as the single most important comparator between compounds. However, while an extended systemic half-life (t1/2) is often desirable, there are many contexts in which a shorter half-life may be appropriate. As a specific example, take ARC183, a potent antithrombin aptamer being developed for use as a heparin replacement in CABG surgery (Bock et al., 1992). In this case, the utility of ARC183 is based largely upon its short (t1/2 Z2 min) pharmacokinetic/pharmacodynamic half-life, allowing rapid reversal of its anticoagulant effect. Similary, for an aptamer delivering a cytotoxic or radionuclide payload to a tumor, it is desirable to limit the overall residence time of the therapeutic aptamer in circulation in order to minimize systemic exposure to the antitumor agent. Overall, the goal is to optimize pharmacokinetic characteristics of a therapeutic aptamer for the anticipated application, through modifications in composition (e.g. backbone, sugar) and/or conjugation (e.g. PEGylation). In general, the two most critical pharmacokinetic characteristics for an ideal therapeutic aptamer are: (1) dose-proportionality, i.e. Cmax and AUC proportional to dose, D; and (2) terminal clearance (Cl) that is independent of dose (D) in range of interest. 17.7.2 Factors Governing Pharmacokinetics and Metabolic Stability of Aptamers

A large body of literature documents the pharmacokinetic and biodistribution properties of phosphorothioate-containing antisense oligonucleotides, which clear rapidly from circulation, and distribute into tissues where elimination occurs slowly as a result of metabolic degradation (Srinivasan and Iversen, 1995; Agrawal and Zhang, 1997; Akhtar and Agrawal, 1997; Crooke, 1997; Grindel et al., 1998; Monteith and Levin, 1999; Peng et al., 2001). In contrast to antisense oligonucleotides, however, aptamers are in general longer (25–40 vs. 10–20 nucleotides), possess different types of stability-enhancing chemical modifications (sugar vs. backbone modifications), and assume complex tertiary structures that are, in many respects, more similar to the three-dimensional forms of globular proteins than to nucleic acids. Given these considerable differences, the in vivo disposition of aptamers is not readily predictable from antisense results. Recent work (Healy et al., 2004; Boomer et al., 2005) has shown that the key factors known to govern the pharmacokinetic and biodistribution of therapeutic aptamers are composition (i.e. length, DNA/RNA, backbone, sugar modifications), conjugation to high-molecular-weight polymers (PEGylation) (Yamaoka et al., 1994, 1995; Kawaguchi et al., 1995; Reyderman and Stavchansky, 1997; Watson et al., 2000; Caliceti and Veronese, 2003; Harris and Chess, 2003) or cell-permeating peptides (Vives et al., 1997; Antopolsky et al., 1999; Zubin et al., 1999;

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imal model. All of these factors have shown significant effects, of comparable magnitude, on the pharmacokinetic profile of aptamers in vivo. The physiological basis of aptamer pharmacokinetic and biodistribution may be broadly attributed to three primary mechanisms. These include degradation by circulating plasma nucleases, renal filtration and subsequent urinary elimination, and hepatic clearance, including metabolism and biliary excretion (Kwon, 2001). The metabolic stability of therapeutic aptamers with respect to both plasma nucleases and liver enzymes such as the cytochrome P450 family and UDPGT (uridine diphosphate glucoronyl transferase) is significantly enhanced by reduction in the fraction of natural ribonucleotides, the use of deoxynucleotides, the incorporation of various chemical modifications to the 2l position of the pentose sugar in some or all nucleoside residues, and the use of inverted 3l–3l caps. As described in Section 17.4.1, typical 2l modifications are 2l-fluoro, 2l-O-methyl, and 2l-NH2 (Green et al., 1995; Ruckman et al., 1998; Geary et al., 2001) stabilizing chemistries. In addition to metabolic clearance, oligonucleotide therapeutics are subject to renal clearance, with a molecular weight cut-off of approximately 30–50 kDa (see, for example, Yamaoka et al., 1995). As such, a typical nucleaseresistant therapeutic aptamer with a molecular weight of 5–15 kDa after intravenous administration exhibits an in vivo pharmacokinetic half-life of I 10 min, unless filtration can be blocked by either facilitating rapid distribution out of the bloodstream into tissues or by increasing the therapeutic aptamer’s apparent molecular weight above the effective size cut-off for the glomerulus. Conjugation of aptamers to high-molecular-weight (i.e. 20–60 kDa) PEG groups (PEGylation) can dramatically lengthen residence times of aptamers in circulation while marginally impacting the ability to bind to protein targets (Kawaguchi et al., 1995; Reyderman and Stavchansky, 1997; Watson et al., 2000), thereby decreasing dosing frequency and enhancing effectiveness against vascular targets. For example, an aptamer with a fixed composition and variable 5l-PEG conjugation (0, 20, 30, and 40 kDa) exhibits a variation in t1/2 (b) of approximately 5- to 20-fold. Figure 17.1 shows the pharmacokinetic profile of a 23-nucleotide fully 2l-Omethyl modified aptamer conjugated to a 40 kDa PEG after intravenous administration to female CD-1 mice at 10 mg/kg (oligonucleotide mass). Groups of three CD-1 mice were dosed intravenously to the tail vein, and blood samples were collected in EDTA-coated tubes at the indicated time-points and processed for plasma. Aptamer concentrations in these plasma samples were assessed by fluorescence of their Oligreenä (Molecular Probes, Eugene, OR, USA) complexes at 520 nm (excitation at 480 nm) by comparison with standard curves. From the figure, the decay in plasma concentration is monophasic, with an elimination halflife, t1/2 z 22 h and a volume of distribution, Vss z 87 mL/kg. This is typical of fully 2l-O-methyl (mRmY) and dRmY composition aptamers, whereas r/mRfY and d/mRd/mY compositions with 20–40 kDa PEG conjugates typically exhibit a biphasic pharmacokinetic profile. This profile compares very favorably with aptamers containing nuclease-susceptible residues such as 2l-hydroxy and 2l-deoxy that can have half-lives as short as 2 h, and enables the possibility of weekly dosing of therapeutic aptamers.


Fig. 17.1 Pharmacokinetic profile of a 23-nucleotide fully 2l-Omethyl aptamer after intravenous administration to female CD-1 mice (n = 3 per time-point) at 10 mg/kg.

In addition to composition and conjugation, two factors that strongly influence the pharmacokinetics and biodistribution of therapeutic aptamer are route of administration and species of animal model. The route of administration will affect the rate and extent of absorption of a therapeutic, that is, its bioavailablity. These pharmacokinetic effects translate directly into the pharmacodynamic effect of the therapeutic, such as the interval between administration and the onset of the desired pharmacologic response and the duration of response. Factors to be considered in choosing the appropriate route of administration for a particular therapeutic aptamer and indication include (1) the dosing volume; (2) the anatomic and physiologic characteristics of the administration site, such as membrane permeability and blood flow; (3) the physiochemical properties of the site, such as pH, osmotic pressure, and the presence of physiologic fluids; (4) interaction of the drug and dosage form at the administration site, including alteration of the site due to the drug or dosage form (e.g. encapsulation of a controlled-release implant); and (5) potential issues with patient acceptance/compliance, such as the advantages of daily oral dosing versus daily intravenous injections (Shargel and Yu, 1999). Routes by which therapeutic aptamers have been administered include intravenous bolus injection and infusion, intraperitoneal injection, subcutaneous injection, oral gavage, and subconjunctival and intravitreal injection. For a given route of administration, the bioavailability of therapeutic aptamers can vary widely across species. For example, the measured bioavailability of an r/mRfY composition aptamer after subcutaneous (SC) administration ranged from approximately 2% in rats to 78% in primates (Tucker et al., 1999). Typical terminal half-lives of aptamers after parenteral administration, such as an intravenous bolus, range from 3 to 22 h in rodent, and 30–60 h in primate, depending on sequence composition, modifications, and conjugation. In contrast, after intravitreal

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injection the terminal half-life of a typical aptamer is approximately 4 days (Drolet et al., 2000; Eyetech Study Group, 2002; Carrasquillo et al., 2003). A final consideration in the planning and interpretation of data from pharmacokinetic and biodistribution studies is the choice of animal model. As all animal models are surrogates for humans, it is imperative to determine as accurately as possible how pharmacokinetic and biodistribution characteristics of a typical therapeutic aptamer will vary across species along the progression from exploratory studies in rodents to definitive safety pharmacology and toxicology studies in primates. The quantitative methodology that has been developed to address the issue of interspecies scaling is sometimes referred to as “allometric” (size-based) scaling. The underlying assumption in allometric scaling is that the physiologic factors and processes (e.g. cardiac output, organ weight, glomerular filtration, metabolic rates) in all relevant animal species scale with body mass or surface area. That is, F(x) = a q xb, where F is the physiologic or pharmacokinetic property, x is the body mass or surface area, and a and b are the allometric coefficient and exponent, respectively. Allometric coefficients and exponents have been tabulated for most common species, and are often used in conjunction with a normalization factor for the difference in maximum lifespan potential (MLP) between species being compared. Despite the extensive body of interspecies scaling data for small-molecule drugs, relatively few data exist for biologics such as peptides, antibodies, and aptamers. Current aptamer data indicate some general trends in scaling between rodent and primate pharmacokinetics but with significant variability possible on an individual aptamer-by-aptamer basis. For example, the measured terminal half-life of several (PEGylated) aptamers has been observed to increase approximately fourfold comparing rat to cynomolgus monkey following intravenous bolus administration. At the same time, subcutaneous bioavailability of therapeutic aptamers has been shown to vary significantly within and across species, ranging from 2 to 80% for aptamers of varying composition (Tucker et al., 1999). The parameter space of the combinatorial factors governing the pharmacokinetic and biodistribution profiles of aptamers is large, and the existing data matrix still relatively sparse. All of these factors have shown significant effects on the pharmacokinetic profile of aptamers in vivo. For example, for a fixed aptamer sequence and 5l-PEG group with variable composition (i.e. varying fractions of ribonucleotide, deoxyribonucleotide, and 2l modified residues) the experimentally observed variation in terminal half-life, t1/2 (b), is as much as 14-fold. Similarly, for a fixed 5l-PEG conjugation (40 kDa) with variable sequence composition, the experimentally observed variation in terminal half-life, t1/2 (b), is approximately six-fold (Archemix Corp). 17.7.3 Biodistribution of Aptamers

Although a small number of studies have investigated the pharmacokinetic properties of PEG-conjugated aptamers (Reyderman and Stavchansky, 1997; Watson et al., 2000; Pagratis et al., 2002), relatively little is known concerning the capacity


of either unconjugated or PEGylated aptamers to escape the vasculature and distribute to organs and tissues in vivo. Knowledge of the rate and extent of extravasation and, in particular, the potential of aptamers or their modified forms to access diseased tissues (for example, sites of inflammation, or the interior of tumors) is expected to better define the spectrum of therapeutic opportunities for aptamer intervention, and to guide the informed choice of new targets for aptamer development (see, for example, Lopes and Mayer, 2002). In an effort to understand the whole-body tissue and cellular biodistribution of aptamers, and further, to assess the relative ability of several aptamer compositions to access inflamed tissues in a murine inflammation model, biodistribution studies utilizing 3H-labeled aptamers have been carried out by both traditional oxidation and liquid scintillation counting (LSC) and quantitative whole-body autoradiography (QWBA) (Healy et al., 2004; Boomer et al., 2005). The biodistribution of several different 3H-labeled aptamers, including 20 kDa PEG-conjugated and unconjugated compositions was assessed in mice at 3, 12, and 24 h post administration. In the QWBA study, the specific focus was to evaluate aptamer distribution to diseased tissues (Boomer et al., 2005). Acute inflammation was therefore induced locally in animal hindlimbs by treatment with carrageenan just prior to aptamer dosing. Aptamer penetration of cells in kidney and liver was also examined at a qualitative level by micro-autoradiography. Figure 17.2 shows two cross-sectional autoradiographic images of a CD-1 mouse 3 h after a single intravenous bolus administration of a 3H-labeled unconjugated 32-nucleotide r/mRfY aptamer. QWBA analysis is superior to traditional LSC analysis in that the whole-body sectional images represent the precise and instantaneous distribution and concentration (quantitated via a standard curve using [3H]-glucose which intersects all slices) of aptamer in all tissues/organs captured in the slice at a single instant in time, whereas with LSC analysis tissues/ organs must be harvested, oxidized/solubilized, and analyzed separately, resulting in the loss of all information about the spatial distribution of aptamer within an organ (Solon and Kraus, 2001; Solon et al., 2002). Visual inspection of these images immediately reveals a great deal of information about the disposition and likely routes of metabolism and elimination of this aptamer. In general, all aptamer compositions studied were distributed widely, although the various compositions exhibited significant variation in the relative concentrations of aptamer detected in organs and tissues. The highest concentrations of radioactivity in whole-body tissues for all animals were observed in the kidney and urinary bladder contents, consistent with urinary elimination as a primary route of clearance. The fully 2l-O-methyl aptamer composition showed the most rapid clearance from circulation, with intact aptamer detectable in urine at 48 h post administration. Relatively little radioactivity was associated with the brain, spinal cord, and adipose tissue. The total level of radioactivity in wholebody tissues was significantly higher for a 20 kDa PEG conjugate than for other aptamers. Comparatively high levels of the 20 kDa conjugate were seen in wellperfused organs and tissues, including liver, lungs, spleen, bone marrow, and myocardium. A fully 2l-O-methyl composition aptamer had the lowest level of

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Fig. 17.2 Quantitative whole-body autoradiograph (QWBA) images of a CD-1 mouse 3 h after a single intravenous bolus adminstration of an unconjugated 3H-labeled aptamer.


radioactivity in whole-body tissues. Interestingly, the 20 kDa PEG-conjugated aptamer showed significantly higher levels of distribution to inflamed paw tissues than did either unconjugated or fully 2l-O-methyl modified aptamers. Cellular distribution of radioactivity was also demonstrated by micro-autoradiography. The presence of labeled aptamer was observed in sinusoidal lining cells, and the proximal and distal tubular cells of the kidney, with different distribution patterns dependent on the aptamer administered. 17.7.4 Bioanalytical Methods for Aptamer Quantification

The concentration of full-length therapeutic aptamer conjugates and their metabolites in plasma and tissue may be quantified by a number of bioanalytical methods. These include radiometric methods such as LSC (see, for example, Reyderman and Stavchansky, 1997; Healy et al., 2004) and QWBA (Boomer et al., 2005), high-performance liquid chromatography (HPLC) (Tucker et al., 1999), capillary (CGE) and non-capillary gel electrophoresis (Reyderman and Stavchansky, 1997), hybridization-based ELISA-type assays (Yu et al., 2002; Healy et al., 2004), quantitative nucleic acid-intercalating dye (QNID) fluorescence assays (Archemix Corp), and mass spectrometry methods such as MALDI-TOF (Ono et al., 1997) or LC-MS (Andrews et al., 1999). These methods vary widely both mechanistically and in the degree of sample preparation or extraction required. Methods such as HPLC, LC-MS, and CGE require extraction of aptamer from the biological matrix (e.g. plasma or tissue) by liquid and/or solid-phase techniques, in addition to stringent desalting, prior to analysis. Similarly, LSC-based radiometric methods generally require modification or removal of biological matrix by solubilization or oxidation prior to analysis, whereas hybridization- and QNID fluorescence-based assay methods are homogeneous in format and do not necessitate extraction or separation steps. As many on the extraction techniques developed for use with antisense oligonucleotides are not applicable to primarily phosphodiester-backbone aptamers, new assay methods, particularly those which do not require extraction or separation steps, continue to be developed. A final critical issue for the bioanalysis of aptamers ex vivo is the determination not only of the amount of full-length oligonucleotide is present in a given biological sample, but the level of activity retained by the detected population of oligonucleotides. While antisense therapeutics may be considered active as long as they remain undegraded, the activity of an aptamer therapeutic is intimately reliant on maintenance of the proper three-dimensional conformation for binding to its cognate target. Several of the bioanalytical methods discussed will verify that an aptamer recovered from a biological sample is intact (i.e. that it is full-length and retains any conjugations such as PEG groups), however, none is capable of differentiatng between active (i.e. intact and correctly folded) and inactive (i.e. intact but improperly folded) full-length aptamer. Thus, in order to ascertain the true pharmacokinetic–pharmacodynamic relationship for an aptamer therapeutic, ex vivo samples must be assayed for both concentration and residual activity.

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An example of a typical pharmacodynamic activity assay for ex vivo samples is a competitive radioimmunoassay (RIA), where unlabeled aptamer from a biological sample competes with a known concentration of radiolabeled aptamer for binding to a known concentration of target. 17.7.5 Summary of Pharmacokinetic and Biodistribution Properties of Aptamers

In general, metabolic and nuclease stability is not limiting with respect to the pharmacokinetic properties of therapeutic aptamers. The terminal half-life of a typical therapeutic aptamers after parenteral administration (e.g. intravenous bolus) ranges from 3 to 22 h in rodent, and 30–60 h in primate, depending on sequence composition, modifications, and conjugation. An aptamer with a fixed composition and variable 5l-PEG conjugation (0, 20, 30, and 40 kDa) exhibits a variation in t1/2 (b) of approximately 5- to 20-fold. For a fixed aptamer sequence and 5l-PEG group with variable composition terminal half-life, t1/2 (b), varies approximately 14-fold, whereas for a fixed 5l-PEG conjugation (40 kDa) with variable sequence and composition t1/2 (b) varies approximately six-fold. Thus composition and conjugation (e.g. PEGylation) have significant and comparable effects on the systemic half-lives of therapeutic aptamers in vivo. Though the primary effect of PEGylation is to retard aptamer clearance, the presence of the 20 kDa moiety appears to facilitate distribution of aptamer to tissues, with sites of local inflammation showing significantly higher levels of accumulation than do either unconjugated or fully 2l-O-methyl modified aptamers with the same sequence. Typical aptamer volumes of distribution, Vss, vary from approximately two- to five-fold larger than the known plasma volume (Z40 mL/kg) in rodent and primate, depending on aptamer composition and PEGylation, suggesting significant distribution into tissues with a moderate degree of binding or sequestration of the aptamer to proteins and/or tissue matrix in the extravascular space. Therapeutic aptamers and their conjugates do not appear to cross the blood/brain barrier at a level greater than 0.1% of the total dose. Urinary elimination is a significant route of clearance for all aptamers. Major organs of accumulation include the liver, kidney, and other highly perfused tissues such as bone marrow. Cellular distribution of aptamers has been demonstrated by micro-autoradiography. The presence of radiolabeled aptamer can be observed in sinusoidal lining cells, and the proximal and distal tubular cells of the kidney following intravenous administration. Thus, the pharmacokinetic parameter space for therapeutic aptamers is large, while the existing data matrix is still relatively sparse. The process of data generation, filling out this matrix with respect to composition, PEGylation, route of administration, and model species, is still under way. All of these factors have been shown to have significant effects of comparable magnitude on the pharmacokinetic profile of aptamers in vivo. Finally, the ideal set of pharmacokinetic properties for therapeutic aptamers are not universal, but depend entirely on the specific indication and anticipated context of use.

17.8 Toxicity Profile of Aptamers

17.8 Toxicity Profile of Aptamers

Therapeutic aptamers do not appear to exhibit any intrinsic toxicity as a class in preclinical assessments. In contrast to many antisense oligonucleotides, which are known to exhibit hepatic and renal toxicities, therapeutic aptamers lack extensive phosphorothioate substitutions. Furthermore, aptamers are generally highly specific for their extracellular protein targets and are not known to exhibit nonspecific binding to other endogenous proteins. Single-dose and/or subchronic repeated dosing toxicity studies with Macugen/ EYE001 were conducted in rats, rabbits, and Rhesus monkeys in order to determine the maximum tolerated dose (MTD) and no-observable-adverse-effect-level (NOAEL) when administered by either intravitreous or intravenous routes (Drolet et al., 2000). Taken together, these studies provided compelling evidence that Macugen has minimal toxic potential. None of the preclinical work on Macugen indicated either a dose-limiting toxicity or a maximum tolerated dose (Drolet et al., 2000; Eyetech Study Group, 2002, 2003). There was no evidence of genotoxicity in the Ames reverse mutation or mouse lymphoma assays, no evidence of immunogenicity in studies in mice, rats, or rabbits, no evidence of complement activation in non-human primates, and no evidence of acute toxicity following single IV doses up to 450 mg/kg in rats and 5 mg/kg in non-human primates. In a 3-month IV rat study, there was no significant target organ toxicity at daily IV doses up to 10 mg/kg, which represents a daily systemic exposure of 1000times the plasma levels observed following an intravitreous bilateral dose of 0.5 mg in rabbits. No compound-related effects were observed in single-dose intravitreous toxicity studies of Macugen in rabbits or non-human primates. The Macugen (pegaptanib) studies represented the first opportunity for evaluation of potential toxicities of the chemical class of phosphodiester-linked 2l-fluoro, 2l-O-methyl-substituted oligonucleotides. Potential for toxicity of the 2l-fluoro nucleoside building blocks of the 2l-fluoro-substituted aptamer class has been evaluated in two species, rat and woodchuck (Richardson et al., 1999). 2l-Fluorouridine or 2l-fluorocytidine was administered to male rats by intravenous injection at doses of 5, 50, and 500 mg/kg/day for 90 consecutive days and to male and female woodchucks at doses of 0.75 and 7.5 mg/kg/day for 90 consecutive days. No adverse effects were observed in either species (Richardson et al., 1999). However, data suggesting the potential for 2l-fluoropyrimidines to be incorporated into nucleic acids of rat and woodchuck tissues following long-term administration (Richardson et al., 2002), and the apparent ability of human polymerases to polymerize 2l-fluoro NTPs in vitro (Richardson et al., 2000) may limit development of 2l-fluoro-substituted aptamers for certain chronic disease indications. Human polymerases cannot polymerize 2l-O-methyl dNTPs in vitro (Richardson et al., 2000).

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17.9 Immunogenicity of Aptamers

Studies in rodents have evaluated the immunogenic potential of aptamers (Vater and Klussmann, 2003; Wlotzka et al., 2002). Five ligands (one with 2l-deoxy nucleosides, one with 2l-NH2 -substituted pyrimidines, and three with 2l-fluoro-substituted pyrimidines with and without PEG) have been tested. The aptamers were administered intradermally, subcutaneously, or intravenously (depending upon the design in each study) in buffered saline each week for 4–8 weeks. Plasma was taken from the animals weekly after the second injection of aptamer. No anti-aptamer antibody could be detected in any of the animals in the treated groups at any time point regardless of whether the oligonucleotide was administered in saline alone or in saline plus Freund’s or Ribi adjuvant (intradermally or subcutaneously). These data indicate that aptamers have essentially no immunogenic potential in rodents. The immunogenic potential of aptamers has also been studied in both primates and humans. No antigenic response was detected in studies of single- and multiple-dose intravitreal injections of the anti-VEGF aptamer Macugen/EYE001 in Rhesus monkeys. Likewise, no antigenic response was observed in the Phase I clinical trial of Macugen for AMD (Eyetech Study Group, 2002).

17.10 Aptamer Manufacture

Aptamers are manufactured by chemical synthesis rather than biological expression as is the case for antibodies and other protein drugs. The current cost of goods for therapeutic aptamers is substantially more than for conventional small-molecule drugs, but is directly competitive with that for monoclonal antibodies. Process development for aptamer synthesis is benefiting from work with both antisense and ribozyme therapeutics. Over the last 10 years, the cost for oligonucleotide synthesis has dropped substantially as process chemistry has improved and as economies of scale have affected the material costs for oligonucleotide precursors. Similar improvements leading to an additional 10-fold drop in synthesis costs are anticipated by 2006. 17.10.1 Contributions to the Costs of Aptamer Synthesis

A handful of therapeutic oligonucleotides are currently under development through mid- and late-clinical phases. While simple DNA-based oligonucleotides can be synthesized for as little as US$200/g, more complex molecules with nonnatural monomers cost up to US$2000/g. Synthesis costs break down approximately 80:20 between raw materials and labor with phosphoramidites representing the major material expense (Z60%). The cost of phosphoramidites is partly

17.10 Aptamer Manufacture

controlled by growth in the oligonucleotide market as a whole and by licensing fees for basic phosphoramidite chemistry. Several key patents governing solidphase synthesis using phosphoramidites (e.g. US Patent 4,458,066, the “Caruthers” patent) will expire in the next few years, potentially widening the supplier base for these key reagents. Improvements in the process of oligonucleotide synthesis are continuing to increase the net yield from oligonucleotide synthesis while reducing the need for excess phosphoramidites to drive each coupling step. Similarly, improvements in solid support chemistries have increased the capacities for oligonucleotide synthesis. By reducing the volume required for a fixed synthesis scale, solvent and other reagents can be correspondingly lowered. Combining lower phosphoramidite costs, continued improvements in coupling efficiencies (overall yield), and support chemistries, the overall cost of oligonucleotide synthesis is predicted to drop to US$50–US$100/g in the next 4 years. 17.10.2 Manufacturing Infrastructure

Several companies, including Avecia, Proligo, and Sirna, have invested significantly in the development of therapeutic oligonucleotide manufacturing capabilities and will provide their expertise on a contract basis. For example, Avecia, currently the world’s leading oligonucleotide manufacturer, has built two large-scale chemical plants devoted to 100 kg/year-scale synthesis of oligonucleotide medicines. With cGMP manufacturing infrastructure and expertise in pharmaceutical synthesis (including the antisense therapeutics from Genta, AVI, and Isis), Avecia is well-positioned to drive further improvements in process chemistry and manufacturing economics. 17.10.3 Advantages of Chemical versus Biological Synthesis

In contrast to monoclonal antibodies, aptamer therapeutics are chemically manufactured rather than biologically expressed. Whereas the process for oligonucleotide synthesis has been shown to scale well from the microgram to the kilogram level, synthesis of proteins in bioreactors is known to change upon scale-up in ways that can fundamentally alter their therapeutic properties (e.g. extent of glycosylation). Markets for several antibody-based therapeutics, most notably Remicade and Enbrel (targeting TNF-a), are currently limited by production rather than demand. To meet increased market demand, several monoclonal antibody companies are making substantial capital investments in manufacturing (e.g. Immunex $500-million plant in West Greenwich, RI, USA). By comparison, building increased capacity in nucleic acid synthesis is relatively inexpensive.

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17.11 Examples of Therapeutic Aptamers in Development 17.11.1 Antithrombin Aptamer ARC183

Heparin has been used extensively, and almost exclusively, as the anticoagulant during cardiac surgery, including CABG procedures. The reasons for its use are related to low cost, ease of monitoring (by activated clotting time (ACT)), reversibility (using protamine) and lack of a suitable anticoagulant replacement. There are, however, several limitations to the use of heparin, including: a long halflife requiring protamine to be used as an antidote; heparin-induced thrombocytopenia in 2–5% of patients exposed; significant non-specific plasma binding; heparin cannot inhibit clot bound thrombin; and heparin has been associated with platelet aggregation and dysfunction (Warkentin and Greinacher, 2003; Adler, 2004). Moreover, protamine has been associated with immune-mediated reactions, and its short half-life (Z5 min) may result in unopposed heparin effects post operatively (Butterworth et al., 2002). The adverse properties and limitations of heparin with protamine illustrate that this combination of agents is not ideal for anticoagulation during cardiac surgery. Consequently, a number of newer, higher cost anticoagulants, such as low-molecular-weight heparins and Angiomax (Bivalirudin), are being developed for this market. However, these compounds have similar side effects and their anticoagulation activity cannot be reversed rapidly. There is a significant unmet medical need for a safe, moderate-cost anticoagulant that does not require a separate reversing agent. The antithrombin aptamer drug ARC183 (Archemix Corp) is a thrombin inhibitor in development for use as an anticoagulant during CABG procedures (Bock et al., 1992). ARC183 is an all-DNA molecule, 15 nucleotides in length, and comprised entirely of G and T residues having the following sequence: 5l GGT TGG TGT GGT TGG 3l. NMR studies performed by Wang et al (1993) indicate that ARC183 folds into a stable “chair-like” structure as depicted in Fig. 17.3.

Fig. 17.3 Folded structure of antithrombin aptamer ARC183.

17.11 Examples of Therapeutic Aptamers in Development

ARC183 exhibits a Kd of 2 nmol/L for thrombin, 50 nmol/L for prothrombin, and binding to other serum proteins or proteolytic enzymes is essentially undetectable (Bock et al., 1992). It is a strong anticoagulant in vitro, and inhibits thrombin-catalyzed activation of fibrinogen, and thrombin-induced platelet aggregation. ARC183 has key advantages in that it is a specific inhibitor with rapid onset, is effective at inhibiting clot-bound thrombin and has a short in vivo half-life that allows for rapid reversal of its effects and the avoidance of the dose-adjusting complications of heparin and protamine (Lee et al., 1995). Neither significant toxicities nor excessive bleeding intraoperatively have been observed in preclinical studies (DeAnda et al., 1994). ARC183-dependent anticoagulation occurs within minutes (Griffin et al., 1993; Lee et al., 1995). It is a potent anticoagulant in dog and monkey models of cardiopulmonary bypass, yielding dose-dependent ACT values of 1500 s, at a 0.5 mg/ kg/min dose (DeAnda et al., 1994). ARC183 exhibits a very short functional half-life in vivo of Z2 min, thus allowing for rapid reversal of the anticoagulant effects (Lee et al., 1995). It is cleared through the action of serum nucleases (Shaw et al., 1995) and through renal elimination. No acute toxicities have been observed, nor did ARC183 show any evidence of genotoxicity in preclinical testing. These findings indicate that the agent will continue to show an improved efficacy and safety profile over that of heparin. Clinical trials of ARC183 began in 2004. 17.11.2 Anticomplement C5 Aptamer ARC187

Complement is a system of i30 plasma and membrane-bound proteins that contributes essential functions to both adaptive and innate immunity. Activation of the complement system (e.g. following infection) can occur by a variety of molecular-recognition events which all converge in the generation of a common set of molecular effectors that mediate the principal activities of complement, including the innate defense against foreign pathogens, the clearing of immune complexes and cellular debris, the stimulation of inflammatory responses and the induction of B-cell proliferation. Natural inhibitors of complement tightly regulate its activity to ensure that healthy host cells are not targeted. However, disease, and the interventions used to treat it, can lead to a breakdown in this control (Makrides, 1998). Three pathways have been described for the early phases of complement activation: classical, lectin, and alternative (Fig. 17.4). The classical pathway is normally activated by antigen–antibody (IgG or IgM) complexes, which bind to the C1q subunit of complement component C1. The lectin pathway is similar to the classical pathway, except that mannose-binding lectin (MBL) takes the place of C1. The alternative pathway lacks a triggering factor analogous to C1 and MBP, but is activated instead through a C3-dependent mechanism. The effector molecules produced upon activation of the complement cascade promote immunity by several different mechanisms. The proteolytic fragments C3a and C5a are anaphyla-

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Fig. 17.4 The three complement activation pathways converge to yield activated C5.

toxins that bind to G protein-coupled receptors on the surfaces of phagocytes, stimulating their activation. C5a is a potent chemoattractant and activator of neutrophils in particular, promoting neutrophil degranulation and extravasation. C3b, in addition to its role as a component of the C3 and C5 convertases, is an opsonin, coating the surfaces of cells to identify them as targets for phagocytosis. C5b also binds to cell surfaces, promoting the assembly of the transmembrane C5b-9 (C5b·C6·C7·C8·C9n) or membrane attack complex (MAC) which has both lytic and proinflammatory activities (for review, see Klickstein et al., 2001). Inappropriate or excessive activation of the complement system can lead to harmful, potentially life-threatening consequences due to severe inflammation. These consequences are clinically manifested in various disease states, including septic shock and multiple organ failure. Anticomplement therapy has been proposed for use in CABG surgery among other possible indications (e.g. for the prevention of transplant rejection and for the treatment of sepsis). Activation of the complement cascade during CABG surgery is associated with significant morbidity and mortality mediated by C5 cleavage products C5a and C5b-9. Biesecker and co-workers (Biesecker et al., 1999; Biesecker and Gold, 2002) originally selected high-affinity aptamers to complement C5 which have been studied extensively at Archemix Corp. ARC186 binds to purified human C5 protein with a Kd of 0.6 e 0.1 nmol/L as determined in nitrocellulose filter assays. The aptamer blocks conversion to C5a and C5b catalyzed by the C5 convertase, and is a potent inhibitor of complement C5 activation in vitro. Following a single IV bolus, a PEG-conjugated derivative of ARC186 sustains an inhibitory effect for up to 12 h in cynomolgus macaques, as determined ex vivo by activation of plasma samples. The properties of ARC187 make it an attractive candidate for the treatment of complement-meditated disease. 17.11.3 Anti-L-Selectin Aptamer

Selectins are a family of calcium-dependent cell surface lectins. Selectins recognize complex anionic carbohydrate ligands, mediate cell adhesion and initiate many cell–cell interactions within the vasculature. In both normal and pathological conditions selectins participate in leukocyte extravasation from the vasculature into tissues. L-Selectin is constitutively expressed on leukocytes. Aptamers tar-

17.11 Examples of Therapeutic Aptamers in Development

geted to L-selectin block L-selectin-dependent lymphocyte trafficking in vivo, and may be useful in treating diseases in which tissue damage is neutrophilmediated. The L-selectin aptamer (1d40) binds to its target with greater specificity and affinity than does sialyl Lewis X, a tetrasaccharide glyconjugate membrane protein, which binds equally well to L-, E-, and P-selectins. 1d40 shows little, if any, affinity for E- and P-selectins (Hicke et al., 1996). It binds to native L-selectin on peripheral blood lymphocytes, and also blocks L-selectin interactions with natural ligands upon endothelial venules. 1d40 also blocks L-selectin-dependent adhesion to neutrophils (Hicke et al., 1996). 1d40 is 34 nucleotides long, is of 2l-ribopurine 2l-fluoropyrimidine (rRfY) composition, 3l-inverted dT-capped and 5l-40 kDa PEG-capped and therefore nuclease resistant. It has a Kd of 450 pmol/L for L-selectin (Watson et al., 2000) and Kdvalues of 150 nmol/L and 750 nmol/L for E- and P-selectin respectively. 1d40 was selected at 37 hC after earlier selections at lower temperatures yielded aptamers that bound poorly when the temperature was raised to 37 hC (Hicke et al., 1996; O’Connell et al., 1996). Selection was against a recombinant human L-selectin–IgG fusion protein in which the protein was immobilized on protein A sepharose beads. Elution was performed with EDTA since the binding of adhesion-blocking aptamers had already been shown to be divalent ion-dependent (Hicke et al., 1996; O’Connell et al., 1996). 1d40 exhibits a pharmacokinetic half-life of 3.8 h when administered intravenously to the rat at 1 mg/kg. In this study the blood concentration of aptamer was still four-fold above the Kd 24 h after administration (Watson et al., 2000). In in vivo experiments that measure the duration of effect (T50) of blocking lymphocyte trafficking in SCID mice, the 1d40 aptamer exhibits a T50 -value of 670 min (Watson et al., 2000). Furthermore, 1d40 blocks L-selectin binding to GlyCAM-1 and prevents lymphocyte binding to endothelial venules (O’Connell et al., 1996). Therapeutic areas for which L-selectin inhibitor 1d40 may potentially be useful include anti-inflammatory and antithrombolytic indications. 17.11.4 Anti-PDGF-BB Aptamer ARC127

Platelet-derived growth factor (PDGF) is a mitogen for fibroblasts, smooth muscle cells, mesangial cells, and a variety of other cell types (reviewed in Heldin and Westermark, 1999). PDGF exists in several disulfide linked hetero- and homodimeric isoforms, the most extensively studied being PDGF-BB, PDGF-AB, and PDGF-AA. The factors activate the homo- and heterodimeric receptor tyrosine kinases PDGFR-bb, PDGFR-ab, and PDGFR-aa. PDGF has been implicated in a number of proliferative disease states including glomerulonephritis, restenosis, proliferative diabetic retinopathy, and cancer (Pietras et al., 2003). Green et al. (1996) selected an aptamer that is highly selective for PDGF-BB and PDGF-AB (Kd of 100 pmol/L) versus PDGF-AA (Kd 70 nM). The aptamer, which was selected using a DNA pool, was modified with 2l-O-methylpurine

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and 2l-fluoropyrimidine residues and capped with 3l-inverted deoxythymidine to enhance its stability to nucleases. A 40 kDa PEG group was appended to the 5l end to increase lifetime in plasma by reducing renal filtration (Floege et al., 1999). The resulting aptamer, designated ARC127, retains PDGF-binding activity and inhibits PDGF-BB-dependent cell proliferation in vitro. ARC127 has demonstrated efficacy in several animal model systems for human proliferative disease. Intimal hyperplasia, or blockage of the vessel lumen, is a common complication of percutaneous coronary intervention (angioplasty) and bypass grafting. Blockage is postulated to be caused by PDGF-mediated proliferative and migratory response of smooth muscle cells on the arterial wall. Leppanen et al. (2000) showed that when administered at a dose of 2 mg/kg/day for 2 weeks to rats that had suffered carotid injury, ARC127 inhibited intimal hyperplasia by 50% compared with vehicle controls. The effect was not sustained, however, 8 weeks after cessation of treatment. Many progressive renal diseases are characterized by PDGF-mediated proliferation of glomerular mesangial cells. In two experimental models of mesangioproliferative glomerulonephritis, ARC127 significantly reduced the number of glomerular mitoses in comparison with rats treated with vehicle or a scrambled, non-binding aptamer sequence (Floege et al., 1999; Ostendorf et al., 2001). Although rats were treated for only 4 days after disease induction, significant beneficial effects were observed at 100 days, including reduced levels of proteinuria, tubulointerstitial damage, and renal extracellular matrix accumulation. ARC127 may have therapeutic value for cancer treatment, given the putative role of PDGF in regulating interstitial fluid pressure (IFP) and, by extension, uptake of chemotherapeutic agents in to tumors. Most solid tumors are characterized by interstitial hypertension, which impairs the transport of solutes, including chemotherapeutic agents, across the capillary membrane into the tumor interstitium. Connective tissues are thought to control IFP by exerting tension on the collagen/microfibrillar network. PDGF is known to upregulate synthesis of collagen and to mediate interactions of anchor proteins such as integrins with extracellular matrix components (Heuchel et al., 1999), suggesting a role for PDGF signaling in mediating IFP. Pietras et al. (2001) demonstrated that ARC127 treatment lowered the IFP in PROb tumors grown subcutaneously in rats by 30% as compared to scrambled aptamer-treated animals. In a follow-up study, the authors investigated the effect of ARC127 treatment on IFP and cytotoxic uptake in KAT-4 tumors, which are stroma-rich subcutaneous tumors derived from a human anaplastic thyroid carcinoma. As observed with PROb tumors, treatment with ARC127 resulted in an approximately 40% decrease in IFP. Animals treated with ARC127 showed a 2.5-fold increase in uptake of [3H]-taxol into tumor tissue. Most importantly, animals treated simultaneously with ARC127 and taxol showed a 35% reduction in tumor volume versus animals treated with the same dose of taxol alone. Analysis of homogenates from the tumors revealed that PDGFR-b receptors in ARC127-treated tumors displayed significantly lower activation than observed in control groups (Pietras et al., 2002). These studies suggest ARC127 may find utility when used in conjunction with approved cytotoxic agents.

17.12 Future Prospects for Aptamer Therapeutics

Anti-PDGF treatment may also be efficacious in the treatment of angiogenesismediated diseases. Angiogenesis is critical for normal development but also plays a very important role in the progression of various pathologies, including cancer and proliferative retinopathies. Angiogenesis requires the proliferation and sprouting of vascular endothelium, which is primarily modulated by VEGF (Ferrara, 2004). In addition to VEGF, PDGF-BB is also involved in regulating vascular maturation as is required for the recruitment of mural cells (i.e. pericytes, vascular smooth muscle cells) to the newly formed vessels, and thereby stabilizes the vessels. Disruption of the PDGF-BB/PDGFR-b receptor system by means of blocking agents or gene knockout results in immature, unstable, and non-functional vessels that lack mural cell coverage (Leveen et al., 1994; Hellstrom et al., 2001). A recent study by Bergers et al. (2003) demonstrated that combination therapy using anti-VEGF and anti-PDGF-BB agents proves more effective in regressing pathological vessels than either therapy alone in the Rip1Tag2 murine cancer model, validating the concept of targeting both endothelial cells and mural cells in anti-angiogenesis therapy.

17.12 Future Prospects for Aptamer Therapeutics

High-affinity aptamers can be generated readily against a variety of therapeutic protein targets, best illustrated perhaps, by the steady progression of aptamers selected against extracellular growth factors (VEGF, PDGF) and components of coagulation (thrombin, Factor IXa) and complement (C5) pathways through preclinical and clinical testing. With remarkable target specificity, versatile pharmacokinetic properties, ease of synthesis, and relatively low manufacturing costs, aptamers are becoming established as a promising new class of medicines. Aptamers can be stabilized and shielded from renal filtration by a variety of chemical and compositional modifications for assessment in in vivo preclinical discovery programs and, ultimately, entry into the clinic. Recent FDA approval of Eyetech/ Pfizer’s aptamer therapeutic (Macugen) for treatment of AMD is extremely encouraging and bodes well for the future of aptamer therapeutics in general. With a number of additional aptamers expected to enter clinical trials over the next year, aptamers appear poised to make a significant contribution to the treatment of acute and chronic diseases.

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17 Properties of Therapeutic Aptamers to factor VIIa. Thromb Haemost 84, 841– 848. Rusconi, C. P., Scardino, E., Layzer, J., Pitoc, G. A., Ortel, T. L., Monroe, D., Sullenger, B. A. (2002). RNA aptamers as reversible antagonists of coagulation factor IXa. Nature 419, 90–94. Rusconi, C. P., Roberts, J. D., Pitoc, G. A., Nimjee, S. M., White, R. R., Quick, G., Jr., Scardino, E., Fay, W. P., Sullenger, B. A. (2004). Antidote-mediated control of an anticoagulant aptamer in vivo. Nat Biotechnol 22, 1423–1428. Saitoh, H., Nakamura, A., Kuwahara, M., Ozaki, H., Sawai, H. (2002). Modified DNA aptamers against sweet agent aspartame. Nucleic Acids Res Suppl, 215–216. Sakamoto, T., Miyazaki, E., Aramaki, Y., Arima, H., Takahashi, M., Kato, Y., Koga, M., Tsuchiya, S. (2004). Improvement of dermatitis by iontophoretically delivered antisense oligonucleotides for interleukin10 in NC/Nga mice. Gene Ther 11, 317–324. Sandrasagra, A., Leonard, S. A., Tang, L., Teng, K., Li, Y., Ball, H. A., Mannion, J. C., Nyce, J. W. (2002). Discovery and development of respirable antisense therapeutics for asthma. Antisense Nucleic Acid Drug Dev 12, 177–181. Santulli-Marotto, S., Nair, S. K., Rusconi, C., Sullenger, B., Gilboa, E. (2003). Multivalent RNA aptamers that inhibit CTLA-4 and enhance tumor immunity. Cancer Res 63, 7483–7489. Seiwert, S. D., Stines Nahreini, T., Aigner, S., Ahn, N. G., Uhlenbeck, O. C. (2000). RNA aptamers as pathway-specific MAP kinase inhibitors. Chem Biol 7, 833–843. Shadidi, M., Sioud, M. (2003). Identification of novel carrier peptides for the specific delivery of therapeutics into cancer cells. FASEB J 17, 256–258. Shargel, L., Yu, A. (1999). Applied Biopharmaceutics and Pharmacokinetics. New York: McGraw-Hill, pp. 108–154. Shaw, J. P., Fishback, J. A., Cundy, K. C., Lee, W. A. (1995). A novel oligodeoxynucleotide inhibitor of thrombin. I. In vitro metabolic stability in plasma and serum. Pharm Res 12, 1937–1942. Shea, R. G., Marsters, J. C., Bischofberger, N. (1990). Synthesis, hybridization properties and antiviral activity of lipid-oligodeoxynu-

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tors of Trypanosoma cruzi and inhibit cell invasion. J Biol Chem 277, 20756–20762. Vaish, N. K., Larralde, R., Fraley, A. W., Szostak, J. W., McLaughlin, L. W. (2003). A novel, modification-dependent ATP-binding aptamer selected from an RNA library incorporating a cationic functionality. Biochemistry 42, 8842–8851. Vant-Hull, B., Payano-Baez, A., Davis, R. H., Gold, L. (1998). The mathematics of SELEX against complex targets. J Mol Biol 278, 579–597. Vater, A., Klussmann, S. (2003). Toward thirdgeneration aptamers: Spiegelmers and their therapeutic prospects. Curr Opin Drug Discov Devel 6, 253–261. Vinogradov, S. V., Suzdaltseva, Y. G., Kabanov, A. V. (1996). Block polycationic oligonucleotide derivative: synthesis and inhibition of herpes virus reproduction. Bioconjug Chem 7, 3–6. Vives, E., Brodin, P., Lebleu, B. (1997). A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J Biol Chem 272, 16010–16017. Wang, H., Cai, Q., Zeng, X., Yu, D., Agrawal, S., Zhang, R. (1999). Antitumor activity and pharmacokinetics of a mixed-backbone antisense oligonucleotide targeted to the RIalpha subunit of protein kinase A after oral administration. Proc Natl Acad Sci USA 96, 13989–13994. Wang, K. Y., McCurdy, S., Shea, R. G., Swaminathan, S., Bolton, P. H. (1993). A DNA aptamer which binds to and inhibits thrombin exhibits a new structural motif for DNA. Biochemistry 32, 1899–1904. Warkentin, T. E., Greinacher, A. (2003). Heparin-induced thrombocytopenia and cardiac surgery. Ann Thorac Surg 76, 2121–2131. Watson, S. R., Chang, Y. F., O’Connell, D., Weigand, L., Ringquist, S., Parma, D. H. (2000). Anti-L-selectin aptamers: binding characteristics, pharmacokinetic parameters, and activity against an intravascular target in vivo. Antisense Nucleic Acid Drug Dev 10, 63–75. White, P. J., Gray, A. C., Fogarty, R. D., Sinclair, R. D., Thumiger, S. P., Werther, G. A., Wraight, C. J. (2002). C-5 propyne-modified oligonucleotides penetrate the epidermis in

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18.1 Evolutionary Selection Techniques

18 Spiegelmers for Therapeutic Applications – Use of Chiral Principles in Evolutionary Selection Techniques Dirk Eulberg, Florian Jarosch, Stefan Vonhoff, and Sven Klussmann

18.1 Evolutionary Selection Techniques

Screening of compound libraries usually starts with either the synthesis or the isolation of individual molecular entities which are subsequently tested for a desired activity or their suitability for a particular purpose. In contrast to this, employing evolutionary selection techniques rather means to act as custodian of a randomly generated, extremely diverse population of artificial chemical entities that can be evolved or even evolve themselves in vitro as if they were organisms that are subjected to natural (Darwinian) selection. In this “artificial biosphere,” the survival or extinction of an individual molecular entity is determined by its fitness – which is imposed to the selection process by the experimenter as desired. Such libraries do not need sophisticated high-throughput technologies for screening; in fact, they are easily obtained and can be selected with basic technology present in any molecular biology laboratory. Two techniques are currently most popular: Phage display for the isolation of peptidic ligands and the SELEX process (systematic evolution of ligands by exponential enrichment) for the generation of nucleic acid-based ligands. In phage display, the target of interest is used to select interacting ligands from up to 1011 bacteriophage particles which differ molecularly on their protein surfaces. From the mid-1980s, engineered filamentous phages such as M13 or fd have been used as libraries of genetic information. Such phage particles are still infectious and able to propagate, thus they can pass along their genotype to the progeny (Smith, 1985). The diversity of such libraries originates in combinatorial chemistry (i.e. DNA of random sequence). The encoded random peptides are then displayed on the surface of the phage. By performing successive rounds of selection with the target of interest and subsequent propagation, phages which specifically interact with the target can be identified (Bass et al., 1990; Scott and Smith, 1990). The in vitro selection of target-binding nucleic acids, also known as SELEX, was established in 1990 independently in three groups (Ellington and Szostak, 1990; The Aptamer Handbook. Edited by S. Klussmann Copyright c 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-31059-2

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Robertson and Joyce, 1990; Tuerk and Gold, 1990). In contrast to phage display, where the phage particle provides the link between genotype and the selected phenotype (the displayed peptide), in the SELEX process the members of the oligonucleotide libraries are directly subjected to the selection step. In fact, oligonucleotides combine the “phenotype” and the “genotype” within one molecule. This means that every individual oligonucleotide not only forms a distinct three-dimensional structure that is able to interact specifically with a potential target by shape complementarity, but at the same time contains the necessary information for its own propagation. Amplification of selected oligonucleotides is easily achieved in vitro by employing natural RNA and/or DNA polymerases (i.e. polymerase chain reaction (PCR) in the case of DNA-SELEX and RT-PCR plus transcription for the RNA-SELEX). The number of individual molecules that are routinely used at the beginning of an oligonucleotide in vitro selection scheme is approximately 105 times higher than with phage display. For the resulting individual binding nucleic acid molecules, Ellington and Szostak coined the term “aptamer” from the Latin word aptus, to fit. Ligands generated by phage display and SELEX have in common that they are polymers built of naturally occurring building blocks: l-amino acids and d-nucleotides, respectively. From a chemical point of view, these l-polypeptide and d-oligonucleotide compounds are stable. However, the omnipresence of degrading enzymatic activities by proteases and nucleases accounts for their rapid decomposition, especially in biological systems. Further, artificial l-polypeptides often induce a vigorous humoral immune response that impairs their use in animals and humans. Much progress has been made over the past few years on the improvement of biostability of nucleic acid aptamers (Lin et al., 1994; Green et al., 1995; Jellinek et al., 1995; Burmeister et al., 2005). The efforts mainly include the use of partially modified libraries (e.g. 2l-fluoro or 2l-amino modifications in pyrimidine nucleotides) which are still accepted by the polymerases that are employed within the SELEX process. These modifications to aptamers are also referred to as pre-SELEX modifications. However, the use of modified nucleotides may affect the efficiencies of the used enzymes and thus influences the selection process. After a successful SELEX experiment, the identified sequences can also be subjected to the so-called post-SELEX modifications. Here, the remaining natural nucleotides (mainly purine nucleotides) are exchanged to more biostable nucleotides (mostly 2l-O-methyl-nucleotides). These modifications have to be introduced very carefully because every nucleotide replacement may destroy the active threedimensional structure that is responsible for the binding event. An elegant possibility to achieve biostability, however, is the use of either mirror-image peptides or mirror-image oligonucleotides. These polymers are not susceptible to nucleases since nature did not evolve any respective enzymatic degrading activities. Consequently, nature did not evolve any anabolic activities either. Nevertheless, by exploiting the principles of stereochemistry, mirror-image polymers can be successfully integrated into evolutionary screening techniques. The basis for this concept is the phenomenon of chirality that was discovered more than 150 years ago (Pasteur, 1848).

18.2 Chirality

18.2 Chirality 18.2.1 Discovery and Consequences of Nature’s Handedness

“Chirality” is derived from the Greek word for hand (cher), meaning the characteristic of particular objects, like the right and left hand of a person, not being able to be brought into congruence with their mirror images. The absence of a symmetric plane or center in a molecule implicates the existence of two (stereo) isomers, which correspond to each other like image and mirror image. They are designated enantiomers or optical antipodes. The asymmetry of chiral compounds is the cause of their optical activity. In 1844, the German crystallographer Eilhard Mitscherlich announced a puzzling fact. Chemically synthesized sodium-ammonium paratartrate and sodiumammonium tartrate isolated from grapes had the same chemical formula and were identical in every respect save one: solutions of the tartrate rotated polarized light to the right, while paratartrate exerted no effect on light. This finding was in contradiction to the proposal of Michel Chevreul, who had postulated in 1823 that “species of compound bodies are identical when the nature, proportion and arrangement of the elements are the same.” According to these criteria, Mitscherlich’s tartrate and paratartrate should have been totally indistinguishable. Louis Pasteur was the one who resolved the enigma in 1848, when he showed that the optical difference between the two sodium-ammonium salts could be correlated with subtle structural difference of the crystals that could be obtained from tartrate and paratartrate, respectively (Pasteur, 1848). By means of careful experimentation, he succeeded in separating two different types of crystals that had grown in the paratartrate solution. Solutions of these two crystalline forms rotated the plane of polarized light in equal but opposite directions, one to the left, and the other to the right. Pasteur concluded that the opposite optical activity of the dissolved crystals was caused by the nature of the molecules themselves. In 1857, Pasteur discovered that a sample of synthetically prepared – originally optically inactive – paratartrate had become optically active after growth of a mold in the solution. Further examining the sample, he recognized that it contained more (–) than (+) tartaric acid. Pasteur deduced that the mold, probably Penicillium glaucum, had metabolized the familiar (+) form, which is the main form present in grapes. What he had discovered was a very elegant method of separating enantiomers by reacting one of them with a chiral catalyst. In this case, the catalyst was a microorganism whose chemistry obviously preferred a specific chirality. Consequently, he proposed in 1874: “The universe is asymmetric and I am persuaded that life, as it is known to us, is a direct result of the asymmetry of the universe or of its indirect consequences.” Later, in 1874, Le Bel and van’t Hoff independently advanced the concept of molecular asymmetry by developing a three-dimensional model for the carbon atom with its four tetrahedric valences (LeBel, 1874; van’t Hoff, 1874). Their theoretical

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predictions of the possible stereoisomers for distinct numbers of asymmetric carbon atoms were experimentally corroborated by Emil Fischer’s work with sugar molecules during the years 1884 to 1908. In his “key and lock” hypothesis he postulated that two chiral partners interact through shape complementarity and are thus stereospecific (Fischer, 1894). In 1933, the importance of stereocomplementarity between receptor–ligand (drug) interactions was recognized and described in a three-point attachment model (Easson and Stedman, 1933): Mirror-image spatial relation of atoms in optical isomers should preclude them from interacting in an identical fashion with three points on a receptor surface. After having studied proteolytic enzymes, Bergmann and Fruton developed the “polyaffinity” theory for enzyme–substrate chiral recognition in 1937 (Bergmann and Fruton, 1937). This theory states that enzymes exhibiting chiral substrate specificity must contain several active groups in a fixed asymmetric arrangement that interact with a similar number of active groups of the substrate. During enzymatic action the active groups of the substrate are forced into a fixed spatial position. The fact that natural proteinases have well-defined binding sites which only recognize l-polypeptides and do not bind the d-enantiomers was finally described in 1967 (Schechter and Berger, 1967). 18.2.2 Mirror-Image Proteins

Except for some lower organisms that use d-amino acids in certain specialized molecules like cell walls and antibiotics (Kleinkauf et al., 1969; Lee and Lipmann, 1977; Lipmann et al., 1941), proteins are always composed entirely of l-residues. For a long term it was merely idle speculation how mirror-image proteins or “Doppelgnger” proteins consisting exclusively of d-amino acids might fold and how they might interact with chiral substrates (Robson, 1996). In 1990 Robert Merrifield’s and Mati Fridkin’s labs demonstrated that it is possible to synthesize functional d-peptides: both stereoisomers of the channel-forming helical antibiotic peptides cecropin, magainin-2 amide, and melittin acted functionally identically in the achiral environment of lipid bilayer and water (Bessalle et al., 1990; Wade et al., 1990). But these peptides were not large enough to exhibit defined globular folds which are characteristic for the native state of larger proteins. With the availability of increasingly powerful methods for the chemical synthesis of peptides, including those containing unnatural amino acids, it has been possible to produce totally synthetic folded proteins (Kent, 1988). Rubredoxin from Desulfovibrio desulfuricans which adopts a short three-stranded antiparallel b-sheet fold with a number of loops was the first protein to be synthesized in all-d configuration (Zawadzke and Berg, 1992). It was intended to generate racemic, centrosymmetrical crystals from a mixture of d- and l-rubredoxin to facilitate derivation of very high-quality electron density maps (Zawadzke and Berg, 1993). They reported that both isoforms showed identical binding of (achiral) Fe2+ and Co2+ ions and that the d-protein half-life in serum was much greater than that

18.2 Chirality

of its l-counterpart. Moreover, it could be shown in an immunogenicity study that only l-rubredoxin elicited an immune response after repeated, adjuvant-assisted administration to mice (Dintzis et al., 1993). It appears that despite their large size, d-proteins may not be recognized by the body, a fact that has caused some researchers to call d-proteins “stealth proteins” (Robson, 1996). The first chemical synthesis of an active enzyme in the d-configuration was achieved by Kent’s group with preparation of the d- and l-form of the 99 amino acid-long HIV-1 protease in 1992 (Milton et al., 1992). As expected, the folded protein–enzyme enantiomers showed reciprocal chiral specificity on peptide substrates (i.e. each enzyme enantiomer cut only the corresponding substrate enantiomer). Moreover, the achiral Evan’s Blue potently inhibited both stereoisomers, whereas a “matching” enantiomeric inhibitor (pseudohexapeptide amide) only worked against its corresponding enantiomeric HIV protease. While solid-phase peptide synthesis is currently limited to sequences of approximately 100 amino acids, longer polypeptides or even proteins can be obtained using peptide-ligation techniques (Borgia and Fields, 2000; Kochendoerfer, 2001; Wehofsky et al., 2003). Virtually unlimited access to mirror-image proteins might finally be achieved using modified cell-free protein-synthesizing systems. It still remains to be demonstrated if this is practicable. To date, cell-free expression systems that employ mutated ribosomes have the ability to incorporate two different d-amino acids into proteins (Dedkova et al., 2003). 18.2.3 Mirror-Image Nucleic Acids

Holy´ and co-workers started synthesizing the first nucleotides of the l-series in 1969 (Holy´, 1972; Holy´ and orm, 1969; Robins et al., 1970). In order to investigate the properties of l-nucleic acids, it was necessary to synthesize either l-RNA or l-DNA oligonucleotides. In 1984, Anderson and co-workers described the preparation of an 18mer consisting of l-deoxyuridine residues (Anderson et al., 1984). He already recognized that the l-oligonucleotide is much more resistant to enzymatic degradation than its d-counterpart of the natural handedness. Soon after, short l-RNA oligonucleotides (not containing all four different nucleotides) were prepared to investigate their interaction with strands of the natural handedness (Visser et al., 1986; Ashley, 1992). It was hypothesized that l-oligonucleotides could be used as antisense molecules to block the activity of natural messenger RNAs (Fujimori et al., 1990a). However, with the availability of all four building blocks and the possibility of synthesizing longer molecules more efficiently, l-oligonucleotides were excluded from being used as antimessenger oligonucleotides (Urata et al., 1992; Garbesi et al., 1993). Besides biophysical techniques such as circular dichroism, little information about the three-dimensional structures of l-oligonucleotides is available. Recently, the crystallographic analysis of a short RNA helix r(CUGGGCGG) · r(CCGCCUGG) totally composed of mirror-image nucleotides was reported (Vallazza et al., 2004). The results were compared with a previously determined

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d-RNA duplex structure of the same sequence. Although enantiomers should display identical chemical and physical features with regard to solvents, the crystallization screens using conditions close to those optimized for the d-RNA duplex surprisingly failed. Conditions could be identified that yielded crystals for both enantiomers, but the d-RNA and l-RNA crystals at each condition differed sharply in size, shape, and diffraction quality. Obviously, the two enantiomers required different conditions to produce crystals good enough for diffraction experiments. The l-RNA duplex adopts a standard A-form conformation with all the helical parameters resembling those of a piece of A-RNA. Some distinguishing features between these l-RNA and d-RNA structures – disappearance of a wobble-like GC+ base pair and the altered hydration in the l-RNA structure – may probably be explained by the different crystallization conditions.

18.3 Mirror-Image Evolutionary Techniques: Selection–Reflection

The general rule that life prefers distinct stereoisomers for its biomolecules applies to all natural biopolymers. Accordingly, interactions between biomolecules are governed by lock-and-key stereocomplementarity (Fischer, 1894; Gilbert and Greenberg, 1984). On the one hand, catalytic activities exist that synthesize these biomolecules from naturally occurring building blocks. On the other hand, metabolizing activities are responsible for degrading the biopolymers to recover the building blocks. Accordingly, artificial biopolymers that may result from evolutionary screening techniques are composed of natural building blocks and are subject to degradation processes as well. However, l-nucleic acids or d-peptides that consist of building blocks of non-natural chirality escape from enzymatic recognition and subsequent degradation. As a result of the same principle nature developed no enzymatic activities to amplify mirror-image nucleic acids or to build mirror-image peptides or proteins. Applying the principles of stereochemistry, however, reveals a method by which oligonucleotides or polypeptides could be introduced into an evolutionary selection scheme, resulting in biostable, mirror-image compounds. Considering a 1:1 complex between a target and a binding molecule and taking into account that each molecule can occur in two configurations, four possible potential pairings could be formed (Fig. 18.1). The most obvious situation is a complex that is formed between an “aptamer” (natural configuration) that binds to a “natural target” (e.g. polypeptide of natural configuration) (Fig. 18.1a). If both interacting partners are synthesized in their respective mirror-image configuration, resulting in a “mirror-image aptamer” and a “mirror-image target”, the same complex is formed as before, but mirror-inverted (Fig. 18.1b). Both (mirror-image) configurations are not “from this world” and therefore are only of limited use if at all. There are two further possibilities how interacting partners of different chiralities could be formed. In the first instance an “aptamer” of the natural configuration binds to a polypeptide of the unnatural configuration, a “mirror-image tar-


Fig. 18.1 Complex formation. There are four different possibilities for formation of a complex between two chiral molecules (a–d). If two molecules of the “natural” configuration – an aptamer and a target – build a complex (a), the same complex – mirror-inverted – could be formed with the same sequences of the “unnatural” or mirror-image configurations (b: mirror-image target recognized by a mirror-

image aptamer). However, a complex can also be formed between a molecule of the “natural” configuration and a molecule of the “unnatural” configuration (c: aptamer binding to a mirror-image target). Again, this complex can be mirrored to yield a mirror-image nucleic acid (Spiegelmer) binding to a natural target (d).

get” (Fig. 18.1c). The other possibility can be gained by generating the mirrorimage of this complex. The result is an aptamer of the mirror-image configuration that binds a natural target (Fig. 18.1d). To differentiate a “mirror-image aptamer” from a “normal aptamer” the term “Spiegelmer” (Spiegel is the German word for mirror) was coined (Klussmann et al., 1996). Through this principle it is possible

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to generate biostable (non-natural) compounds that bind to a natural target of interest. 18.3.1 D-Peptide Aptamers

In 1996, Peter Kim and co-workers demonstrated that the so-called mirror-image phage display is not only a theoretical option (Schumacher et al., 1996). They started with the chemically synthesized d-enantiomer of the Src homology 3 (SH3) domain of chicken Src. This non-natural target was then used in a phage display experiment employing a conventional library with ten randomized amino acid positions. The selected peptide sequences of the natural configuration were chemically synthesized as d-peptides, which bound to the natural l-SH3 domain. The same group also generated d-peptides targeting the HIV envelope gp41 protein, which promotes viral entry into the host cell (Eckert et al., 1999). The identified d-peptides were biologically active in the micromolar range with respect to inhibition of membrane fusion. Such d-peptide inhibitors might be useful for development and identification of a new class of orally bioavailable anti-HIV drugs. Non-natural stereoisomers of prokaryotic cell surface carbohydrates, synthesized on solid supports, have also served as targets for mirror-image phage display (Kozlov et al., 2001). Both 3-deoxy-a-l-manno-2-octulosonic acid (l-KDO) and l-sialic acid, as well as an l-sialo-disaccharide were used for screening of a phage-displayed single-chain antibody library. The screening process yielded ligands with dissociation constants in the submicromolar range. Such d-peptides capable of binding sugars and saccharides might provide a new approach to developing antibiotics and drug delivery systems. More recently, Willbold’s research team – using the mirror image of amyloid peptide Ab(1–42) – identified a d-peptide that was capable of binding to l-Ab(1–42) with a dissociation constant in the submicromolar range (Wiesehan et al., 2003). The compound may be suitable for detection of amyloid plaques associated with Alzheimer’s disease in living humans or could be used in animal models to identify compounds that are suitable for therapy. 18.3.2 Functional Mirror-Image Oligonucleotides: Spiegelmers

Analogously to the strategy used in mirror-image phage display, which facilitates identification of d-peptide ligands, mirror-image in vitro selection provides biostable mirror-image aptamers. Again, the method relies on the synthesis of the identified target (natural target, Fig. 18.2a) in its non-natural configuration (mirror-image target, Fig. 18.2b). Then combinatorial libraries of unmodified RNA or DNA libraries are prepared and used in successive cycles of selection and amplification (Fig. 18.2c). After enrichment of binding molecules, individual sequences


Fig. 18.2 Spiegelmer technology. A target is defined (a) and synthesized in the mirrorimage configuration (b). Carrying out the SELEX process (c), aptamers of the natural configuration are selected to bind to the artificial mirror-image target molecule (d). The identified aptamer sequences will then be

synthesized in the opposite chirality to give mirror-image oligonucleotides, so-called Spiegelmers, that will recognize the natural target molecule (e). The products of this “doublemirroring-process” are of the non-natural configuration and therefore biostable.

– aptamers (Fig. 18.2d) – are determined and ranked with regard to their biophysical qualities towards the selection target (mirror-image target, Fig. 18.2d). The best candidates are finally synthesized using l-phosphoramidite building blocks in order to generate l-RNA- or l-DNA-oligonucleotides. By rules of symmetry, these l-oligonucleotides, the Spiegelmers, bind to the target in its natural configuration (Fig. 18.2e).

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Fig. 18.3 Circular dichroism (CD) spectra of enantiomeric RNA molecules. SELEX-derived oligonucleotides are usually highly structured molecules. Therefore, aptamers (d-oligonucleotide) as well as Spiegelmers (l-oligonu-

cleotide) show distinct but mirror-inverted CD spectra. (Reprinted with permission from Wiley VCH, Highlights in Bioorganic Chemistry. Methods and Applications, 2004, ed. Schmuck, C. & Wennemers, H.)

Like small-molecule enantiomers, nucleic acid enantiomers do not differ in physical and chemical properties except their interaction(s) with other chiral molecules or polarized light. The structures of nucleic acid enantiomers are exact mirror-images of each other and therefore not superimposable. Accordingly, circularly polarized light is able to interact specifically with the shape or conformation of these macromolecules, yielding wavelength-dependent absorption values that are equal in value for both enantiomers but opposite in sign. Since aptamer (d-oligonucleotide) as well as Spiegelmer (l-oligonucleotide) molecules show distinct three-dimensional structures, very clear mirror-image circular dichroism spectra can be obtained (Fig. 18.3).

18.3.2.1 Proof of Principle The proof of principle for a functional mirror-image oligonucleotide was achieved with two small-molecule targets: the nucleoside adenosine (Klussmann et al., 1996) and the amino acid arginine (Nolte et al., 1996). As expected, the Spiegelmers exhibited outstanding biostability. This was demonstrated using a d-adenosine specific RNA Spiegelmer. It shows no evidence of degradation in human serum, even after 60 h of incubation at 37 hC, a result of its extremely high resistance to nuclease attack (Fig. 18.4). The authors recognized that the exceptional stability of l-nucleic acid ligands suggests a utility analogous to monoclonal antibodies as ligands for affinity chromatography or biosensors, for diagnostic applications or as pharmaceuticals.


Fig. 18.4 Biological stability. A 58mer RNAoligonucleotide in the d-configuration and the same sequence of the l-configuration were incubated in buffered human serum at 37 hC, samples were withdrawn at the given times and analyzed on denaturing polyacrylamide

gels. Whereas the RNA of the d-configuration is subject to rapid degradation within seconds, the RNA of the l-configuration is stable for days. (Reprinted with permission from the Nature Publishing Group, Nat Biotechnol 1996, 14, 1112–1115.)

18.3.2.2 Catalytically Active Spiegelmers: Spiegelzymes Ribozymes are RNA molecules with catalytic properties. They occur in nature but can also be identified artificially by in vitro selection techniques. A new class of ribozymes which catalyze enantioselective bond formation are the Diels-Alderase ribozymes. The Diels–Alder reaction is one of the most important carbon–carbon bond-forming processes available to organic chemists. The reaction creates two carbon–carbon bonds and up to four new stereocenters. The selection of DielsAlderase ribozymes from an RNA library had been described in 1999 (Seelig and Jschke, 1999). These ribozymes accelerate the carbon–carbon bond formation between anthracene – which is covalently bound to the ribozyme – and a biotinylated maleimide. In the course of the reaction, two product enantiomers are generated. As RNA is a homochiral polymer, a Diels–Alder ribozyme should therefore not only accelerate the reaction but also influence the enantiomeric distribution. In order to prove this, a 49-nucleotide common structural motif that had been shown to be active towards a covalently tethered anthracene was synthesized from d- and from l-phosphoroamidite building blocks, respectively. Whereas the uncatalyzed reaction yielded, as expected, a racemic product mixture, the d-ribozyme produced the two compounds with an enantiomeric excess value of 95%. In agreement with the principles of stereochemistry, the mirror-image l-ribozyme not only showed similar rate acceleration but also the opposite product ratio compared with the “natural” d-ribozyme (Seelig et al., 2000).

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18.3.2.3 Domain Approach All mirror-image evolutionary techniques have in common that the mirror image of a given target molecule needs to be synthetically available. Therefore, peptides as well as small protein targets are easy to work with since they can be obtained by standard solid-phase peptide synthesis methods relatively quickly. However, many of the pharmacologically important targets are larger proteins which cannot be synthesized as full-length entities by solid-phase peptide synthesis. For such targets, protein domains have to be identified that can be accessed be chemical synthesis. Most preferred are domains that are either functional or close to functional sites such as exposed regions that are supposed to interact with other pharmacologically relevant molecules (e.g. receptors). This approach directly guides the oligonucleotide inhibitor to the most important target site and disrupts pathological protein–protein interactions. As demonstrated by Purschke and co-workers, even very small peptides can serve as selection targets and are addressable to identify aptamers to generate

Fig. 18.5 Domain approach. In order to access larger protein targets peptide domains within the context of the full-length protein need to be defined. This has been successfully demonstrated by identifying Spiegelmers to the protein target SEB (a). First, a mirrorimage peptide domain has to be synthesized

(b). Employing SELEX (c), aptamer sequences are identified that bind to the mirror-image peptide domain (d). After converting the aptamer sequences into mirror-image oligonucleotides, the resulting Spiegelmers bind to the full-length SEB protein (e).


Spiegelmers (Purschke et al., 2003). In this case a 25-amino-acid domain of the staphylococcal enterotoxin B (SEB), which is a protein that consists of 239 amino acids, was identified as the point of attack (Fig. 18.5a). The 25mer was synthesized in the non-natural d-configuration to give the mirror-image peptide domain (Fig. 18.5b). Employing the SELEX process (Fig. 18.5c), aptamer sequences were identified that recognize the mirror-image peptide domain with high affinity (Fig. 18.5d). After converting the aptamer sequences into Spiegelmers, the l-DNA oligonucleotides not only showed the expected binding behavior towards the 25mer l-peptide (natural configuration) but also recognized the fulllength SEB protein with high affinity (Fig. 18.5e). This concept has been broadened to other protein targets (unpublished data).

18.3.2.4 Bioactive Spiegelmers Vasopressin Williams et al. showed in 1997 that “selection-reflection” is a viable means of producing bioactive and nuclease-resistant ligands for bioactive target molecules (Williams et al., 1997). Selecting for the all-d form of vasopressin – a cyclic nonapeptide – they finally generated a 55-nucleotide l-DNA that binds to the natural vasopressin with equal (low micromolar) affinity. This DNA Spiegelmer was active in an assay using cultured renal cells which expressed the V2 receptor, antagonizing vasopressin-stimulated receptor activation with a calculated IC50 in the lower micromolar range. An l-DNA control sequence with identical base composition and the same putative secondary structure was virtually inactive in the assay. Gonadotropin-releasing hormone Another peptide hormone that had been addressed by a “selection-reflection” approach is gonadotropin-releasing hormone I (GnRH; synonyms: gonadoliberin, luteinizing hormone-releasing hormone, LHRH) (Leva et al., 2002). GnRH is a key peptide hormone in the regulation of mammalian reproduction. It is a decapeptide that is released from hypothalamic neurons in a pulsatile manner, binding to receptors on gonadotrophic cells on the pituitary. Here, GnRH stimulates the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which in turn trigger the production of sexual steroids. Therefore, GnRH and its receptor have been identified as therapeutic targets for sex steroid-dependent conditions such as prostate and breast cancer, endometriosis, as well as in assisted-reproduction techniques. For the first time, RNA as well as DNA Spiegelmers were identified for the same target molecule. The oligonucleotides exhibit dissociation constants between 50 and 100 nmol/L in equilibrium dialysis assays. As demonstrated with surface plasmon resonance experiments, both Spiegelmers showed very high specificity for GnRH, since even the exchange of a single amino acid, as in chicken GnRH (Arg8pGln), resulted in a dramatic loss of binding affinity. The unrelated

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peptide hormones vasopressin or oxytocin, which are of similar molecular weight, were not recognized by the Spiegelmers at all. Cell culture experiments with transfected Chinese hamster ovary (CHO) cells that stably expressed the human GnRH receptor revealed that the Spiegelmers inhibit binding of l-GnRH to its receptor with an IC50 of 200 and 50 nmol/L for the 48-nt RNA and the 60-nt DNA Spiegelmer, respectively. Calcitonin gene-related peptide An RNA Spiegelmer directed to the migraine-associated molecule calcitonin gene-related peptide 1 (CGRP) resulted from a mirror-image selection against rat d-CGRP (Vater et al., 2003). This selection was performed using the so-called “Tailored-SELEX” process which relies on in vitro selection of a primerless RNA library and custom-designed primers/adapters that are added by ligation before and removed within the amplification processes. The calculated affinity (Kd = 2.5 nmol/L at 37 hC) of the best Spiegelmer almost reaches the binding of the neuropeptide to its receptor (EC50 = 1 nmol/L). The potential of this molecule to act as a CGRP antagonist in vivo and its feasibility in the treatment of migraine is currently under investigation (Karl Messlinger, personal communcation). Nociceptin Another pain-related target is the endogenous neuropeptide nociceptin/orphanin FQ (N/OFQ), which plays a prominent role in the regulation of several biological functions such as pain and stress. High-affinity RNA Spiegelmers were shown to antagonize the interaction of N/OFQ with its receptor ORL1 in a binding competition assay exhibiting an IC50 of 110 nmol/L (Faulhammer et al., 2004). Furthermore, the identified Spiegelmers inhibited N/OFQ-induced GTPgS incorporation into cell membranes of a CHO-K1 cell line expressing the human ORL1 receptor and showed an antagonistic effect to the N/OFQ–ORL1 receptor system that was functionally coupled with G protein-regulated inwardly rectifying K+ channels in Xenopus laevis oocytes. Substance P The mirror image of the neuropeptide l-substance P served as a target to demonstrate the robustness of an automated in vitro selection protocol (Eulberg et al., 2005). Substance P is an 11-amino-acid peptide that belongs to the tachykinin family and has a wide variety of biological activities, such as peripheral vasodilation, smooth muscle contraction, pain transmission, and neurogenic inflammation. The identified 44-nucleotide Spiegelmer ligand binds substance P with high affinity (Kd = 40 nmol/L at 37 hC) and also inhibits substance P action in a bioactivity assay using substance P-sensitive human AR42J pancreatic cells (IC50 z 45 nmol/L).


18.3.2.5 Spiegelmer Activity in Vivo Gonadotropin-releasing hormone The functional activity of mirror-image oligonucleotides in an animal model was demonstrated first with a GnRH-inhibiting DNA Spiegelmer (Wlotzka et al., 2002). In an effort to maximize the pharmacological response of the active Spiegelmer molecule by slowing down its renal clearance from the bloodstream, the lDNA-oligonucleotide was modified with a 40 kDa branched polyethylene glycol (PEG) moiety to its 5l end. PEGylation of the Spiegelmer had no effect on its binding behavior towards GnRH, as both the PEGylated and the non-PEGylated versions showed the same activity in cell culture assays with an IC50 of approximately 20 nmol/L. The PEGylated Spiegelmer was further studied in a widely used rat model of GnRH regulation in which the serum LH concentration in orchidectomized (castrated) animals represented the efficacy parameter: due to orchidectomization, the lack of testosterone production led to an increase in the frequency of GnRH pulses resulting in high LH plasma levels. Neutralization of the GnRH activity in turn leads to a normalization of the LH plasma concentration to the level of intact untreated animals. The DNA Spiegelmer was administered 8 days after castration, when the animals displayed stably high LH levels. The subcutaneously administered non-PEGylated Spiegelmer (100 mg/kg) showed maximal GnRH antagonism 1.5 h after administration, which levelled off during the following hours. The effect of the PEGylated Spiegelmer, given intravenously at 150 mg/kg, lasted longer and was comparable to that of the current gold-standard Cetrorelixä, a GnRH receptor antagonist, administered at a dose of 100 mg/kg to control groups. Ghrelin Ghrelin was discovered in stomach extract by Kangawa and co-workers (Kojima et al., 1999). The 28-amino-acid peptide is the only endogenous ligand for the growth hormone secretagogue receptor 1 (GHS-R1a) known to date. Receptor binding and activation requires the presence of a post-translationally added octanoic, decanoic, or decenoic acid modification at Ser3. This modification is essential for bioactivity, whereas more than 20 amino acid residues may be deleted from the C-terminus without compromising receptor binding in vitro. RNA Spiegelmers were generated that bind with extremely high specificity to the octanoylated form of ghrelin (Helmling et al., 2004). The best binding candidate NOXB11 displays a binding affinity in the low nanomolar range (Kd z 35 nmol/L). NOX-B11 also inhibits ghrelin-mediated stimulation of cells expressing GHSR1a (IC50 z 5 nmol/L). Furthermore, the Spiegelmer clearly differentiates between the octanoylated and desoctanoylated forms of ghrelin and requires only the N-terminal five amino acids of bioactive ghrelin for binding, as demonstrated in competition experiments. The in vivo activity of the Spiegelmer was studied in several animal models. Since ghrelin is known to induce growth hormone (GH) release as well as

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acute food intake, these parameters were analyzed in the first instance. After stimulating rats with 3 nmol of (octanoylated) ghrelin, an increase of GH in the serum could be detected. This effect was dose-dependently alleviated by NOXB11, which was administered 15 min before the ghrelin stimulus. A 5- to 10fold molar excess of NOX-B11 over ghrelin blocked GH release completely. Two similar surrogate studies in rats were carried out to monitor the effects of NOX-B11 on the parameter acute food intake as well as neuronal activation in the arcuate nucleus (Kobelt et al., 2005). Spiegelmer NOX-B11 was able to reduce ghrelin-induced (3 nmol per animal) food intake dose-dependently. Whereas at a dose of 1 nmol per animal no effect was observed, 30 nmol of NOX-B11 per rat totally abolished effects of exogenous ghrelin. A comparable result was obtained by monitoring ghrelin-induced c-Fos-like immunoreactivity in the arcuate nucleus. NOX-B11 specifically reduced neuronal activation as compared to a nonfunctional control Spiegelmer. These data confirm the potential therapeutic utility of Spiegelmers in the living organism. They further indicate that treatment with a ghrelin-binding Spiegelmer may provide an approach to the treatment of obesity and obesity-related diseases through blocking of peripheral, endogenous ghrelin. First results indicate that ghrelin blockade by chronic infusion of NOX-B11 leads to weight loss in dietinduced obese mice (L. P. Shearman, personal communication).

18.3.2.6 Pharmacological Properties of Spiegelmers Immunogenicity of Spiegelmers A major safety concern in the development of novel high-molecular-weight entities (e.g. proteins) is their immunogenic potential (Amin and Carter, 2004; Schellekens, 2002). An immune response is mainly elicited by product heterogeneities such as deamidated or oxidized amino acids, modified carbohydrate patterns, and fragment impurities (Chirino and Mire-Sluis, 2004). The general immunogenic potential of Spiegelmers was assessed in Zimmermann rabbits employing a standard immunization protocol over 6 weeks, followed by two additional monthly boosts (Wlotzka et al., 2002). PEGylated as well as non-PEGylated anti-GnRH Spiegelmers were given with and without adjuvant subcutaneously in phosphate-buffered saline on days 0, 7, 14, 28, 35, 63, and 91. As positive control, an additional group was immunized with Spiegelmer (either PEGylated or non-PEGylated) conjugated to cationic BSA (cBSA) in combination with adjuvant. Blood samples were drawn before each administration and analyzed for Spiegelmer-specific antibodies by ELISA. The time-course of antibody titer development was monitored over a period of 98 days (Fig. 18.6, data shown for the PEGylated Spiegelmer). In the groups that had only received Spiegelmer with or without adjuvant, no Spiegelmer-specific antibodies could be detected. A very low titer (1:1000–1:3000) was observed in the groups immunized with the cBSA–Spiegelmer conjugate on day 63 and 91 but not on the last day 98; the same sera of the third (control) group exhibited


Fig. 18.6 Immunogenicity of Spiegelmers (Wlotzka et al., 2002). Spiegelmer immunogenicity was tested in a rabbit model. Pure Spiegelmer (black bars), Spiegelmer plus adjuvant (white bars), and Spiegelmer conjugated to cBSA (gray bars for analysis of Spie-

gelmer-specific antibodies and hatched bars for analysis of bovine serum albumin-specific antibodies) was administered subcutaneously. Blood samples were drawn at the indicated times (before each boost) and the serum was analyzed by ELISA.

cBSA-specific titers of >1:200 000, indicating a strong response to the cBSA moiety (or its fragments). These results suggest that Spiegelmers (PEGylated or non-PEGylated) have only weak, if any, immunogenic potential (data for nonPEGylated Spiegelmer not shown). Toxicity of Spiegelmers Besides immunogenicity, potential side effects of medicines are very important issues in the drug development process due to deficiencies in absorption, distribution, metabolism, elimination, or toxicological problems (so-called ADMET). Because of these reasons, 50% of all drugs in development fail to enter the market. Indeed, ADMET deficiencies are the leading cause of attrition during drug development (Hodgson, 2001). For aptamers and Spiegelmers, few toxicological data are available as yet. The aptamer that has been studied clinically most intensively is the inhibitor of the vascular endothelial growth factor, Macugenä. Macugen is an RNA-based aptamer in which all pyrimidine nucleotides are exchanged to 2l-deoxy-2l-fluoro nucleotides and all but two adenosines are exchanged to 2l-O-methyl building blocks; in addition, the 3l-terminal nucleotide is a deoxythymidine 3l–3l-linked to the chain and the 5l end is modified with a 40 kDa PEG moiety. For this modified RNA molecule, toxic effects have been reported neither in preclinical studies (Drolet et al., 2000), nor in Phase I trials (Eyetech Study Group, 2002). For Spiegelmers, only data from acute toxicology studies are available to date. Doses up to 1 g/kg in mice have not shown any adverse effects (unpublished data). In many efficacy studies, chronic dosing schemes for more than 4 weeks have not shown any observable side effects (unpublished). It is assumed that

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due to their high stability and their high target specificity, Spiegelmers will not show any major toxicological effects if at all.

18.3.2.7 Production of Spiegelmers Building blocks Key intermediates for the synthesis of l-DNA and l-RNA oligonucleotides are the respective l-configured nucleosides, which, unlike their natural d-enantiomers, do not exist in nature. Therefore, since the beginning of the first investigational studies on L-nucleosides in the 1970s several synthetic routes for l-DNA and l-RNA nucleosides have been devised (Holy´, 1972; Holy´ and orm, 1969; Robins et al., 1970; Spadari et al., 1992). To establish Spiegelmers as new class of drug substances, robust and economic manufacturing routes for the production of l-nucleosides, their protected phosphoramidite analogs, and of the l-oligonucleotide itself are prerequisite. Here, we briefly illustrate a synthetic route for the kilogram-scale production of l-adenosine, l-cytidine, l-guanosine, and l-uridine and their protected analogs which are required for solid-phase l-RNA synthesis. The most convenient starting material for the production of l-nucleosides is l-ribose, which itself does not naturally exist. l-Ribose used to be a very expensive pentose, but has become a chemical commodity in recent years. Triggered by the development of l-ribose derived antiviral drugs such as clevudin, levovirin, and maribavir, the traditional manufacturing route using the molybdenum-catalyzed epimerization of l-arabinose to l-ribose has been scaled-up to produce ton-quantities of l-ribose per year (Fig. 18.7A) (Okano and Ueda, 2005). l-Arabinose is a


Fig. 18.7 (A) Outline of the manufacturing route of l-ATBR starting from l-arabinose. (B) Synthesis of N-acetyl-2l-tert-butyldimethylsilyl5l-(4,4l-dimethoxytrityl)-l-adenosine-3l-(2-cyanoethyl-N,N-diisopropyl) phosphoramidite. (a) Bis-N,O-trimethylsilyl-acetamide (BSA), trimethylsilyl triflate (TMSOTf); (b) methylamine; (c) trimethylsilyl chloride, pyridine; acetyl chloride; (d) dimethoxytrityl chloride (DMT-Cl),

pyridine; (e) tert-butyl-dimethylsilyl chloride, imidazole; (f) cyanoethyl N,N,Nl,Nl-tetra-isopropyl phosphane, benzylthio tetrazole (BTT). (C) Synthesis of N-acetyl-2l-tert-butyldimethylsilyl-5l-(4,4l-dimethoxytrityl)-l-cytidine-3l-(2cyanoethyl-N,N-diisopropyl) phosphoramidite. (a) BSA, TMSOTf; (b) methylamine; (c) acetic anhydride, dimethylformamide (DMF); (d) DMT-Cl, pyridine.

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Fig. 18.7 (e) tert-butyl-dimethylsilyl chloride, imidazole; (f) cyanoethyl N,N,Nl,Nl-tetraisopropyl phosphane, BTT. (D) Synthesis of N-acetyl-2l-tert-butyldimethylsilyl-5l-(4,4l-dimethoxytrityl)-l-guanosine-3l-(2-cyanoethylN,N-diisopropyl) phosphoramidite. (a) BSA, TMSOTf; (b) methylamine; (c) acetic anhydride, pyridine, DMF; acetyl chloride; (d) 10N NaOH, tetrahydrofuran; (e) DMT-Cl, pyridine;

(f) tert-butyl-dimethylsilyl chloride, imidazole; (g) cyanoethyl N,N,Nl,Nl-tetra-isopropyl phosphane, BTT. (E) Synthesis of 2l-tert-butyldimethylsilyl-5l-(4,4l-dimethoxytrityl)-l-uridine3l-(2-cyanoethyl-N,N-diisopropyl) phosphoramidite. (a) BSA, TMSOTf; (b) methylamine; (c) DMT-Cl, pyridine; (d) tert-butyl-dimethylsilyl chloride, imidazole; e) cyanoethyl N,N,Nl,Nltetra-isopropyl phosphane, BTT.

cheap and abundant raw material which is isolated from sugar beet or wood. Recently, an alternative three-step biotransformation route to l-ribose starting from d-glucose has been described which can be operated at pilot-plant scale (Okano and Ueda, 2005). Starting from l-ribose the intermediate 1-O-acetyl-2,3,5-tri-O-benzoyl-l-ribose (l-ATBR) is obtained in three steps (Fig. 18.7A) (Recondo and Rinderknecht, 1959). l-ATBR is the common starting material for all four l-nucleosides. In the subsequent Vorbrggen nucleosidation reaction the individual l-nucleosides are produced by trimethylsilyl triflate-catalyzed reaction of l-ATBR with in situ silylated nucleobase followed by deacylation with methylamine (Fig. 18.7B–E for individual nucleosides) (Pitsch, 1997; Vorbrggen, 1996; Vorbrggen and Bennua, 1981). While the reaction conditions of the nucleosidation can be controlled to give regio- and stereoselectively, l-adenosine, l-cytidine, and l-uridine, for the coupling of guanine a mixture of N7- and N9-connected nucleoside is obtained. From this mixture l-guanosine can be isolated in high purity by crystallization from water. The further chemical processing of the four individual l-nucleosides to their respective oligonucleotide building blocks is directed by the choice of chemistry used to assemble an l-RNA oligonucleotide. There is a wide variety of exocyclic N-protecting groups and more importantly of 2l-protecting groups described in

18.4 Summary

the literature (Mueller et al., 2005). Moreover, the coupling of the reactive nucleotide can be achieved by various methods using phosphoramidite, phosphortriester, or phosphonate chemistry. The synthesis schemes (Fig. 18.7B–E) illustrate the processing of l-nucleosides to their respective 2l-TBDMS-protected phosphoramidites, which is achieved by identical conditions to the well-established manufacturing of their d-configured enantiomers (Damha and Ogilvie, 1993). l-Oligonucleotides In general, l-DNA and l-RNA oligonucleotides can be produced by employing the same synthesis and purification methods that have been described for their d-configured enantiomers. Since the 1980s short l-DNA and l-RNA oligonucleotides up to 20 nucleotides long have been synthesized by the phosphotriester (Anderson et al., 1984; Visser et al., 1986; Fujimori et al., 1990b) and phosphoramidite approach (Asseline et al., 1991; Damha et al., 1991; Urata et al., 1991). With increased availability of l-nucleic acid building blocks and improved synthetic methods, the length and produced quantity of reported l-oligonucleotides has increased from the mid-1990s onwards (Garbesi et al., 1998; Klussmann et al., 1996; Pitsch, 1997; Williams et al., 1997). Today, RNA Spiegelmers of a length of up to 70 nucleotides are routinely produced by solid-phase synthesis using phosphoramidite chemistry (Scaringe et al., 1990; Usman et al., 1987) followed by base/silyl-deprotection and HPLC or PAGE purification (Wincott et al., 1995). Production of multi-gram quantities of RNA Spiegelmers intended for preclinical and clinical programs is analogous to the well-established manufacturing process for ribozymes and aptamers. The most prominent example of the latter class of aptamers is Macugenä, which was approved recently by the FDA for the treatment of age-related macular degeneration. Due to siRNA being the latest emerging class of potential oligonucleotide drugs, the manufacturing of RNA oligonucleotides is raising increased interest by biotechnology companies and a growing number of CMOs (contract manufacturing organizations) specialized in the synthesis of oligonucleotides.

18.4 Summary

The introduction of chiral principles into evolutionary selection techniques is an elegant approach to create functional (i.e. target-binding) mirror-image peptides as well as oligonucleotides (Spiegelmers) that display extraordinarily high biostability. Biostability combined with the molecules’ property of high target specificity makes Spiegelmers an interesting and promising substance class for drug discovery and drug development. The observed biostability is based on the fact that homochirality is a common theme throughout nature: all nucleic acids that are involved in cellular processes consist of so-called d-sugars, whereas all amino acids of proteins that are directly synthesized by the ribosome contain amino acids of the l-configuration. As a con-

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sequence, organisms show strong enantiomeric selectivity in their molecular components (Cairns-Smith, 1986) so that Spiegelmers, composed of building blocks of the unnatural configuration, escape from degradation by abundant nucleases and benefit from a “stealth effect.” The perception that biologically active molecules interact in a “lock-and-key” fashion based on the principle of stereocomplementarity and the ability to synthesize “biopolymers” of the unnatural configuration made it possible to integrate chiral principles into the powerful techniques of evolutionary in vitro selection. The SELEX process has been shown to deliver very potent oligonucleotidebased inhibitors, called aptamers (Ellington and Szostak, 1990; Tuerk and Gold, 1990). The first aptamer, Macugenä, has already made its way through clinical development and is a viable drug on the market. It has been shown that Spiegelmers with outstanding characteristics can be generated to a wide variety of pharmacologically important target molecules; for an overview see Table 18.1. Like aptamers or antibodies, Spiegelmers usually show low nanomolar to picomolar dissociation constants to their respective targets. Due to their unique properties, Spiegelmers have the potential to directly

Table 18.1 Overview of mirror-image peptides/oligonucleotides that were obtained by using chiral principles in evolutionary selection techniques Target

Substance class

Biophysical data

Bioactivity (cell culture)

In vivo Reference efficacy

Amyloid Ab(1–42)

d-Peptide

+

n.d.

n.d.

Wiesehan et al., 2003

HIV gp41

d-Peptide

+

+

n.d.

Eckert et al., 1999

Sialic acid/KDO

d-Peptide

+

n.d.

n.d.

Kozlov et al., 2001

Src SH3 domain

d-Peptide

+

n.d.

n.d.

Schumacher et al., 1996

Adenosine

l-RNA

+

n.d.

n.d.

Klussmann et al., 1996

Arginine

l-RNA

+

n.d.

n.d.

Nolte et al., 1996

CGRP

l-RNA

+

+

+

Vater et al., 2003; Messlinger et al., pers.comm.

Ghrelin

l-RNA

+

+

+

Helmling et al., 2004; Kobelt et al., 2005

GnRH

l-DNA/ RNA

+

+

+

Leva et al., 2002; Wlotzka et al., 2002

N/OFQ

l-RNA

+

+

n.d.

Faulhammer et al., 2004

SEB

l-DNA

+

n.d.

n.d.

Purschke et al., 2003

Substance P

l-RNA

+

+

n.d.

Eulberg et al., 2005

Vasopressin

l-DNA

+

+

n.d.

Williams et al., 1997

References

progress from the bench to the animal model. At NOXXON, research programs are active to develop Spiegelmers as drug candidates for clinical testing in several indications.

Acknowledgments

We thank Jenny Fischer and Esther Brimacombe for critical reading of the manuscript and Christian Mihm for preparing the artwork for Figs 18.1, 18.2, 18.5, and 18.7.

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19.2 Rationale for Targeting VEGF

19 Applications in the Clinic: The Anti-VEGF Aptamer Tony Realini, Eugene W.M. Ng, and Anthony P. Adamis

19.1 Introduction

So far in these pages, we have described the technical aspects of aptamers and aptamer-like molecules as potential therapeutic agents. This promise has now been realized. In December 2004, pegaptanib sodium – an antivascular endothelial growth factor 165 (VEGF165) aptamer – became the first aptamer to be approved by the US Food and Drug Administration (FDA) for human therapeutic use. Pegaptanib is indicated for the treatment of neovascular age-related macular degeneration (AMD), a leading cause of blindness worldwide (Resnikoff et al., 2004), and is marketed under the trade name Macugenr. This chapter will recount the story of the journey of Macugen from the laboratory to the clinic.

19.2 Rationale for Targeting VEGF

Angiogenesis – the growth of new blood vessels – is a central component of many human disease processes (Witmer et al., 2003). There are several examples that have been areas of very active research. As solid tumors grow they require an ever-increasing supply of oxygen and nutrients, and hence develop their own vascular beds in order to meet this requirement. In patients with diabetes mellitus, chronically elevated blood glucose levels result in damage to small blood vessels and resultant end-organ ischemia distal to these sites of damage. Subsequently, compensatory vascular beds adjacent to these sites of damage emerge in an effort to maintain tissue perfusion. In neovascular AMD, tufts of neovascular tissue grow from the choroid underlying the macula (the central portion of the retina) in response to metabolic distress. These neovascular tufts leak fluid and bleed, leading to loss of central vision. Clearly, an agent capable of inhibiting angiogenesis may have broad applications (Kerbel and Folkman, 2002). However, inhibiting angiogenesis requires a The Aptamer Handbook. Edited by S. Klussmann Copyright c 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-31059-2

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Fig. 19.1 Cascade of events in angiogenesis (Ferrara et al., 2003). Adapted with permission from the Angiogenesis Foundation website. EC, endothelial cell; ECM, extracellular matrix.

complete understanding of what drives this process in order that a molecular target can be identified. Fortunately, research over the last 30 years has led to the discovery of a druggable target, namely VEGF (Ferrara et al., 2003), and the subsequent approval of the use of anti-angiogenesis (anti-VEGF) agents for treatment of solid tumors and ophthalmic disorders. Angiogenesis is comprised of a complex, multistep cascade of events that occurs in response to a stimulus (Fig. 19.1) (Angiogenesis Foundation, 2005). In the 1940s and 1950s, this hypothetical stimulus was named “Factor X”. Factor X was theorized to be released by ischemic tissue in order to stimulate angiogenesis, as well as to direct vascular growth towards regions of ischemia (Michaelson, 1948; Ashton, 1957; Wise, 1956). Ischemia could arise from either vascular damage, as in diabetes, or outgrowth of existing vascular beds, as in tumors. Once released, Factor X was believed to diffuse through local tissues to nearby vascular beds and stimulate the growth of new vessels, which in turn grew along the Factor X concentration gradient back to the original site of ischemia. The Factor X analog in tumor biology was coined tumor angiogenesis factor by Folkman (1971). Both terms referred to a hypothetical angiogenic factor. Vascular

19.3 VEGF and Human Disease

permeability factor (VPF) was isolated from guinea-pig ascites in 1983 (Senger et al., 1983) and found to be 50 000 times more potent than histamine at inducing vascular permeability (Senger et al., 1990). Six years later, VPF was cloned and rechristened “vascular endothelial growth factor” when additional experiments revealed that it also stimulated potently the growth of vascular endothelial cells and induced angiogenesis in vivo (Leung et al., 1989). When VEGF encounters vascular endothelial cells in vitro, it stimulates angiogenesis by binding to VEGF receptors located on these cells (De Vries et al., 1992; Millauer et al., 1993). The cells then migrate and proliferate, forming new vascular buds. At the same time, matrix metalloproteinases are stimulated and break down basement membranes, hence permitting vascular ingrowth into surrounding tissues (Ferrara et al., 1992).


In more recent years, the role of VEGF in human disease has become much better understood, and VEGF has emerged as a key candidate for therapeutic inhibition (Ferrara et al., 2003). Angiogenesis plays an important role in many diseases, therefore providing a unique opportunity to develop agents against a single target for a diverse set of applications in many fields of medicine. Therapeutic success has been found in the eye for several reasons. First, angiogenesis is central to a number of ocular diseases; second, the eye is an easily accessible and relatively closed system, allowing direct delivery of therapeutic levels of drug to exactly where it is required; and third, the eye permits direct, in vivo examination of blood vessels, which facilitates preclinical and clinical drug development. 19.3.1 Cancer

As described above, a rapidly growing solid tumor may quickly outgrow native vascular beds in its tissue of origin. This will result in necrosis and tumor death unless an alternate oxygen delivery system can be established. Hence, the ability to stimulate angiogenesis is a critical part of the neoplastic process, which would be self-limited without such ability. VEGF drives this angiogenesis process in tumors. Indeed, studies demonstrated that anti-VEGF antibody suppressed tumor growth in rodents by inhibiting tumor angiogenesis (Kim et al., 1993). In these animal models, VEGF inhibition was effective in suppressing the growth of several types of solid tumors. The same antibody was then used in humanized form for human cancers. Subsequently, Phase II and III randomized clinical trials demonstrated prolongation of survival times in patients with renal (Yang et al., 2003) and colon (Hurwitz et al., 2004) cancer, respectively. Based on these results, anti-VEGF antibody (Avastin, Genentech, Inc.) in combination with 5-fluorouracil was approved by the

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FDA as first-line therapy for metastatic colorectal cancer. Interestingly, this same agent had no effect in trials of late-stage breast cancer (Rugo, 2004; Miller et al., 2005) possibly due to issues such as suboptimal trial design, poor penetration into the target tissues, or potential redundancy of angiogenic pathways in human breast cancer. Ultimately, successful anti-angiogenesis therapy may consist of multiple agents, each affecting different targets at different points in the angiogenesis pathway. 19.3.2 Age-Related Macular Degeneration

AMD is a chronic and progressive disease of the eye that primarily affects older and Caucasian populations. AMD is in fact the leading cause of blindness in people above 50 years of age in the developed world (Resnikoff et al., 2004). AMD affects the central portion of the retina, which is called the macula. The macula is important for central vision. Patients who lose central vision have dif-

Fig. 19.2 The layers of the retina.


ficulty with activities of daily living, such as reading, driving, meal preparation, and housework (Dong et al., 2004). Photoreceptors are light-detecting cells that exist within the retina, and are most densely packed in the macula. Visual resolution is therefore highest in the macula. The inner layers of the retina receive oxygen and nutrients via blood vessels in the retina. The outer layers of the retina do not have a direct blood supply, but receive nutrients and waste removal services via the retinal pigment epithelium (RPE), upon which the outer retina is directly apposed. Photoreceptors are tightly interdigitated with RPE cells (Fig. 19.2). Beneath the RPE lies the choroid, a vast bed of capillaries that supplies oxygen to the outer layers of the retina. The RPE and choroid are separated by a layer of connective tissue called Bruch’s membrane. The earliest changes observed in AMD are the presence of drusen, small clumps of material at the level of Bruch’s membrane that likely represent the residua of degenerating RPE cells (Ambati et al., 2003). Drusen are visible clinically as yellow spots in the macula (Fig. 19.3) (Machemer, 2005). Additional RPE abnormalities may also be present (areas of death and/or hyperpigmentation). This early stage of AMD is called dry, or non-exudative AMD. A subset of patients with dry AMD will progress to an advanced stage of disease called wet AMD, also known as exudative or neovascular AMD (Ambati et al., 2003). In wet AMD, new blood vessels budding from capillaries of the choroid emerge through breaks in the degenerating Bruch’s membrane. These buds of fibrovascular tissue form neovascular membranes that grow in the sub-RPE and/or subretinal planes (Fig. 19.4). Choroidal neovascular membranes leak fluid and bleed, thus destroying the delicate architecture of the macula and causing severe central vision loss. Ocular neovascularization is mediated by VEGF. High VEGF messenger ribonucleic acid (mRNA) levels and increased VEGF receptor levels have been observed in areas of ischemic retina in primates (Miller et al., 1994). Injecting VEGF inhibitors into the vitreous cavity blocks the neovascularization process in primates (Tolentino et al., 2004). In humans, VEGF levels are elevated in the vitreous cavity of eyes with subretinal neovascularization (Wells et al., 1996) and in surgically excised neovascular membranes from eyes with AMD (Lopez et al., 1996; Frank et al., 1996; Asayama et al., 2000).

Fig. 19.3 The presence of drusen in a patient with neovascular age-related macular degeneration (Machemer, 2005). Copyright, Online Journal of Ophthalmology, used with permission.

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19 Applications in the Clinic: The Anti-VEGF Aptamer Fig. 19.4 Neovascular membranes grow into the area under the retinal pigment epithelium.

19.3.3 Diabetic Retinopathy

Like AMD, diabetic retinopathy represents a serious public health problem. It is the leading cause of blindness in of individuals between the ages of 25 and 74 in the United States (National Eye Institute, 2005), a segment of the population that makes up the majority of the workforce. Long-term exposure to elevated blood glucose levels causes damage to pericytes, small cells that surround and support the capillaries of the retinal vascular bed (Resnikoff et al., 2002). Loss of pericytes leads to focal weak spots developing along retinal vascular walls. These weak areas bulge outward, forming microaneurysms, which leak (causing retinal edema) and eventually burst (causing retinal hemorrhage). This stage of diabetic retinopathy is called background or nonproliferative retinopathy because most diabetics develop at least mild microvascular changes even with relatively good long-term blood glucose control. But the disease does not always stop there. If enough small vessels are affected, retinal tissue distal to these ruptured microaneurysms are deprived of blood and oxygen, and therefore become ischemic. Retinal ischemia promotes retinal angiogenesis, and hence “proliferative” diabetic retinopathy ensues. This form of the disease is far more visually debilitating because these patches of fibrovascular proliferation are hemorrhagic and often destroy normal retinal architecture in their effort to “rescue” distal ischemic tissue beds. VEGF drives diabetic retinopathy. In a mouse model of diabetes, the expression of VEGF mRNA was more than three-fold higher in diabetic eyes than in non-diabetic eyes (Qaum et al., 2001). Retinal vascular damage similar to diabetic retinopathy can be induced by injecting VEGF into primate eyes (Tolentino et al., 1996). In humans, vitreous VEGF levels are higher in patients with diabetic macular edema than in both normal subjects and diabetics without retinopathy (Funatsu et al., 2002). Increased VEGF levels have also been found in both the aqueous

19.4 The VEGF Therapeutic Dilemma

(Aiello et al., 1994) and vitreous (Adamis et al., 1994) of human eyes with proliferative diabetic retinopathy.

19.4 The VEGF Therapeutic Dilemma

The evidence presented above strongly supports the hypothesis that VEGF is a viable target for inhibition of pathologic angiogenesis in a number of diseases. However, VEGF inhibition must be undertaken with caution because VEGF is also necessary for the physiologic angiogenesis that underlies normal human development and organ maintenance. 19.4.1 VEGF and Human Physiology

The embryo is dependent upon VEGF for the development of its vascular system. Deleting both alleles of the VEGF gene in mice results in early fetal death (Shalaby et al., 1995). The amount of VEGF in the system during embryogenesis is critical as well – too much or too little results in fetal demise. Case in point, deleting a single allele of the VEGF gene also led to fetal demise in mice (Carmeliet et al., 1996) as did exposure to a twice-normal level of VEGF during fetal development (Miquerol et al., 2000). Various functions in the female reproductive tract are also angiogenesis dependent (Ferrara et al., 1998) as are bone growth (Gerber et al., 1999) and wound-healing (Nissen et al., 1998); all of these are impaired in the setting of VEGF inhibition. VEGF and VEGF receptors are expressed constitutively in normal eyes, suggesting strongly that there are homeostatic functions of VEGF in the eye. Preliminary evidence supports a role for VEGF in the growth and maintenance of the fine capillary beds of the choroid which maintain blood flow to the RPE and photoreceptors (Kim et al., 1999). In addition, researchers are exploring the role of VEGF as a trophic factor for neural cells in the retina, such as retinal progenitor cells and photoreceptors. Evidence also suggests that VEGF may have neuroprotective properties in the eye and other parts of the nervous system (Jin et al., 2000; Storkebaum and Carmeliet, 2004; Shima et al., 2004). Therefore, the dilemma in the eye and elsewhere lies in how to selectively inhibit pathologic angiogenesis while preserving physiologic angiogenesis. For example, Avastinr (bevacizumab, Genentech, Inc.), which blocks all isoforms of VEGF, can result in adverse events such as thromboembolism, hypertension, and proteinuria (presumably due to injury to the blood vessels of the glomerulus) (Kim et al., 1993; Yang et al., 2003; Hurwitz et al., 2004; Rugo, 2004). These adverse events may perhaps arise from interference of the homeostatic functions of VEGF on the systemic vasculature. A distinct advantage of treating ocular neovascularization is that local delivery of a VEGF inhibitor directly into the eye is possible, which can minimize extraocular VEGF inhibition and its potentially adverse sequelae.

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19.4.2 Overcoming the Dilemma

A safe and effective anti-VEGF aptamer must inhibit pathologic neovascularization while leaving physiologic neovascularization unaffected. To understand how this was accomplished through the specificity of aptamer binding requires greater familiarity with the VEGF molecule. VEGF exists as a family of molecules (Kerbel and Folkman, 2002). The molecule referred to in angiogenesis and vascular maintenance is called VEGF-A (for simplicity, the term VEGF will be used in this discussion). Other family members include VEGF-B, whose function is not yet known; VEGF-C and VEGF-D which are involved in lymphangiogenesis; VEGF-E which like VEGF-A, is involved in angiogenesis; and placental growth factor which mediates angiogenesis and inflammation. In turn, VEGF-A (VEGF) exists as a small group of isoforms, which differ in size and action, but all of which arise from the same VEGF gene (Kerbel and Folkman, 2002). The gene consists of eight exons and seven introns. During transcription, variable splicing of exons generates VEGF molecules of varying lengths (Kerbel and Folkman, 2002). These isoforms are named for the number of amino acids in the translated protein. The human VEGF isoforms are VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206. The properties of these isoforms vary with their length. With increasing length, VEGF isoforms become more pH basic, more bound to heparin, and less diffusible in tissue. VEGF isoforms are key to solving the therapeutic dilemma. They are differentially expressed in physiologic and pathologic neovascularization (Kerbel and Folkman, 2002) providing the potential to selectively inhibit pathologic neovascularization.

19.5 VEGF Inhibition

Inai and colleagues studied the effects of non-selective inhibition of all VEGF isoforms with VEGF-TRAP (a pan-VEGF antibody) on neovascularization associated with solid tumors in mice (Inai et al., 2004). Within 24 h of treatment, neovascular sprouting of new vessels from existing capillary beds within the tumors was suppressed. More exciting was the observation that blood flow was reduced or arrested in some existing vessels. This suggested that anti-VEGF therapy not only blocked angiogenesis, but could also cause regression of existing tumor vessels. This was at once a blessing and a curse: while it would be greatly beneficial to promote regression of existing vessels in solid tumors in order to starve them to death even faster, what effects might vascular regression have on the normal vasculature? In fact, by 7 days after treatment, vascular density within the tumors had decreased by 70%, but unfortunately the vascular density of the normal tracheal vascular bed in treated animals also decreased by 55%.

19.5 VEGF Inhibition

To overcome this therapeutic dilemma limiting the viability of targeted antiVEGF therapy, Ishida and co-workers undertook a novel series of studies in rats to investigate the importance of VEGF isoforms in pathologic versus physiologic neovascularization (Ishida et al., 2003a). They employed an animal model of retinopathy by exposing full-term newborn rats to 80% oxygen for the first 10 days of life to induce an avascular retina, then moving them to room air for 7 days, during which time proliferative retinal neovascularization developed. A control group of full-term rats was raised in room air from birth. During the first 10 days of life, VEGF levels (as measured by retinal RNA levels) in the control rats were higher than in the rats exposed to 80% oxygen, corresponding to the occurrence of normal retinal vascularization in the control animals compared with no vascularization in the rats who received oxygen (Ishida et al., 2003a). During the next 7 days when the rats who originally received oxygen were exposed to room air, VEGF levels spiked to well above normal as pathologic neovascularization developed. The research team (Ishida et al., 2003a) not only assessed total VEGF levels, but also fractionated the isoforms. A pattern of differential isoform expression emerged that offered hope for overcoming the dilemma. In the rats exposed to high levels of oxygen, the ratio of VEGF164 (the rat equivalent of human VEGF165) to VEGF120 (the rat equivalent of human VEGF121) was more than 12fold higher during their pathologic neovascularization period than in the normal rats during their physiologic neovascularization period (Fig. 19.5). Ishida and colleagues (Ishida et al., 2003a) speculated that perhaps VEGF164 drove pathologic neovascularization, while VEGF120 in part drove physiologic neovascularization. If so, isoform-specific molecular targeting would overcome the safety issue. All they needed was a method of selectively inhibiting VEGF164 to test their theory.

Fig. 19.5 VEGF164 (165) is preferentially elevated in pathological neovascularization (Ishida et al., 2003). VEGF, vascular endothelial growth factor.

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19.6 Enter Macugen

Fortunately, a specific inhibitor of VEGF165 already existed. Pegaptanib, an antiVEGF165 aptamer, is an oligonucleotide that binds with high affinity and specificity to the region of human VEGF165 protein encoded in exon 7 of the VEGF gene (Bell et al., 1999). Prior to being called pegaptanib, it was referred to as EYE001. The molecule was initially developed by a team led by Larry Gold and Nebojsa Janjic at Nexstar, and the molecule was licensed by Eyetech in April of 2000 and given the trade name Macugen. Pegaptanib does not bind to VEGF120/121 (Ishida et al., 2003a) and effectively neutralizes VEGF165(164) (Ishida et al., 2003a; Bell et al., 1999; Eyetech Study Group, 2002). Ishida and colleagues used pegaptanib to explore the theory that different VEGF isoforms may drive different types of neovascularization. They returned to the rat models described above (Ishida et al., 2003a), and this time treated some with pegaptanib to selectively inhibit VEGF164, and treated others with an anti-VEGF fusion protein that non-selectively blocked all VEGF isoforms. They found that non-selective blockade of all VEGF isoforms inhibited both physiologic neovascularization in the room-air rats and pathologic neovascularization in the rats exposed to high levels of oxygen, thus confirming Inai and colleagues’ findings that pan-VEGF blockade was of limited value therapeutically. But remarkably, Ishida’s group found that selective VEGF164 blockade with pegaptanib blocked pathologic neovascularization in the rats exposed to 80% oxygen but had no effect on physiologic neovascularization in the room-air rats (Ishida et al., 2003a). These experiments paved the way to human applications for pegaptanib. The FDA generally requires preclinical testing and three stages of clinical testing before a drug can be considered for regulatory approval. Safety, pharmacokinetics, and potential efficacy signals are established in a small group of healthy volunteers or appropriate patients in Phase I testing. Evidence of efficacy and optimal dosing are assessed in a larger group of volunteers or patients in Phase II testing. If a drug demonstrates safety and potential efficacy in these smaller trials, then large Phase III trials (the FDA requires two confirmatory trials which are commonly run concurrently) are undertaken to establish efficacy and safety of the drug (Feinsod and Chambers, 2004). 19.6.1 Preclinical Studies

In preclinical studies, Macugen induced almost complete attenuation of VEGFmediated leakage in the cutaneous vascular permeability (Miles) assay (guineapig model of vascular leakage) (Eyetech Study Group, 2002). Similarly, in a model of diabetic blood–retinal barrier breakdown (BRB) Macugen led to almost complete suppression of BRB (Eyetech Study Group, 2002). In a rodent model of corneal neovascularization, systemic treatment with Macugen resulted in signifi-

19.6 Enter Macugen

cant inhibition (65%) of VEGF-dependent angiogenesis compared with placebo. In a murine model of retinopathy of prematurity (ROP), systemic treatment with Macugen resulted in 80% reduction in retinal neovascularization compared with placebo (Eyetech Study Group, 2002).

19.6.2 Macugen Clinical Trials Phase I A multicenter, open-label dose-escalation trial was conducted in eyes with neovascular membranes growing directly under the center of the macula (Eyetech Study Group, 2002). Fifteen patients were enrolled with neovascular AMD. Participants received single intravitreal injections of Macugen ranging from 0.25 mg to 3 mg. From a safety standpoint, no significant ocular or systemic safety issues were observed. Although Phase I studies are not designed to evaluate efficacy, at 3 months after Macugen injection, 12 of 15 eyes (80%) demonstrated stable or improved visual acuity, including four patients (27%) who gained the ability to read an additional three lines on the standardized eye chart, compared with their baseline pre-treatment visual acuity (Eyetech Study Group, 2002). Since it was expected that Macugen would stabilize vision – not necessarily improve it – these preliminary data were encouraging. Phase II In an effort to better characterize the visual benefits of Macugen therapy, a Phase II study was performed in 21 patients with AMD and neovascular membranes under the center of the macula (Eyetech Study Group, 2003). Patients in this open-label, Phase II study received an intravitreal injection of Macugen 3 mg/ 100 mL every 28 days for a total of three injections. A subset of patients whose choroidal neovascular membrane was morphologically favorable (i.e. the angiographic subtype was predominantly classic) was allowed to receive photodynamic therapy (PDT) with Visudyner (verteporfin, Novartis Pharmaceuticals) in addition to Macugen. PDT with Visudyne is an ablative therapy approved for the treatment of patients with predominantly classic neovascular membranes. In the trial, 10 patients received Macugen alone, and 11 received Macugen as well as PDT with Visudyne. Safety analysis revealed that none of the 21 patients experienced a serious drugrelated adverse event in this study (Eyetech Study Group, 2003). Of the eight patients who received Macugen alone and who completed the 3-month study, 87.5% had stable or improved visual acuity, including 25% who gained at least three lines of vision on the standardized eye chart. In comparison, of the 10 patients receiving both Macugen and PDT with Visudyne who completed the study, 90% had stable or improved visual acuity, including 60% who experienced a gain of three or more lines on the eye chart.

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These Phase II data provided further encouragement that Macugen was safe and effective. Therefore, two large Phase III randomized clinical trials were initiated. Phase III The two Phase III clinical trials were prospective, randomized, double-masked multicenter, dose-ranging, placebo-controlled trials that ran concurrently and the data pooled for analysis (Gragoudas et al., 2004). Once again, generally healthy patients aged 50 and above with AMD, a subretinal neovascular membrane growing under the center of the macula and visual acuity 20/40 or worse were eligible for inclusion. In both studies combined, a total of 1208 patients were enrolled, of whom 1186 met the criteria to be included in the final analysis. Patients were randomly assigned to receive either an intravitreal injection of Macugen 0.3 mg, 1 mg, or 3 mg every 6 weeks for 54 weeks (nine injections total) or a sham injection in which the hub of the syringe (without a needle attached) was pressed to the anesthetized eyeball so that the patient could not discern that an injection was not given (Gragoudas et al., 2004). The three doses of Macugen were included in the study in order to determine the optimal drug dosage. There were approximately 300 patients in each of the four groups (three drug dose levels and placebo) (Table 19.1).

Table 19.1 Patient Characteristics at Baseline (Gragoudas et al., 2004) Macugen (n = 892)

Usual care (n = 298)

42%

40%

Gender Male Female

58%

60%

Mean age (years)

76.0

75.7

In study eye

51.5

52.7

In fellow eye

55.7

55.9

Predominantly classic

26%

26%

Minimally classic

36%

34%

Occult

38%

40%

I 4 disc areas

58%

52%

j 4 disc areas

41%

47%

Mean visual acuity (letters)

Angiographic lesion subtype

Angiographic lesion size

19.6 Enter Macugen

The primary efficacy endpoint in the Phase III trials was the proportion of patients losing less than three lines of vision on the standardized eye chart (Gragoudas et al., 2004). Overall, all three dosages of Macugen provided statistically better visual outcomes than placebo at 54 weeks: 55% of patients in the placebo group lost fewer than three lines of vision, compared with 70% of patients in the 0.3 mg group (P I 0.001), 71% of the 1 mg group (PI 0.001), and 65% of the 3 mg group (P = 0.03). Analysis of secondary efficacy endpoints revealed that visual acuity remained unchanged or improved in 33%, 37%, and 31% of the 0.3 mg, 1 mg. and 3 mg treatment groups, respectively, compared with only 23% in the sham-treated group. These were all statistically significant. Larger proportions of patients treated with 0.3 mg Macugen gained 5, 10, or 15 letters (approximately 1, 2, and 3 lines on the study eye chart, respectively) of visual acuity (Fig. 19.6) (Gragoudas et al., 2004). In addition, the sham group was significantly more likely than any of the three treatment groups to experience severe vision loss of six or more lines on the eye chart: this occurred in 22% of sham eyes versus 10%, 8%, and 14% in the 0.3 mg, 1 mg, and 3 mg treatment groups, respectively (Fig. 19.7) (Gragoudas et al., 2004). These, too, were also statistically significant. There were no serious adverse events considered to be attributable to Macugen itself in either of these two concurrent Phase III studies (Gragoudas et al., 2004). Non-serious adverse events were generally transient, mild to moderate in severity, and tended to be attributable to the injection process rather than the study drug. These included eye pain, vitreous floaters, punctate keratitis, cataracts, vitreous opacities, anterior chamber inflammation, visual disturbances, eye discharge, and corneal edema.

Fig. 19.6 Percentage of patients who maintained or gained visual acuity at 54 weeks in patients receiving Macugen 0.3 mg or sham in the Phase III pivotal trials (Gragoudas et al., 2004).

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Fig. 19.7 Percentage of patients losing j 6 lines (j 30 letters) at week 54 in patients receiving Macugen 0.3 mg or sham in the Phase III pivotal trials (Gragoudas et al., 2004).

Of the adverse events attributable to the injection procedure (Gragoudas et al., 2004), the most serious was endophthalmitis, an intraocular infection often associated with significant loss of vision. Overall, 12 patients (all in the Macugen injection groups) developed endophthalmitis, but only one patient experienced severe vision loss as a result. Interestingly, this patient’s enrollment was, in retrospect, a protocol violation because of an active periocular soft tissue infection at the time of enrollment. In fact, 9 of the 12 patients with endophthalmitis experienced injection-related protocol violations, the most common being failure to use a lid speculum to keep the bacteria-laden eyelashes out of the way during the injection. In addition to endophthalmitis, five patients experienced traumatic injury to the lens from the needle used in the injection, and six more patients in the injection groups experienced retinal detachment. Only one of these 11 patients experienced severe vision loss greater than six lines on the eye chart.

19.7 The Future

Macugen is the second anti-VEGF therapy to enter the marketplace, but is the first aptamer approved in the United States for human therapeutics. In the clinical trials performed to date, Macugen has demonstrated significant efficacy in preserving vision in eyes with neovascular AMD, and has also exhibited a very favorable safety profile. But the Macugen story is far from complete. These clinical trials showed that the most significant safety issues associated with Macugen arise from the method

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S.J. (2004). Intravitreal injection of vascular endothelial growth factor small interfering RNA inhibits growth and leakage in a nonhuman primate, laser-induced model of choroidal neovascularization. Retina 24, 132–138. Wells, J.A., Murthy, R., Chibber, R., Nunn, A., Molinatti, P.A., Kohner, E.M., Gregor, Z.J. (1996). Levels of vascular endothelial growth factor are elevated in the vitreous of patients with subretinal neovascularisation. Br J Ophthalmol 80, 363–366. Wise, G.N. (1956). Retinal neovascularization. Trans Am Ophthalmol Soc 54, 729–826. Witmer, A.N., Vrensen, G.F.J.M., Van Noorden, C.J.F., Schlingemann, R.O. (2003). Vascular endothelial growth factors and angiogenesis in eye disease. Prog Retin Eye Res 22, 1–29. Yang, J.C., Haworth, L., Sherry, R.M., Hwu, P., Schwartzentruber, D.J., Topalian, S.L., Steinberg, S.M., Chen, H.X., Rosenberg, S.A. (2003). A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer. N Engl J Med 349, 427–434.

The Beginning References

Epilogue A Personal Perspective: Aptamers after 15 Years Larry Gold

The Beginning

In 1990 Craig Tuerk and I published the first paper (Tuerk and Gold, 1990) on a new technology, which we called SELEX (for systematic evolution of ligands by exponential enrichment). The technology described the entirely in vitro combinatorial chemistry method in which the materials in the compound library were single-stranded nucleic acids. While the first experiment used only 65 000 different RNA sequences, it was clear immediately that single-stranded nucleic acid libraries could be larger than any other combinatorial chemistry library (because of nucleic acid amplification techniques). One important lesson from SELEX is that library size can be very important, an idea I will return to at the end of this chapter. Since our publication (and the near-simultaneous publication by Ellington and Szostak, 1990, in which the aptamer products of selection, rather than the method, were named), more than 2000 aptamer publications have accumulated in the literature (Tim Fitzwater, personal communication). Craig and I were very fortunate (Gold et al., 1997). The selection Craig did (for the preferred eight loop nucleotides within the hairpin of the translational operator of the bacteriophage T4 gene 43 mRNA) yielded two winners (from the 48 input sequences) – one was the identical sequence to that carried by wild-type phage, while the other contained four “mutations”, the very kind of sequence alteration that might never be observed in nature and, to our knowledge, has never been (Pavlov and Karam, 2000, and Jim Karam, personal communication as well as http://phage.bioc.tulane.edu). The “major variant” (as we called it) had such a different apparent structure from the wild-type RNA (two extra base pairs at the top of the stem and a hairpin loop half the length of the wild type) that we instantly invented (as the extrapolation of that amazing fact) the uses of SELEX that had been until that moment obscured by our conventional thinking: we proposed that such bewildering structural diversity in RNAs and singlestranded DNAs could lead to “nucleic acid antibodies” of equivalent richness to real antibodies, except that the chemistry of the binding molecules – the soon The Aptamer Handbook. Edited by S. Klussmann Copyright c 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-31059-2

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A Personal Perspective: Aptamers after 15 Years

to be named aptamers – would be nucleic acids rather than proteins. As Craig Tuerk reported in a recent unfortunate interview (“unfortunate” in that the interviewer apparently had an agenda) (Archemix Press Release, 2004), he thought he had “looked into the mind of God” (while the interviewer was looking in the toilet).

The First Patent

We wrote an extensive description of the selection method and obvious variations in those methods, as well as a lengthy list of the expected biochemical and functional properties of the molecules that would come from the SELEX method. We wrote this description in a patent application (Gold and Tuerk, 1995) rather than in the shorter Science paper that came out in August of 1990. Many scientists of course never read the patent literature, assuming that the primary scientific literature contains everything of interest (although always a little behind the patent literature). In this case Craig and I poured our hearts into the patent application, and predicted many properties of the molecules that would come from SELEX. We were correct about everything, largely because we had been thinking about single-stranded nucleic acids (as translational operators on various T4 mRNAs, and also as ribosome binding sites; Lemaire et al., 1978; Gold et al., 1981; Winter et al., 1987) for a long time. Our first SELEX data opened the floodgates in our brains. I am still astounded at how much of the next 15 years we saw correctly. People have frequently asked me why we wrote a patent application at all. In fact Craig and I had many conversations about the usefulness and appropriateness of doing that. I had been part of an early biotech company – Synergen – and I was clear that patents are critical if one hopes to see commercial entities invest in and develop a technology. Thus we chose that course, and in fact a small fortune has been spent on aptamer commercial applications. I believe that in the absence of our patent application no concentrated commercial effort to develop aptamers would have occurred, and commercial efforts always are needed to facilitate better health care.

Creation of NeXagen and NeXstar

Because of my Synergen experience I had become friendly with people at the venture firm of Warburg-Pincus. Craig and I approached Rod Moorhead, a director of their biotech efforts, and after appropriate due diligence a small company – NeXagen – was set up. Rod and his team had to negotiate with the University of Colorado (who owned the patent application), and one of the chief WarburgPincus people, Pat Mahaffy, moved from New York to be our CEO. Because the patent application was so broad, and because the opportunities for aptamers in health care seemed so rich, we spent some time at NeXagen exploring the technology quite deeply. At the same time others (largely academics) began to see

Diagnostic Imaging

what they could select as well, using the methods we had described. An early aptamer paper, aimed at thrombin, was done by people at Gilead, probably spurred by the fact that the Gilead team included scientists who had been in training at the University of Colorado when Craig Tuerk did his work and defended his PhD thesis. The Gilead thrombin aptamer now is undergoing clinical development (under the direction of Archemix) (Archemix Press Release, 2004) after all this time. The early work at NeXagen was just wonderful. The scientists we hired were great, and they thought of themselves as pioneers. They were on a mission to create an entirely new class of therapeutic drug. Many protein and non-protein targets were run through the SELEX process, almost always successfully (that is, leading to aptamers with low Kd-values and very high specificities toward the intended target molecule) (Eaton et al., 1995). We studied the length requirements in the random regions of a SELEX library and learned how to make aptamers stable against most nucleases. In short we did what one must do with a new technology, leading to many additional patents and an enviable intellectual property position (Thiel, 2004). One reason for the strong patent position was that many people felt that aptamers had little or no commercial “legs” – people were convinced that antibodies had to be better than aptamers for most applications, a view that was conventional and in keeping with the brilliant history of molecular biology that had occurred at the MRC laboratories in Cambridge (Judson, 1980). No single piece of writing is as strong as the Horace Freeland Judson book in describing why antibodies (as proteins) were thought to be vastly superior to aptamers, more or less independent of the biochemical data we had accumulated for aptamers. That is, we all learned to think of proteins as the astounding molecules in cells, and to think of the four-letter nucleic acid code as chemically narrow compared with the 20 amino acids in proteins. This mind set – RNA and DNA are informational, proteins are globular and do “work” – was played out earlier when Tom Cech and his colleagues discovered ribozymes. It was Tom’s ability to think for himself that led to his great work. After Tom won the Nobel Prize, I wrote about the difficulties one has fighting strong conventional ideas (especially if they are mostly right, as is the Central Dogma). That paper, “Catalytic RNA: a Nobel Prize for small village science” (Gold, 1990), made the case that Tom was fortunate to have discovered ribozymes in Boulder, Colorado, a place where no one would say to him “but that cannot be.” Having other scientists, often ones friends, start sentences with “Yes, but…” is not a lot of fun!

Diagnostic Imaging

Meanwhile, the work continued at NeXstar (after a corporate move that merged NeXagen with Vestar to create NeXstar). An early agreement gave Schering AG the right to aptamers for in vivo diagnostic imaging. An aptamer candidate in

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the tumor imaging field aimed at tenascin-C (Daniels et al., 2003, and references therein) is now in clinical development. The advantages of aptamers over antibodies for these applications might be substantial: aptamers bind very tightly to their target analytes, can clear from the blood more quickly than antibodies, and can penetrate (by diffusion) a pathological location faster than antibodies – these three parameters alone could provide better signal-to-noise for aptamers than is possible with imaging antibodies.

Aptamer Therapeutics

The scientific staff at NeXstar were faced with an interesting problem. As a group we were largely academic (in beliefs, in approaches, in politics, all of it) and we were trying to make drugs using a new technology. We were facing a pharmaceutical industry that had not yet learned that injected drugs (like therapeutic antibodies or other biologicals) were a legitimate idea and, on top of that, aptamers had a short academic history. The scientists at NeXstar were asked to choose a protein target for therapeutic intervention, identify a neutralizing aptamer with high affinity and specificity, and convince Pat Mahaffy and me that the commercial opportunity for that aptamer was significant. We were lucky to have on the scientific staff Nebojsa Janjic, a scientist more than up to those tasks. Early on, Nebojsa had decided that angiogenesis was often pathological and should be a target for therapeutic intervention, and as such he and his team began successful aptamer development programs aimed at (among others) VEGF, bFGF, and PDGF. Nebojsa’s VEGF aptamer, then called NX1838, became the lead therapeutic aptamer at NeXstar, and went into the clinic in 1998 for the treatment of age-related macular degeneration. The Phase I clinical trial started only about 8 years after the invention of the technology, a remarkable event for which Nebojsa deserves enormous credit. Of course we now know NX1838 as Macugen, the first aptamer drug approved by the FDA. Macugen is owned by Eyetech (under a license agreement with Gilead), and has been partnered with Pfizer for worldwide development and distribution. Other drug discovery technologies also are being aimed at VEGF and its receptors. For many years I thought that “me too” drugs within the pharmaceutical industry are a waste of effort and money, and I still do; however, I do appreciate the value of a therapeutic target validated in humans. Macugen (and Avastin, for oncology) validate the VEGF pathway in diseases that depend upon increased angiogenesis. After NeXstar was sold in 1999 to Gilead (who did a good job with its aptamer assets), a new company – Archemix – ended up with the therapeutic rights to all aptamers other than those aimed at VEGF. Archemix is building additional opthamological aptamers for Eyetech and has moved some of the legacy aptamers from NeXstar and even Gilead (the thrombin aptamer) into clinical development. Archemix is working through the issues around aptamer “cost of goods” so that indications other than within the eye can be helped by novel aptamers. Like NeXstar before it, Archemix continues to focus on extracellular therapeutic targets, choos-

Do Natural Aptamers Exist?

ing not to tackle the additional difficulties in getting aptamers across cell membranes.

Aptamer-based Diagnostics at SomaLogic

Around 1997, the NeXstar management team concluded that diagnostics might be another strong application for aptamers. Earlier we had tried to develop diagnostics with Becton-Dickenson, but had not seen a differentiated path toward diagnostic products that took advantage of the differences between aptamers and antibodies. Thus at the time of the Gilead acquisition NeXstar still owned all the diagnostic rights, which Gilead was willing to sell to a small group of scientists and investors. Thus in 2000 we started SomaLogic, a small company aimed at using aptamers for all manner of ex vivo measurements (see www.somalogic.com for some details). We realized that a differentiated path did exist, using photoaptamers rather than classic aptamers. Through an analogy with sandwich formats using pairs of antibodies, we realized that an aptamer that contained a photo-activated cross-linking nucleotide could provide a second element of specificity so that very rare proteins (in serum or plasma) might be cross-linked and measured quantitatively (Golden et al., 2000; Petach and Gold, 2002; Gander et al., 2003; Gander and Brody, 2005). In our view the use of photoaptamers was a form of kinetic proofreading, as described by Hopfield (1974), and we expected to be able to develop extreme specificity without sandwiches in complex matrices such as urine, tissue homogenates, serum, or plasma. We have been developing panels of photoaptamers for in vitro diagnostics, and have done some early biomarker discovery as well as mathematics aimed at aggregating information from independent markers. At this point we have only done nine clinical studies on serum from various people and their controls, and the data are quite strong for the seven studies in which we discovered markers. SomaLogic has made a distribution agreement with Quest Diagnostics that should allow us to get these multiplexed proteomic arrays into use for patient care.

Do Natural Aptamers Exist?

Over the last 15 years, as the biochemical qualities of aptamers have been appreciated more, various scientists began to find natural aptamers in extant creatures, as had been predicted (Gold et al., 1997, 2002). We thought natural aptamers would be found if one looked properly, and those thoughts diminish in no way the stunning discoveries from Ron Breaker’s laboratory (Mandal and Breaker, 2004; Mandal et al., 2004; Serganov et al., 2004); predictions are easy, and discovery is very hard work! Through careful genome scanning of many bacterial species, Breaker and his colleagues were able to identify conserved RNA structures that suggested “natural aptamers” and to then show that the predicted aptamers

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were signaling metabolite concentrations and driving an appropriate physiological response to those metabolic demands. “Breakerism” must exist in mammals and other eukaryotes also but the sequence gazing that successfully identifies natural aptamers in the eubacteria is more difficult in eukaryotes (as Breaker has himself suggested (personal communication), and see Sudarsan et al., 2003); we simply do not have enough whole genome sequences of mammals to infer conserved RNA structures that play a regulatory role. I continue to believe that mammalian organisms and genomes are messy, similar to Rube Goldberg machines. The perfectly trimmed mammalian genome might evolve after more planetary time, as probably exemplified by many eubacterial and bacteriophage genomes. Genomes today reflect the confluence of evolutionary pressures and time, and 5 billion years on the earth is not really a long time (given replication rates for mammals).

Conclusions – SELEX Lessons for Drug Discovery

Aptamers are discovered through a combinatorial chemistry paradigm. At the bench, with something like 1015 potential single-stranded DNA or RNA aptamers in a test tube, various target molecules are used to screen the library to find aptamers that bind most tightly and selectively. The paradigm is decidedly not a rational drug design method. Within the large numbers of molecules in the library one asserts that one or more sequences fold into an “attractive” globular shape for binding with high affinity and specificity to the target. No attempt has been made (in spite of much pressure to do so) to “design” the oligonucleotide pools to be target “friendly” – when that was tried, nothing profound happened. Craig Tuerk and I understood one big thing, which was that the intricate threedimensional shapes of single-stranded oligonucleotides, including the vast structural possibilities within the unpaired nucleotides, were sufficient to think of aptamers as nucleic acid antibodies. However, one might like to do SELEX with oligonucleotide libraries that include better chemistry; that has been achieved through the beautiful pyrimidine chemistry of Bruce Eaton (Eaton, 1997). One might imagine that the future of SELEX will include five position-modified pyrimidines such that the oligonucleotides can still be amplified (and thus SELEXed) but that the resulting chemistries will be even more “protein-like.” In fact, this is a large part of what we do at SomaLogic. SELEX is not typing; aptamers must be identified at the bench, even though the SELEX paradigm can be routine. There are drug discovery paradigms that are typing – antisense, ribozymes, and now siRNAs. For a long time people have hoped that a “typing” paradigm would work, and the latest candidate technology – siRNAs – is now in its euphoric stage. In the final analysis typing-based oligonucleotides (and aptamers as well) will struggle with the cost of goods, with aptamers (aimed at extracellular targets, sensibly) having an advantage over those molecules that must enter a cell to function (such as the “typed” drugs,

Conclusions – SELEX Lessons for Drug Discovery

which are always aimed at oligonucleotide targets). Aptamer therapeutics will succeed or fail more because of the cost of oligonucleotide synthesis (which will come down over time) than because of the intrinsic affinity or selectivity of aptamers – aptamers have wonderful affinity and specificity. More interestingly, SELEX and aptamers may have something profound to say about combinatorial chemistry methods aimed at finding orally active small molecules. Combinatorial chemistry is not in as much favor today as it once was, probably because no one has made large enough libraries to screen, and no one has figured out how to screen efficiently. Can huge libraries, well screened, yield better orally active compounds than we are used to seeing? (After all, aptamers compete well with antibodies, and in many cases have higher affinities and specificities – is that because SELEX uses huge libraries or because oligonucleotides have better chemistries than proteins? Probably aptamers with terrific properties are identified because of the starting library size and not the functional advantages of RNA or DNA over proteins; as Bruce Eaton once noted (personal communication), RNA and DNA chemists should have “side group” envy toward the amino acids.) There is a lesson for finding small, classic, orally active drugs. One might select orally active compounds from enormous libraries or one might quickly identify some weak “hit” present in smallish libraries and ask the medicinal chemists to develop nanomolar and picomolar compounds based on those indifferent compounds. Today’s chemists work with focused libraries that are built from weak hits, largely because no one can change the game by giving the chemists better hits as their starting materials. SELEX provides both high-quality compounds (with picomolar Kd-values) and a useful paradigm for finding better small molecules as hits. The dual requirements for terrific drugs are high affinity and specificity, which can be obtained even with the limited surface interactions available from a compound with a molecular weight around 700 or so. It is not axiomatic that small molecules must have low affinity and specificity (and thus unexpected side effect profiles from non-target interactions) – we can look at biotin binding to avidin and think that our screening protocols are more limiting than is chemistry itself. The available shape space for medicinal chemists is adequate for better drugs than we are used to seeing. While SELEX may be a source of strong aptamer therapeutics for many years to come, SELEX also has taught us that large libraries have better winners than smaller libraries, a deep and obvious lesson from the SELEX experience of the last 15 years.

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Acknowledgments

SELEX could not have happened without Craig Tuerk. Nebojsa Janjic is the person who facilitated, more than anyone else (even as others contributed), the proof that aptamers could be drugs. Aptamer development on a broad scale was a function of many friends and colleagues at NeXstar and more recently at SomaLogic, Archemix, NOXXON, and Eyetech.

References Archemix Press Release (2004). Archemix and Nuvelo announce inititaion of Phase I trial for thrombin inhibitor ARC183. 9 August 2004. Daniels, D. A., Chen, H., Hicke, B. J., Swiderek, K. M., Gold, L. (2003). A tenascin-C aptamer identified by tumor cell SELEX: systematic evolution of ligands by exponential enrichment. Proc Natl Acad Sci USA 100, 15416–15421. Eaton, B. E. (1997). The joys of in vitro selection: chemically dressing oligonucleotides to satiate protein targets. Curr Opin Chem Biol 1, 1–16. Eaton, B. E., Gold, L., Zichi, D. A. (1995). Let’s get specific: The relationship between specificity and affinity. Chem Biol 2, 633–638. Ellington, A., Szostak, J. (1990). In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822. Gander, T. R., Brody, E. N. (2005). Photoaptamer chips for clinical diagnostics. Expert Rev Mol Diagn 5, 1–3. Gander, T. R., Brody, E. N., Mehler, R. E., Heilig, J. S., Singer, B. S., Gold, L. (2003). Driving forces in cancer diagnostics. MLO Med Lab Obs 35, 10–16, 20; quiz 20–11. Gold, L. (1990). Catalytic RNA: a Nobel Prize for small village science. New Biol 2, 1–4. Gold, L., Tuerk, C. (1995). US patent Number 5,475,096 (December 12, 1995). Gold, L., Pribnow, D., Schneider, T., Shinedling, S., Singer, B. S., Stormo, G. (1981). Translational initiation in prokaryotes. Annu Rev Microbiol 35, 365–403. Gold, L., Brown, D., He, Y.-Y., Shtatland, T., Singer, B. S., Wu, Y. (1997). From oligonucleotide shapes to genomic SELEX: Novel biological regulatory loops. Proc Natl Acad Sci USA 94, 59–64.

Gold, L., Brody, E., Heilig, J., Singer, B. (2002). One, two, infinity: genomes filled with aptamers. Chem Biol 9, 1259–1264. Golden, M. C., Collins, B. D., Willis, M. C., Koch, T. H. (2000). Diagnostic potential of PhotoSELEX-evolved ssDNA aptamers. J Biotechnol 81, 167–178. Hopfield, J. J. (1974). Kinetic proofreading: a new mechanism for reducing errors in biosynthetic processes requiring high specificity. Proc Natl Acad Sci USA 71, 4135– 4139. Judson, H. F. (1980). The Eighth Day of Creation: Makers of the Revolution in Biology. New York: Simon & Schuster, 1980. Lemaire, G., Gold, L., Yarus, M. (1978). Autogenous translational repression of bacteriophage T4 gene 32 expression in vitro. J Mol Biol 126, 73–90. Mandal, M., Breaker, R. R. (2004). Gene regulation by riboswitches. Nat Rev Mol Cell Biol 5, 451–463. Mandal, M., Lee, M., Barrick, J. E., Weinberg, Z., Emilsson, G. M., Ruzzo, W. L., Breaker, R. R. (2004). A glycine-dependent riboswitch that uses cooperative binding to control gene expression. Science 306, 275–279. Pavlov, A. R., Karam, J. D. (2000). Nucleotidesequence-specific and non-specific interactions of T4 DNA polymerase with its own mRNA. Nucleic Acids Res 28, 4657–4664. Petach, H., Gold, L. (2002). Dimensionality is the issue: use of photoaptamers in protein microarrays. Curr Opin Biotechnol 13, 309– 314. Serganov, A., Yuan, Y. R., Pikovskaya, O., Polonskaia, A., Malinina, L., Phan, A. T., Hobartner, C., Micura, R., Breaker, R. R., Patel, D. J. (2004). Structural basis for discriminative regulation of gene expression by

References adenine- and guanine-sensing mRNAs. Tuerk, C., Gold, L. (1990). Systematic evoluChem Biol 11, 1729–1741. tion of ligands by exponential enrichment: Sudarsan, N., Barrick, J. E., Breaker, R. R. RNA ligands to bacteriophage T4 DNA (2003). Metabolite-binding RNA domains polymerase. Science 249, 505–510. are present in the genes of eukaryotes. Winter, R. B., Morrissey, L., Gauss, P., Gold, RNA 9, 644–647. L., Hsu, T., Karam, J. (1987). Bacteriophage Thiel, K. (2004). Oligo oligarchy-the surprisT4 regA protein binds to mRNAs and preingly small world of aptamers. Nat Biotechvents translation initiation. Proc Natl Acad nol 22, 649–651. Sci USA 84, 7822–7826.

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Index a abzymes (catalytic antibodies) 56, 211-213 acute pancreatitis 157 acute respiratory distress syndrome (ARDS) 148 adaptive evolution 31 adaptive landscape 57 adenovirus 131 adjuvant 154, 398 ADP 72 ADP-ribosylation factors (ARFs) 283 affinity 371 affinity capillary electrophoresis 335, 338 affinity chromatography 324-325, 328, 330, 426 – affinity capillary chromatography 327 – affinity ligand 325 – analyte capture 326 – buffer composition 331 – capillary internal diameter 331 – chiral separation 332, 340 – chiral stationary phase 332, 334 – chromatographic support 325 – column temperature 332 – EDTA 331 – EDTA gradient 327 – eluent pH 332 – elution conditions 325 – immobilized ligand 325 – immunoaffinity columns 325 – ionic strength 331 – mass transfer kinetics 340 – microbead affinity chromatography 328 – mobile phase pH 331 – particle diameter 331 – protein capture 327 – recovery 327 – salt concentration 332 – stationary phase 325-326, 328-329, 340 – streptavidin sepharose support 326

affinity purification 122-123, 214 affinity tag 122, 125, 128 African trypanosomiasis (African sleeping sickness) 366 age-related macular degeneration (AMD) 368, 387, 405, 443, 446, 453, 464 – degenerating RPE cells 447 – drusen 447 – dry, or non-exudative AMD 447 – lines of vision 455 – neovascularization 457 – ocular neovascularization 447 – photodynamic therapy (PDT) 453 – proliferative retinal neovascularization 451 – regression of neovessels 457 – wet AMD, also known as exudative or neovascular AMD 447 agonists 270 alcohol dehydrogenase (ADH) 78, 219-220 allometric coefficients 392 allosteric deoxyribozymes 290 allosteric ribozymes; see aptazyme, allosteric (deoxy) ribozymes allosteric ribozymes 290 AMD; see age-related macular degeneration (AMD) analyte–ligand interaction 325 angiogenesis 140, 274, 349, 368, 443 antibody 268-269, 303, 324-325, 349, 363, 366-367, 369-370, 383, 398-399, 426, 438, 463- 464 antibody fragments (scFv, Fab) 345, 348 anticoagulation 149 anticoagulation and thrombosis 368 antidote 136, 151, 369, 383 antigen–antibody complexes 335 antisense 170, 186, 235, 268-269, 319, 353, 355-357, 381-382, 384-386, 388-389, 395, 398, 466 – 99mTc-labeled antisense oligonucleotide 350


472

Index antiterminator 180 anxiety 160 aptamer-competition screens 314 aptamer conversion assay 316 aptamer displacement 317 aptameric sensor; see biosensor (aptasensor, sensor) aptamers as ligands for affinity chromatography 326, 330, 334 – d-adenosine 332 – adenosine 326, 330-331 – adenosine and analogs 326 – amino acids and derivatives 326 – d-arginine 334, 340 – enantiomers 326 – flavin mononucleotide (FMN) 326, 331-332 – HCV RNA polymerase and replicase 326 – non-target analytes 326 – non-target proteins 326 – proteins 326 – L-selectin 326-327 – small molecules 326 – thrombin 326, 328 – l-tyrosinamide 332 – d-tyrosine 334 – tyrosine 334 – vasopressin 326, 332 aptamers as ligands in affinity capillary electrophoresis – antithrombin III 336, 338 – HIV type 1 reverse transcriptase (HIV-1 RT) 336-337 – IgE 336-337 – thrombin 336-339 aptamers for in vivo imaging 343, 346 – radioiodinated (123I) aptamer 359 – tenascin-C 350, 359 aptamers to – activated protein C 365 – adenine 75 – d-adenosine 332 – l-adenosine 75 – adenosine 74, 95, 305, 319, 326, 330-331 – adenosine and analogs 326 – adenosine-containing ligands 8 – adenosylcobalamin 98 – S-adenosyl homocysteine (SAH) 97 – S-adenosyl methionine (SAM) 97-98, 204 – adhesion molecules, receptors, and other cell surface proteins 364 – ADP 304-305, 318-319 – amino acids 74, 84, 95, 99

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

amino acids and derivatives 326 aminoglycosides 119, 124 amyloid beta 4 (Ab4) 365 angiogenin 364 angiopoietin-1 (Ang1) 373 angiopoietin-2 (Ang2) 137, 142, 274, 364, 369, 373 anti-AChR antibody, mAb198 370 anti-insulin receptor antibody MA20 138, 152 antibiotics 116 antibodies 365-366 antibodies/immunoglobulins 152 antithrombin III 336, 338 d-arginine 75 l-arginine 11, 75, 99-101, 196, 333-334 ARNO/cytohesin-2 283 aspartame 380 ATP 6, 8-9, 11, 74-75, 79, 95-97, 292, 300, 302, 380 ATP analogs 76 autoantibodies 370 B12 (cyanocobalamin) 6, 75, 98 B52 (splicing factor) 272, 368 bacteriophage T4 DNA polymerase 461 basic fibroblast growth factor (bFGF) 137, 142, 364, 369, 372, 374, 464 biotin 75, 99 caffeine 6, 75 cAMP 97, 306 carbohydrates 95, 102 cCMP 306 CD4 364 CD28 380 cell surface carbohydrates 104 cell surface receptor/cell adhesion molecules 155 cell surface receptors 366 cell surface targets 366 cellobiose 103 cellular components 95 cellulose 103 cGMP 296, 306 chloramphenicol (peptide antibiotic) 118, 119, 125 cholic acid 108 Cibacron Blue 3GA 109, 110 l-citrulline 11, 75, 99, 196 coagulation cascade components 365 coagulation factor IXa (FIXa) 369 cobalt ions 306 cocaine 107-108 coenzyme A (coA) 76-77, 97-98

Index – coenzymes 74 – cofactors 97 – complement C5 139, 157, 365, 371, 401-402, 405 – CTLA-4 138, 154, 274, 364, 367 – cyanocobalamin 6, 75, 98 – 3l, 5l cyclic-monophosphates 296 – 3l5l-cyclic-GMP 297 – cytohesin 1 271 – cytohesin 1 (Sec7 domain) 282-283, 314, 317, 365, 368 – cytokine/growth factors 140, 364, 375 – cytotoxic T-cell antigen 4 (CTLA-4) 138, 154, 274, 364, 367 – l-dopa 101 – dopamine 101 – E2F1 protein 146 – extracellular domain of HER3 375 – extracellular membrane proteins 139 – extracellular regulated kinase 2 (ERK2) 314, 365 – factor VIIa (FVIIa) 138, 151, 365, 369 – factor IX (FIX) 151 – factor IXa 138, 274, 365, 405 – fibronectin 156 – flavin 79, 98 – flavin moiety 99 – flavin mononucleotide (FMN) 95, 98-99, 112, 292-294, 300, 306-307, 326, 331-332 – flavin-adenine diphosphate (FAD) 11, 77, 98-99 – fluorescein 110-111 – fluorescent dyes 95, 109 – G protein-coupled receptor kinase 2 (GRK2) 314 – gonadotropin-releasing hormone (GnRH) 274 – growth factors 375 – GTP 6, 11-13, 375 – guanine 75, 96, 204 – guanosine 75 – guanosine monophosphate (GMP) 11 – HCV NS5B 365 – HCV RNA polymerase and replicase 326 – heparin binding targets 363 – heparin-binding proteins 372 – heparin sulfate 156 – hepatitis C DNS3 138 – hepatitis C NS3 138, 147, 282 – hepatitis C virus (HCV) RNA polymerase and replicase 328 – HER3, extracellular domain 364, 367, 375 – HIV gp120 365

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

HIV integrase 365 HIV-1 regulatory protein 367 HIV-1 Rev ARM 299 HIV-1 Rev peptide 6 HIV-1 Rev protein 137, 271, 281, 285, 303-304, 307, 314, 316-317, 365 HIV-1 reverse transcriptase (HIV-1 RT) 137, 145, 281, 285, 336-337, 365, 375 HIV-1 surface envelope glycoprotein 120 (gp120) 367 HIV-1 Tat 137, 144, 271, 365 HIV-2 Tat 137 Hoechst dye 33258 110-111, 121 hTSA 364 human epidermal growth factor receptor-3 (HER3) 367 human neutrophil elastase (HNE) 138, 148, 365, 382 Human non-pancreatic secretory phospholipase A2 (hnps-PLA2) 139, 157 human OSM 373 ICAM-1 375 Immunoglobulin E (IgE) 138, 153, 336-337, 365, 369, 376 aLb2 -integrin, cytoplasmic domains 271 interferon-g 137, 142 intracellular targets 367 isoleucine 83 l-isoleucine 101 kanamycin A (aminoglycoside) 124 kanamycin B (aminoglycoside) 124 keratinocyte growth factor (KGF) 364, 372, 377 laminin 156 LFA-1 (CD18) 364 lipoproteins 139 lividomycin (aminoglycoside) 124 lumiflavin 98 lysozyme 307 mAb 20 (anti-insulin receptor antibody) 365, 370 mAb G6–9 (anti-DNA antibody) 365 mAb198 (anti-AChR antibody) 153, 365 malachite green 6, 110-111 MAP kinase ERK 270 MAP kinase, pERK 270 MAP kinase ERK2 304 MCP-1 364, 369 metal ions 74 monoclonal antibody to acetylcholine receptor 138 MS2 coat protein 6 NAD 97-98

473

474

Index – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

– – – – – – – – – – – – – – – – – – –

NAD+/ATP 77 NAD+ 74 NAD(P) 95 natural products 105 neomycin 117, 119 neomycin B (aminoglycoside) 124 neuropeptide Y 364 NFkB 137, 146-147, 271, 380 NGF 364 nicotinamide cofactors 98 NMN 98 NMN/NAD 75 non-target analytes 326 non-target proteins 326 nucleic acid binding proteins 144 nucleic acid structures 167 nucleocapsid (NC) protein of HIV-1 282 nucleotide 95 nucleotide factors 74 oncostatin M (OSM) 364, 369 organic dyes 109 organic molecules 281 paromomycin 124 peptide antibiotic viomycin 119 peptide hormones 366 l-phenylalaninamide 102 l-phenylalanine 101 pigpen 364, 366 platelet-derived growth factor (PDGF) 137, 143, 273-274, 276, 313, 315, 349, 364, 372, 374, 377, 403, 405, 464 porphyrins 74 prion protein PrPSc 139, 158 prostate-specific membrane antigen (PSMA) 139, 156, 350, 364, 367, 375 protein 131, 281, 299, 326 Raf-1 RBD 365 Reactive Blue 4 110 Reactive Brown 10 110 Reactive Green 19 110 Reactive Red 120 110 Reactive Yellow 86 110 riboflavin 75, 98 RNA polymerase II 281 Sec7 domain of cytohesin-1 282-283, 314, 317, 365, 368 L-selectin 138, 326-327, 350, 364, 367, 371-372, 374-375, 377, 402-403 L-selectin/IgG fusion protein (LS-Rg) 155 P-selectin 138, 155, 364, 367, 371-372, 374 selectins 363 sephadex 95 sephadex G-100 104

– – – – – – – – – – – – – – – – – – – – – –

– – – – – – – – – – – – – – – – –

– – – – –

serine proteases 147 sialyl Lewis X (sLex) 104 sialyllactose 104, 381 signaling mediators 365 small molecules 95, 326 small organic dyes 6 small peptides 74 special elongation factor (Se1B) 281 splicing factor B52 272, 281, 368 streptomycin (aminoglycoside) 117, 119, 122, 128 substance P 365, 371 subunit p50 147 sulforhodamine B 110-111 T4 DNA polymerase 132, 211 TAR RNA 171-172 target binding 45 tenascin-C (TN-C) 157, 274, 346, 350, 353, 359-360, 364, 367 tetracycline (peptide antibiotic) 118-119, 121 TGFb1 364 TGFb2 364 theophylline 17, 75, 105-106, 112-113, 292, 296-297, 300, 306-308 thrombin 6, 138, 149, 274, 300-301, 326, 328, 330, 336-339, 349, 353, 359, 365, 369, 373, 378, 383, 389, 400-401, 405, 463-464 tobramycin 124, 128 transcription Factor E2F 145 transcription factor NFkB 282 thrombospondin 156 TNF, tumor neurosis factor 274 tobramycin (aminoglycosides) 117 transcription factor E2F 137 Trypanosoma brucei (flagellar pocket protein) 364 Trypanosoma brucei VSD protein 364 Trypanosoma cruzi 139, 156 Trypanosoma cruzi cell surface receptor 364 l-tryptophan 101 l-tyrosinamide 102, 332 d-tyrosine 101 l-tyrosine 101 l-valine 75, 101 vascular endothelial growth factor (VEGF) 137, 140, 305, 314, 349, 364, 368, 371-372, 374, 377, 381, 383-384, 387, 405, 443, 464 vasopressin 326, 332, 364 VEGF-A 274 viomycin (peptide antibiotic) 117, 126 viral coat proteins 366 viral proteins 365

Index – viruses 281 – Vitamin B12 204 – wheat germ agglutinin (WGA) 313-315 – xanthine 96 – YPEN-1 cells 366 aptamers with catalytic properties – biphenyl isomerization 213 – cholesterol esterase activity 213 – porphyrin metallation 213 aptasensor; see biosensor (aptasensor, sensor) aptazyme, allosteric (deoxy) ribozymes 16, 56, 79, 290, 303-304, 318 – allosteric hammerhead ribozyme 106 – aptamer-controlled ribozyme 17 – array 306-307 – ATP-dependent ligase 79 – Boolean logic gates 306 – cAMP 79 – cGMP 79 – communication module 294 – deoxyaptazyme 301 – effector activation 295 – FMN aptamer motif 99 – hammerhead ribozyme pool 296 – molecular beacon 106 – molecular computation 306 – rational design 292 – ribozyme ligase 106 – scheme for an effector-activated aptazyme 295 – self-cleaving aptazyme 18 – theophylline aptamer 17, 106 – theophylline-dependent ligase 79 – use in vivo 308 aptazyme effector – adenosine 305, 319 – ADP 304-305, 318-319 – ATP 292, 300-302 – cAMP 306 – cCMP 306 – cGMP 296, 306 – cobalt ions 306 – 3l, 5l cyclic-monophosphates 296 – 3l5l-cyclic GMP 297 – Cyt18-dependent 299 – flavin mononucleotide (FMN) 292-294, 300, 306-307 – HIV-1 reverse transcriptase (RT) 285 – lysozyme 307 – MAP kinase ERK2 304 – non-nucleic acid effectors 291 – non-oligonucleotide effectors 292 – oligonucleotide 291

– – – – – –

peptide 292, 296, 300 protein 292, 296, 299 Rev ARM 299 Rev protein 285, 303-304, 307 small organics 292, 296 theophylline 17, 106, 292, 296-297, 300, 306-308 – thrombin 300-301 aptazyme, catalytic domain – cleavases 305 – deoxyribozyme ligase 302 – 8–17 deoxyribozymes 292, 305 – 10–23 deoxyribozymes 292 – group I ribozyme 292 – hairpin 292, 300 – hammerhead 292, 296-297, 301, 303-304, 306-307 – HDV 292 – L1-ligase 293-294, 296-297, 306-307 – ligase 292-293, 299, 305 – self-splicers 305 – signaling aptazyme 303 – trans-acting hammerhead 292 – X-motif ribozymes 292 aptogenicity 371 ARC 127; see PDGF-BB aptamer ARC 127 ARC183; see thrombin aptamer ARC183 ARC187; see complement C5 aptamer ARDS 157 area under the concentration–time curve, AUC 388 arthritis 148 asthma 369 atherosclerosis 356 ATP 72 autoimmune disease 142, 368-369 autoimmunity 152, 373 autoradiography 303 avidity 194

b bacterial peritonitis 157 base stacking 312 beacons 318 bifunctional kissing aptamer 177-178 biliary excretion 390 binding analysis 133 binding kinetics 373 binding pocket 196, 200, 213 bioanalytical methods – capillary gel electrophoresis (CGE) 395 – hybridization-based ELISA-type assays 395 – liquid scintillation counting (LSC) 393

475

476

Index – lipophilic moieties 389 – short, positively charged cell-permeating peptides composed of polyarginine (Arg7) 379 Chagas’ disease 156 chelator 352 – bifunctional 353 – DO3A 354, 359 – DOTA 352, 354, 356, 358 – DTPA 350-352 – EDTA 376 – 68Ga-DO3A-TTA1 360 – HYNIC 350-351 – MAG2 359 – MAG3 350-352, 356 – peptidergic 352 chemical interference 176 chemical trapping 214 chemokines 369 chemotactic growth factor 143 chemotherapeutics 276 chimeric RNA expression system 281 c CABG; see coronary artery bypass graft (CABG) chimeric tRNA–RRE aptamer 145 cancer 141-142, 403 chiral inversion 334 – VEGF antibody (avastin) 445 chiral principles 417 capillary electrochromatography (CEC) 328, chirality 212 331, 340 – asymmetric carbon 420 capillary electrophoresis (CE) 324 – homochiral polymer 427 – laser-induced fluorescence detection 335 – homochirality 437 – mobility 337 – lock-and-key stereocomplementarity 422 – separation efficiency 337 – optical antipodes 419 cardiopulmonary bypass (CPB) 149 – polyaffinity theory 420 cardiovascular diseases 350 – shape complementarity 420 – atheriosclerosis 145 – stereocomplementarity 420 – cardiac hypertrophy 145 – stereoisomers 420 – cardiac intimal hyperplasia 145 circular dichroism (CD) 426 catalysis 212 circulation time 349 catalytic activity 4 clearance 149, 349, 388-390 catalytic antibody 56, 211-213 coagulation cascade 149, 151, 369 catalytic center 213 coenzyme 72, 80-83, 191, 204 catalytic pocket 214 coenzyme A 72 cell adhesion 271 colorectal cancer 369 cell membranes 349 combinatorial chemistry 461, 467 cell trafficking 155 combinatorial library 4, 229 cell–cell adhesion 366 compartmental modeling 388 cellular biodistribution 393 competition assay 313 cellular uptake 348, 368 competition problem 55 – 13-amino-acid fragment (Tat) of the HIV competitive approach 325 Tat protein 379 complement activation 157 – 16-amino-acid sequence derived from the complement C5 aptamer ARC 187 401-402 third helix of the Drosophila antennapedia complement system (Ant) homeotic protein 379 – alternative pathway 401 – cell-permeating peptides 389 – classical pathway 401 – LSC 395 – mass spectrometry methods such as MALDI-TOF 395 – quantitative nucleic acid-intercalating dye (QNID) fluorescence assays 395 – quantitative whole-body autoradiography (QWBA) 393, 395 – radioimmunoassay (RIA) 396 bioavailability 135, 347, 349, 391 biodistribution 346, 387-390, 392, 396 bioinformatic approach 196, 201 biomarker discovery 465 biosensor (aptasensor, sensor) 107-108, 112, 240, 285, 290, 303, 305-306, 318, 324, 426 biostability 418, 437 biosynthesis of nucleotides 219 blindness 446 breast cancer 159, 429 Bruch’s membrane 447 bypass vein grafts 146

Index – lectin pathway 401 confocal microscope 328 conformation 136 controlled-release – encapsulation 391 – poly(lactic-co-glycolic) acid (PLGA) 384 cooperative binding 203 coronary artery bypass graft (CABG) 373 – CABG 383, 400, 402 – CABG surgery 389 Creutzfeldt–Jakob disease 158 crystallization 422 crystallography; see X-ray crystallography cyclic AMP 72 cytochrome P450 family 390 cytokines 369 cytomegalovirus (CMV) promoter 185 cytoskeletal rearrangement 271 cytoskeleton 283 cytotoxins 366

d Darwinian evolution 14, 20 Darwinian selection 30 decoy 131, 144-145 delivery 368 deoxyribozyme (DNAzyme) 3, 7, 220, 228, 229 – ATP-utilizing DNAzyme NTP-A1 250 – bipartite DNAzymes 245 – bipartite I 245 – bipartite II 245 – class I capase deoxyribozyme 251-252 – 8-17 deoxyribozyme 233-234, 240-244, 257, 292 – 10-23 deoxyribozyme 233-238, 292 – deoxyribozyme ligase 302 – deoxyribozymes for thymine dimer repair 253 – DET22-18 245-246 – DNA enzymes catalyzing the formation of phosphorothioester bond 252-253 – DNA enzymes with foreign functionalities 254 – DNA ligase 301 – DNA ligating DNAzyme 231, 252 – DNA-cleaving deoxyribozyme 248 – DNA-cleaving DNA enzymes 248 – DNA-modifying DNA enzymes 249 – 8-17 DNAzymes 235 – DNAzyme-catalyzed DNA ligation 252 – DNAzymes for thymine dimer repair 254

– fluorescence-signaling DNAzyme DET22-18 245-246 – 10-28 N-glycosylase DNAzyme 248 – histidine-dependent DNAzyme 245 – ligase deoxyribozyme, 7Q10 247 – Mg2+-dependent RNA-cleaving DNA enzyme 230 – Mg2+-dependent RNA-cleaving DNAzyme 233 – Mg5 deoxyribozyme 242 – Pb2+-dependent RNA-cleaving DNAzymes 230 – porphyrin metallating DNAzymes 233 – RNA cleavage reaction 229 – RNA-cleaving deoxyribozyme 925 -11 256 – RNA-cleaving deoxyribozymes 243, 245-246, 255 – RNA-ligating deoxyribozymes 246-247 – self-capping (or self-adenylating) DNAzymes 251 – self-phosphorylating deoxyribozymes Dk1, Dk2 251 – self-phosphorylating DNAzymes 250 – UV1C 254 – Zn2+/Cu2+-dependent DNA-ligating DNAzymes 231 depot 384 diabetes 444 diabetic retinopathy 141-142, 403, 457 – fibrovascular proliferation 448 – loss of pericytes 448 – microaneurysms 448 – microvascular changes 448 – proliferative diabetic retinopathy 448 – proliferative retinal neovascularization 451 – retinal edema 448 – retinal hemorrhage 448 Diels-Alderase 224 – diastereoselectivity 222 – enantioselective transformations 221 – enantioselectivity 221 – immobilization 223 – mirror-image l-ribozyme 221 – modeled three-dimensional structure 223 – probing experiments 223 – refined secondary structure 223 – secondary structure 222 dimerization initiation site (DIS) 172 dimerization signal 168 direct protein inhibitor 265 discrimination 329

477

478

Index fitness landscape 54, 57-58, 60-62, 64, 85 l-DNA 421, 434, 437 fitness value 57 – l-deoxyuridine 421 DNA enzyme; see deoxyribozyme (DNAzyme) flow cytometry 324 fluorescence signaling 246, 319 DNA nanomotor 239 fluorescence intensity (FI) 314 DNA shuffling 6 fluorescence lifetime (FLT) 314 DNA–RNA–protein world 219 fluorescence polarization (FP) 314, 316 domain approach 428 fluorescence resonance energy transfer Doppelgnger proteins 420 (FRET) 303-304, 319 Drosophila melanogaster 180 Food and Drug Administration (FDA) 363, drug development 265 383-384, 405, 443, 446, 452 drug discovery 265 formulations 383 drug screening 287 – polyalginate/polylysine microparticles 386 drug target 268-269 FRET; see fluorescence resonance energy drugability 280 transfer (FRET) dynamic nanomaterials 240 function landscape 56, 85 e functional sequence variant 13 E2F DNA decoy aptamer – ex vivo pressure-mediated transfection 146 g – bypass vein grafts 146 gene expression 268, 272 – intimal hyperplasia 146 gene knockout 268 – phase III study 146 gene regulation 193 electrochromatography 325 gene therapy 168 electron transfer 220 genetic algorithm 57 electrophoretic mobility shift assay 174, 184 genetic engineering 290 ELISA 324, 432 genotoxicity 397 ELONA 324 genotype and phenotype 30 emphysema 148 genotype–phenotype map 39 enantioselectivity 332 ghrelin-Spiegelmer NOX-B11 endometriosis 159, 429 – acute food intake 432 3l-endo sugar pucker 371 – c-Fos-like immunoreactivity 432 energy function 57 – GH release 432 entropy 14 – specificity to the octanoylated form epitopes 281, 321 of ghrelin 431 equilibrium dialysis assays 429 – weight loss in diet-induced obese equilibrium dissociation constants 370 mice 432 error prone 31 glioblastoma U251 360 error threshold 30, 68-70 glioblastoma 359, 366 error threshold of replication 56 glomerular filtration 392 evolutionary adaptation 56 glomerulonephritis 403 evolutionary design 35, 42, 46 – experimental 144 – breeding of molecules 49 – mesangioproliferative 143 – rational design 49 glutamyl-tRNA synthetase 84 – replication assays 49 glycosylation 383 excretion 347 GNRA tetraloop receptor 168 expression vectors 236 gram-negative organisms 196 extracellular targets 349 gram-positive organisms 192, 196 green fluorescence protein (GFP) 121 f guanine nucleotide exchange factors (GEFs) FAD 72 282 FDA; see Food and Drug Administration guanine riboswitch aptamer (FDA) – complex with hypoxanthine 197 Fischer, Emil 420 – purine metabolism 198

Index – secondary structure 197 – three-dimensional structure 197

h half-life 366, 378-379, 381, 383, 387-390, 392, 396, 420 Hamming distance 31, 33, 35, 49 handedness 421 hemorrhage 151 heparin 143, 152 hepatic clearance 390 hepatitis 147 hepatitis C virus (HCV) 179 heteropolymeric macromolecules 15 heteropolymers 22 hexitol nucleic acid (HNA) 181-183 high-performance liquid chromatography (HPLC) 340 high-throughput screening (HTS) 284, 286, 311, 319-321 Hill coefficient 201 Hill equation 201 l-histidine 78 HIV 131 – replication 144-145, 304, 314 HIV therapy 145 HNA; see hexitol nucleic acid (HNA) HNA–RNA chimeras 182-183, 185 HNA–RNA chimeric 184 homopurine–homopyrimidine tracts 168 Hoogsteen 167 HTS; see high-throughput screening (HTS) hydrogen bonding 312

i IFP; see interstitial fluid pressure (IFP) imaging modalities – computed tomography (CT) 343, 361 – magnetic resonance imaging (MRI) 343-344 – optical imaging (OI) 343-344 – positron emission tomography (PET) 343-345, 349, 351-358, 360-361 – single photon emission computer tomography (SPECT) 343, 345, 351-354, 356-357, 360 – ultrasound 343-344 – X-Ray/CT 344 imidazole 77 immune responses 349 immune system 213 immunogenicity 134, 159, 397-398, 421, 432-433

inflammation 350, 359, 368-369, 393 inflammatory disease 142, 155 informational complexity 12 in silico evolution – fitness landscape 44-45 – flow reactor 42 – fluctuations 44 – optimization process 44 – optimization trajectory 44 – population size 44 – simulated population 43 in silico models 312 interference RNA; see siRNA (small interference RNA, RNAi) interferon 131 interferon response 269 internal ribosome entry site (IRES) 179 internalization 348 interstitial fluid pressure (IFP) 273, 275 intimal hyperplasia 146 intrabodies 268, 281 intracellular proteins 284 intracellular targets 271 intramer to 280-281, 284 – ARNO/cytohesin-2 283 – B52 368 – cytohesin-1 282-283 – HIV-1 reverse transcriptase (RT) 281 – LFA-1 (CD11a/CD18) 368 – NS3 of hepatitis C virus (HCV) 282 – nucleocapsid (NC) protein of HIV-1 282 – Rev protein 281 – RNA polymerase II 281 – Se1B 281 – Sec7 domain of cytohesin-1 368 – special elongation factor 281 – splicing factor B52 281 – transcription factor NFkB 282 intramer expression system 281 inverse folding algorithm 50 in vitro evolution 84, 219, 257 in vitro selection 168, 173, 204, 229, 241, 243, 245-246, 248-249, 253, 255, 290, 299-301, 305, 324, 430 – allosteric selection 17 – automation 281 – 10B-containing aptamers 380 – biased (or doped) selection 371 – build species cross-reactivity 373 – by binding to transition state analogs 56 – by compartmentalization 18, 20 – chimeric approach 112 – chiral principles 437

479

480

Index – – – – – – –

counterselection 96, 122, 283 covalent inhibitor 148 cross-SELEX approach 373 degenerated RNA library 282 de novo creation of ribozymes 213 direct selection (techniques) 232, 256 direct selection with linker-coupled reactants 214-215, 221 – DNA-SELEX 418 – encapsulated selection 22 – Fluorescence-activated cell sorting (FACS) based selection 18-20 – in vitro aptazyme selection 293 – mirror-image in vitro selection 424 – mirror-image evolutionary techniques: selection–reflection 422 – multiple-turnover catalyst 18 – of protein enzymes 19 – partitioning 374 – 5-(1-pentynyl)-2l-deoxyuridine 380 – phosphorodithioate-containing aptamers 380 – phosphorothioate SELEX 379 – photo-activated cross-linking nucleotide 465 – post-SELEX 371 – process of discovery 266 – restrictive selection protocol 96 – RNA-SELEX 418 – scheme 5, 120, 229, 231-232, 234 – scheme for an effector-activated aptazyme 295 – SELEX 72, 101, 118, 132-133, 144-146, 149, 154, 156-157, 167, 169-170, 172, 174, 181, 184, 211, 213, 220, 276, 281, 314, 324, 340, 346-347, 349-350, 359, 363, 366-367, 369-371, 373-375, 380, 417-418, 425-426, 428-429, 438, 461-462, 466-467 – sequential SELEX 373 – subtractive SELEX process 372 – tailored-SELEX 159 – tailored-SELEX process 430 – TNA selection 21 – toggle approach 150 – toggle SELEX 136, 367 – TSA approach 213 in vivo diagnostics (in vivo imaging) 463, 465 – 18F-fluorodeoxyglucose (FDG) 344 – anatomical imaging 344 – computed tomography (CT) 343, 361 – contrast agents 344 – intracellular targets 347

– magnetic resonance imaging (MRI) 343-344 – molecular imaging 343 – oncological PET imaging 360 – optical imaging (OI) 343-344 – positron emission tomography (PET) 343-345, 349-361 – signal-to-noise ratio 345-348 – single photon emission computer tomography (SPECT) 343-345, 351-354, 356-357, 359-360 – spatial resolution 361 – tumor-to-blood ratio 345, 357 – tumor-to-normal tissue ratios 345 – tumor-to-organ ratio 348 – ultrasound 343-344 – X-Ray/CT 344 in vivo imaging; see in vivo diagnostics (in vivo imaging) internal ribosome entry site (IRES) 185 in vivo validation 267 ionic interactions 312 iontophoreisis 385 ischemia-reperfusion 148 ischemia-reperfusion injuries 146 isoelectric points 371 isotopic assays 314

k key and lock hypothesis 420 kinetic folding 33 kinetic model 30 kinetic parameters 195 kinetic theory of molecular evolution kissing complexes 186 Kupffer cells 381 Kuru disease 158

l label-free techniques 303 lariat RNA molecules 247 leukemia 143 leukocyte adhesion 317 library design 8 ff., 11-12, 155 – 2l-amino-modified RNA 151 – 2l-aminopyrimidine-modified RNA 153-154 – backbone modifications 376 – communication module 294 – coupled of CoA to 5l-end 219 – degenerated RNA library 282 – direct selection with linker-coupled reactants 214-215

30

Index – model of diabetic blood–retinal barrier breakdown (BRB) 452 – murine model of neuroblastoma 142 – neovascular AMD 456 – neovascularization 453 – no-observable-adverse-effect-level (NOAEL) 397 – ocular neovascularization 141 – 40 kDa PEG moiety 433 – Phase I 398, 433, 452-453, 464 – Phase II 141, 387, 452-453 – Phase III 141, 387, 452, 454 – plasma residence time 381 – 5l-40 kDa-polyethylene glycolterminated 387 – preclinical studies 452 – primary efficacy endpoint 455 – retinal neovascularization 141 – retinopathy of prematurity (ROP) 453 – safety profile 456 – secondary efficacy endpoint 455 – single dose 397 – specificity 450 – subchronic repeated dosing toxicity studies 397 – vascular endothelial growth factor (VEGF) 364, 368, 371 – VEGF inhibition 450 – VEGF pathway 464 – VEGF165 452 – visual acuity 453 major histocompatibility complex (MHC) 142 malachite green aptamer – aptamer in tandem 112 m – aptameric sensor 112 Macugen (pegaptanib sodium) 140, 160, 363, – chromophore-assisted laser-mediated 369, 377, 382-384, 405, 438 inactivation (CALI) of RNA transcripts 111 – adverse events 455-456 MALDI-TOF analysis 328 – age-related macular degeneration (AMD) manufacturing 133, 141, 387, 398, 443, 464 – Caruthers patent 399 – antigenic response 398 – cGMP manufacturing infrastructure 399 – deoxythymidine 3l–3l-linked 433 – cost of phosphoramidites 398 – diabetic retinopathy 457 – large-scale chemical plants 399 – endophthalmitis 456 – material costs 398 – FDA 437 – solid support chemistries 399 – genotoxicity 397 mathematical model 29 – immunogenicity 397 matrix metalloproteinases 445 – intravitreal injection 141, 398, 453 maximum lifespan potential (MLP) 392 – lines of vision 455 maximum observed plasma – liposome-anchored 381 concentration, Cmax 388 maximum tolerated dose (MTD) 397 – macular edema 457 mechanism-of-action 265, 270 – maximum tolerated dose (MTD) 397 medicinal chemistry 265, 313 – Miles assay 452

– 2l-fluoropyrimidine-modified RNA 150, 155, 157 – hammerhead ribozyme pool 296 – internal stem–tetraloop 13 – L1-N50 pool 299 – length requirements in the random regions 463 – libraries of 2l-modified oligonucleotides 376 – modular design 293 – partially designed library 13 – 5-(1-pentynyl)-2l-deoxyuridine 380 – phosphorodithioate-containing libraries 379 – photo-activated cross-linking nucleotide 465 – primerless RNA library 430 – random region 132, 169 – stable internal tetraloop 12 – tailored SELEX 430 life – ancestors of 14 – definition of 14 – physical chemistry of 14 2l-5l linkages 247 lipofection 284 LNA–DNA chimeras 182-183, 185 locked nucleic acid (LNA) 181-183 luciferase reporter gene 283 lung inflammation 148 lymph node metastasis 344 lymphocyte trafficking 367, 403 lymphoma 143

481

482

Index melting curve 183 mesangioproliferative glomerulonephritis 404 metabolic stability 389 metabolism 363, 390 metabolite-binding RNA; see riboswitch (metabolite-binding RNA) metal requirement 7 – Ca2+ 251 – Cu2+ 248, 250 – cupric ion-dependent 220 – divalent lead 229 – divalent metal cofactor 229 – divalent metal ions 239, 246 – magnesium 169, 173, 184, 251, 331 – Mg2+-dependent RNA-cleaving DNA enzyme 230 – Mn2+ 250 – Pb2+ ions 241 – Pb2+-dependent RNA cleavage activity 240 – Pb2+-dependent RNA-cleaving ribozymes 229 – potassium 254 – Zn2+ RNA structure 101 – Zn2+/Cu2+-dependent DNA-ligating DNAzymes 231 Michaelis–Menten constant 232 Michaelis–Menten kinetics 221 microdialysis 331 microRNA (miRNA) 286 migraine 159, 430 Miles assay 140, 452 minimal binding motif 127 minimal cell model – autocatalytic metabolic cycle 54 – chemoton 54 – replicative ribozymes 54 minimal free energy 32 mirror-image phage display 424 mirror-image nucleic acids 418, 421 mirror-image peptide (d-peptide) – cecropin 420 – magainin-2 amide 420 – melittin 420 – rubredoxin 420 – HIV-1 protease 421 mirror-image peptide (d-peptide) aptamer to – amyloid peptide Ab(1–42) 424, 438 – 3-deoxy-a-l-manno-2-octulosonic acid 424 – HIV envelope gp41 protein 424, 438 – prokaryotic cell surface carbohydrates 424 – l-sialo-disaccharide 424 – sialic acid/KDO 438

– Src homology 3 (SH3) domain of chicken Src 424, 438 mirror-image proteins (d-protein) 420 mitogen 143 Mitscherlich, Eilhard 419 modifications 140, 183, 376, 382 – alkylamine 379 – 3-(aminopropynyl)-7-deazadeoxyadenosine 255-256 – 2l-amino 8, 142, 153, 180, 347, 390, 418 – 13-amino-acid fragment (Tat) of the HIV Tat protein 379 – 16-amino-acid sequence derived from the third helix of the Drosophila antennapedia (Ant) homeotic protein 379 – 5-amino analog 380 – 5-aminoallyl-dU 255 – 5l-amino modifier phosphoramidite 379 – antennapedia peptides 382 – backbone modifications 265, 376, 378 – base modifications 380 – 3l biotin 330, 359 – 5l-biotin 231, 234, 326 – biotin 366 – C5-imidazole deoxyuridine 255 – capping the 3l End 347, 378 – capping the 5l End 379 – cell-penetrating peptides 382, 389 – cholesterol 152, 379, 381 – 3l-cholesteryl phosphorothioate 381 – conjugation 389 – 3l end-blocking 359 – 2l-deoxy-2l-fluoro 433 – 5-(1-pentynyl)-2l-deoxyuridine 380 – fatty acids 382 – fluorine 305 – 2l-fluoro 8, 142-143, 180, 270, 340, 347, 359, 387, 390, 397, 404, 418 – 5l-(a-P-borano) GTP 380 – high-molecular-weight polymers 389 – highly positively charged peptides 382 – 8-histaminyl-dA 255 – hydroxymethyl group 305 – inverted 3l–3l caps 390 – 3l-inverted deoxythymidine (3l-idt) 135, 387, 403-404, 433 – 3l-3l inverted nucleotides 236 – 3l-inverted thymidine (3l-idT) 378 – 3H-labeled 393-394 – lipid-modified 381 – lipophilic moieties 389 – liposome surface 135 – LNA-2l-O-methyl chimeric derivative 186

Index – locked nucleic acid (LNA) 181, 236, 347, 353, 378 – MAG3 -aminohexyl chelator 350 – methyl phosphonates 381 – methyldiester 347 – methylphosphonate linkages 380 – 2l-modification 135, 376, 378 – non-nucleotide caps 378 – non-nucleotide linker 179 – non-nucleotide modifications 378 – nuclease-resistant analogs 180 – nucleobase-pairing systems 21 – 2l-O-methyl 8, 135, 140, 143, 180-181, 185, 236, 270, 340, 347, 353, 355, 359, 371, 377, 385-387, 390-391, 393, 396-397, 403, 418, 433 – P-borano linkages 380 – 5l-PEG conjugation (0, 20, 30, and 40 kDa) 396 – PEG linker 177, 214-215 – peptide nucleic acids (PNAs) 347 – peptide tags 382 – phosphate substitutions 379 – phosphoramidate 185 – 3l-phosphorimidazolide 231 – phosphorodithioate 379 – phosphorothioate 180, 236, 347, 355, 379, 381 – photo-activated cross-linking nucleotide 465 – photocleavable o-nitrophenyl group 215 – PNAs 356-357 – polyarginine 382 – polycations 379 – polyethylene glycol (PEG) 135, 273, 276, 347, 370, 379, 381, 384, 389, 392, 395, 432 – 20 kDa polyethylene glycol (PEG) 266, 395 – 30 kDa polyethylene glycol (PEG) 143, 266 – 40 kDa polyethylene glycol (PEG) 266, 390, 404, 431, 433 – 5l-40 kDa polyethylene glycol (PEG) 387, 403 – polyethylene glycol (PEG) link 177, 214-215 – polylysine 382 – post-SELEX modification 347, 376-378, 418 – post-synthesis coupling 379 – pre-SELEX modification 418 – primary amine 380 – 5-propyne 385 – proteins 379 – pyrene 378

– – – –

pyrimidine chemistry 466 radiolabeled 346, 349-351 radiolabeling 347, 351 2l-ribopurine 2l-aminopyrimidine (rRaY) 377 – 2l-ribopurine 2l-fluoropyrimidine (rRfY) 376, 403 – short, positively charged cell-permeating peptides composed of polyarginine (Arg7) 379 – site-specific conjugation of a bifunctional chelator 351 – split synthesis method 380 – sugar modifications 389 – Tat 382 – 5-thiol analog of dU 381 – thiol residues 380 – thiol-modified DNA 305 – 5l-(a-P-borano) UTP 380 modular nature of RNA 118 molecular beacon 180 molecular clock 42 molecular computation 240, 306 molecular modeling 167 mRNA (messenger RNA) 269, 280 multiple turnover 234, 242, 245 multivalent aptamer 368 mutagenized library 11 mutagenized pool 98 mutant 60-61, 65 – mispair 63 mutant distribution 30 mutation – compensatory 56 – multiple 60 – neutral 56 – one- or two-mutant molecules 68 – single 60, 68 myasthenia gravis (MG) 153, 370 mycoplasma 14 myocardial infarction 146

n

N6 -ribosyladenine 78 N-K model 57 NAD 72 NADP 72 nanoparticles, DNA-functionalized 305 nanorobotics 240 natural aptamer 192, 465-466 NECEEM; see non-equilibrium capillary electrophoresis of the equilibrium mixture (NECEEM)

483

484

Index neointima formation 146 neovascularization 141-142, 447, 451-453, 457 nephritis 147 neurogenic inflammation 430 neutral evolution 42, 45 neutral network 29, 39-45 neutrophil infiltration 148 neutrophil–neutrophil adhesion 367 nitrocellulose filter assays 402 NMR; see nuclear magnetic resonance spectroscopy (NMR) no-observable-adverse-effect-level (NOAEL) 397 nociception 160 non-compartmental analysis (NCA) 388 non-equilibrium capillary electrophoresis of the equilibrium mixture (NECEEM), single-exponential function 338 non-small cell lung cancer (NSCLC) 344 NOX-B11; see ghrelin-Spiegelmer NOX-B11 nuclear magnetic resonance (NMR) 11, 96, 167, 172 nuclear magnetic resonance spectroscopy (NMR) 111, 126, 174, 176, 200, 284, 312 – l-arginine 101 – theophylline aptamer 105 nuclease degradation 135, 284 nuclease resistance 140, 183, 377, 380 NX1838; see Macugen (pegaptanib sodium) NX1975; see PDGF aptamer NX1975

o obesity 432 l-oligonucleotides 333 oncology 350, 368 organic synthesis 224 origins of life 14 ff. – DNA–protein world 69 – first biopolymers 15-16 – genetic alphabet 81 – genetic code 56 – non-enzymatic replication 80 – nucleic acid world 77 – primordial metabolism 80 – protein world 77, 79 – replicability 81 – replicative fidelity 81 – RNA organism 17 – translation 56 orthogonal cleavage 214

p pain 160, 430 Pasteur, Louis 419 PCR; see polymerase chain reaction (PCR) PDGF aptamer NX1975 377 PDGF-BB aptamer ARC 127 277, 403 – 2l-fluoropyrimidine 404 – 3l-inverted deoxythymidine 404 – 40 kDa PEG group 404 – combination therapy using anti-VEGF and anti-PDGF-BB agents 405 – human proliferative disease 404 – interstitial fluid pressure (IFP) 404 – mesangioproliferative glomerulonephritis 404 – proliferative retinopathies 405 – treatment of angiogenesis-mediated diseases 405 pegaptanib; see Macugen (pegaptanib sodium) d-peptide; see mirror-image peptide (d-peptide) d-peptide aptamer to; see mirror-image peptide (d-peptide) aptamer to peptide bond formation 218 peptide library 300 peptide world 219 peptide-ligation techniques 421 permeation enhancers – sodium caprate 386 – sodium chenodeoxycholate 386 – sodium laurate 386 – sodium ursodeoxychelate 386 phage display 382 – bacteriophage particles 417 – fd 417 – filamentous phages 417 – M13 417 – random peptides 417 pharmacokinetics 135, 149, 269, 345, 349-350, 354-355, 381, 387-390, 392, 396 – half-life 267, 403 pharmacological probe 267, 269-270, 273 pharmacology 270 phenotype 268 photoaptamer 465 photodynamic therapy (PDT) 141, 453 phylogenetic conservation 195 phylogeny 196, 199, 203 plasma concentration 388, 390 platelet aggregation 149 platelet–neutrophil adhesion 367 platelet–neutrophil association 374

Index polymerase chain reaction (PCR) 133, 215, 229, 231, 294, 418 population genetics 31 post-transcriptional modification 72 post-translational modification 280 production 349 prostate cancer 159, 429 d-Protein; see mirror-image protein (d-protein) protein kinase (PKR) pathway 269 protein space 57 protein synthesis 203 protein translation 269 protein–protein interactions 366 purine efflux pump 199 Pwo DNA polymerase 381

q

Qb replicase 29 quantitative whole-body autoradiograph (QWBA) 394 quartz crystal microbalance (QCM) 303 quasi-species 30, 68

r radioisotope 347, 366 – 3H (H-3) 393, 394 – 11C (C-11) 353 – 13N (N-13) 353 – 15O (O-15) 353 – 18F (F-18) 343-345, 350, 352-356 – 64Cu (Cu-64) 352, 354, 356, 358-359 – 68Ga (Ga-68) 343, 352-354, 360 – 76Br (Br-76) 353 – 99mTc (Tc-99m) 343, 345-348, 350-352, 356-357, 359-360 – 111In (In-111) 343, 352-353 – 123I (I-123) 343, 352-353 – 124I (I-124) 352 – 125I (I-125) 346, 353 random library 9 random sequence 5 rational design 35 rational drug design 312 reactions – acyl-transfer reactions 6 – acylation 216 – S-acylation 217 – aldol reactions 212 – alkylation 6, 216 – N-alkylation 216 – S-alkylation 217 – amide bond formation 6, 211, 216

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

asymmetric carbon–carbon bonds 15 biphenyl isomerization 213, 216 s-bond rotation 212 C-C-bond formation 6, 211, 216 C-metal bond formation 218 C-N bond formation 216 C-O bond formation 217 C-S bond formation (by Michael addition) 216 C-S-bond formation 211, 217 carbonate ester hydrolysis 217 cholesterol esterase activity 213 cleavage of amide bonds 211 cleavage of carboxylic ester bonds 211 cleavage of phosphodiester in trans 220 cyclization 71 cycloadditions 212, 221 Diels–Alder reaction 6, 212-214, 216, 221, 224, 427 DNA ligation 231, 252 DNA phosphorylation 250 DNA self-modification reactions 249 DNA depurination 216 b-elimination 249 excision 71 formation of a phosphodiester bond with a deoxynucleotide 220 formation of amide bonds 216 forming a glycosidic linkage 219 N-glycosidic bonds 216 N-glycosylation of a guanine base followed by b-elimination 249 hydrolysis and ligation of phosphodiester bonds 211 3l-5l ligase activity 247 ligation 71 ligation of DNA strands with 3l phosphorothioates 301 metallation of N-methylmesoporphyrin 220 Michael additions 212, 215, 221 S-Michael reaction 217 oxidation and reduction 218 oxidation of benzyl alcohol 214 P-O bonds 217 peptide bond formation 72 phosphoanhydride formation 217 phosphodiester bond cleavage 6 phosphodiester bond formation 6 phosphorylation 217 5l phosphorylation of DNA 249 5l-phosphothioester linkage 252 photoreaction 253

485

486

Index – porphyrin metallation 213, 218, 232 – redox reaction 111, 212, 216, 218, 221 – RNA cleavage 220 – RNA ligation 217, 220, 246 – RNA/DNA cleavage 217 – 5l-self-capping reaction 251 – thymine dimer repair 254 – trans-aminoacylation of a tRNA 219 – transesterification 217, 248 – transesterification reactions 215 – transfer of a g-phosphate 249 reduced sets of nucleotides 7 renal clearance 135 renal elimination 379 renal filtration 381, 405 replication–mutation matrix 30 replicator 56 reporter ribozyme 284-286 respirable antisense oligonucleotides (RASONs) 386 restenosis 403 restenosis/intimal hyperplasia 349 retinal pigment epithelium (RPE) 447 retroviral frameshift element 99 Rev response element (RRE) 145 reverse Hoogsteen 167-168 rheumatoid arthritis 142, 369 ribosomal (rRNA) 72 ribosome 16, 121, 126, 218-219, 462 – antibiotics 119 – peptide bond formation 125 – peptidyltransferase loop 125 – X-ray crystal structure 124 riboswitch (metabolite-binding RNA) 11, 76, 79, 117, 191, 290 – control of transcription termination 192 – designed 47 – expression platform 193, 199, 201 – FMN 79 – genetic control 192, 196, 198, 203 – glucosamine 6-phosphate (GLcN6P) 79 – intrinsic terminator 193, 199 – natural aptamer 47, 192, 466 – negative feedback loop 192 – regulation of transcription termination 79 – RNA aptamer-regulated mRNA translation 16 – self-cleavage 203 – self-induced RNA switch 47 – tandem arrangement 201 – thiamine 79 – three-dimensional structure 197 – transcription termination 193, 196

– translation initiation 79, 193 riboswitch specific for – S-adenosylmethionine (SAM) 192, 195 – adenine 192, 194, 196, 199 – adenosylcobalamin (AdoCbl) 195 – coenzyme B12 194, 204 – FMN 79, 194-195, 204 – glucosamine 6-phosphate (GlcN6P) 79, 192, 194, 203 – glycine 192, 194-195, 201-203 – guanine 192, 194, 196-197, 204 – hypoxanthine 196 – lysine 192, 194 – SAM I 194, 204 – SAM II 194, 204 – TPP 193-195 ribozyme 3, 6-8, 19, 39, 41, 45-46, 56, 58, 61, 64, 68, 72, 77-80, 82-83, 95, 118, 170, 228, 290, 292-293, 300, 319, 398, 463, 466 – adenine-dependent 77 – ADH 78 – aptamer-controlled 17 – capping ribozyme 219 – cis-acting 127 – class I ligase 9, 13, 17 – class I ligase-based polymerase 20 – cofactor-assisted 56, 74 – Diels-Alderase 212, 220-221, 224, 305, 427 – group I intron 17, 58, 60, 76, 99, 116, 122, 126-127, 215, 291-292, 300, 308 – group II intron 58, 215 – hairpin 58, 59-61, 63, 76, 78, 215, 286, 292, 300 – hammerhead 17-18, 58, 79, 215, 229, 291-292, 296-297, 301, 303-304, 306-308 – hepatitis delta virus (HDV) ribozyme 58, 61, 126, 215, 224, 291-292, 300 – L1 ligase 293-294, 296-297, 306-307 – ligase 293, 299 – mirror-image l-ribozyme 221 – Neurospora varkud satellite (VS) ribozyme 58-59, 60-61, 63-64, 66, 69-71, 127, 215 – nucleolytic 60 – Pb2+-dependent RNA-cleaving ribozymes 229 – peptide bond formation 72 – peptidyltransferase ribozymes 305 – processive polymerase activity 20 – redox active ribozyme 220-221, 305 – replicase 20 – replicase activity 20 – reporter ribozyme 285

Index – – – – – – – – – – –

ribonuclease P 54, 58, 83, 215 ribosome 218 ribox02 305 RNA ligase 49 RNA self-splicing 54 RNA-dependent RNA polymerase 11 self-alkylating 99 self-replicating ligase structures 17 synthesis of acyl-coA 76 synthesis of ribonucleotide monomers 16 template-directed primer-extension activity 9 – tools in organic chemistry 211 – trans-acting 127 – trans-cleaving 58 – X-motif ribozymes 292 l-RNA 334, 421, 434, 437 – l-adenosine 434 – l-cytidine 434 – l-guanosine 434 – l-uridine 434 RNA interference (RNAi) 269 RNA peptidation 83 RNA polymerase 192 RNA polymerization 29 RNA replication 16 RNA replicators 84 RNA template 29 RNA world 4, 50, 56, 69, 79, 84, 95, 102, 204, 212, 218, 220, 228 – nucleotide coenzymes 54 RNA–antibiotic interactions 118 RNA-based metabolism 74 RNA-guided metabolism 204 RNAi; see siRNA (small interference RNA, RNAi) RNase footprinting 170, 179 route of administration 382, 389, 391 – intravenous administration 390-392 – intravitreal injection 387, 398, 453 – oral delivery 385 – oral dosing 391 – parenteral administration 382-383, 391 – pulmonary delivery 386 – topical administration 385 – topical delivery 384

s S-adenosylmethionine 72 safety pharmacology 388, 392 sandwich approaches 325 sandwich-type assay 303 scFv, Fab, see antibody fragments (scFv, Fab)

SCID mice 155 secondary structure 38, 40, 46, 109, 120, 174, 197, 222, 234, 245, 248-249, 251-252, 255, 291, 299, 40 – A=U base pair 39 – active conformation 14 – adenosine platform 167 – Arrhenius-type kinetics 37 – base pair stacking 39 – base pairing 36, 47 – base triplet 47 – bipartite RNA structure 96 – bulge 65, 170, 240, 285 – cloverleaf structure 41-42 – conformation space 47 – conformations (meta)stable 47 – DNA folding 38 – DNA structures 38 ff. – double helices 39 – double kissing complex 177 – folding problem 45 – free energy levels 37 – G-quartet 98, 250, 312, 328-329 – GNRA tetraloop 48 – GTP-binding aptamers 12 – hairpin 12, 36, 83, 127, 169, 170-171, 176, 186, 461 – hairpin loop 39, 99 – hexameric loop 171 – in-line probing 196, 201 – individual folding trajectory 37 – internal bulge 99 – internal loop 186 – internal stem–tetraloop 13 – inverse folding 35, 42, 49 – kinetic folding 36, 37 – kissing complex 170, 172-173, 176, 178, 184 – loop–loop helix formation 178 – loop–loop interaction 170, 173, 176, 178, 186 – m-fold 64 – minimum free energy (MFE) 33-34, 40, 47, 64, 67 – non-canonical interactions 100, 170, 200 – pseudo-knot 11, 34, 47, 58, 64, 99, 127, 180, 196, 282, 371 – RNA folding 35, 45 – RNA folding algorithm 58 – RNA sequence–structure mapping 32 – RNA structures 33 ff. – RNA–DNA cofolding 38 – RNase S1 analysis 126

487

488

Index – secondary structure prediction 35 – stable internal stem–tetraloop 12 – stable tetraloop 11, 48 – stem–loop structure 192 – stems 11 – steric hindrance 39 – suboptimal conformation 36-37 – three stem junction 196 – two-hairpin structure 177 – unpaired loop 285 – Vienna RNA package 35, 64 – wobble pair 47 – wobble-type pairings 11 selectivity 107 sense–antisense duplex 184 sensor; see biosensor (aptasensor, sensor) septic shock 148, 157 sequence space 3, 13, 15, 31, 33, 39-40, 57 serine protease 149, 151 serum-stabilized aptamer 265 shape space 31 Shine–Dalgarno sequence 193 sialyl Lewis X (sLeX) 155, 403 side-effects 313 signal transduction 271 single-chain antibody fragments (scFvs) 269, 370 siRNA (small interference RNA, RNAi) 170, 268, 284, 388, 437, 466 small-molecule drugs 311 solid-phase peptide synthesis 421, 428 specificity 135, 270, 372, 375, 450 Spiegelmer 181, 333, 355-356, 417, 423-425, 429, 433 – adenosine 426, 438 – arginine 426, 438 – d-arginine 334, 340 – biological stability 427 – calcitonin gene-related peptide 1 (CGRP) 159, 430, 438 – catalytically active spiegelmers 427 – Ghrelin 139, 158, 431, 438 – gonadotropin-releasing hormone (GnRH) 139, 159, 429, 431-432, 438 – immunogenic potential 432 – lock-and-key stereocomplementarity 422 – neuropeptide calcitonin gene-related peptide 1 139 – neuropeptide nociceptin/orphanin FQ 139 – nociceptin/orphanin FQ (N/OFQ) 160, 430, 438 – peptides 158

– production 434 – l-ribose 434 – SEB 438 – Spiegelmer technology 425 – staphylococcal enterotoxin B (SEB) 429 – Substance P 430, 438 – toxicity 433 – d-tyrosine 334 – vasopressin 429, 438 spliceosome 16 splicing 116, 272 stacking 33 stereochemistry 418, 427 stereoselectivity 212 stochastic corrector model 54-56 stochastic model of species evolution 44 streptavidin–agarose 214 streptavidin–biotin 325, 330 streptomycin 116 StreptoTag 123 surface plasmon resonance (SPR) 303 surface plasmon resonance measurement 171 systemic administration 348

t T7 RNA polymerase 377, 380 Taq polymerase 20 TAR RNA 99-100, 131, 144, 170, 176, 181, 184 target validation 265, 267, 271, 273, 287 – in vivo 266 – disease pathology 266 target-to-hit 265 Tat 131 Tat–TAR interaction 378 Taxol 276 tenascin aptamer TTA1 – clinical trials 360 – fecal excretion 347 – 68Ga 360 – 68Ga-DO3A-TTA1 360 – images of lung and breast tumors 360 – intestinal uptake 347 – 125I-labeled 346 – 99mTc-labeled 346, 348 – labeled with 99mTc 360 – LNA modification 347, 353 – MAG2 chelator 353 – 2l-O-methyl 353 – 2l-O-methyl modification 347 – murine tenascin-C 360 – PET 360

Index – renal clearance 348 – SPECT 360 – Tenascin-C 364, 367 – tumor imaging in mice 350 – tumor-to-organ ratio 348 tetracycline aptamer – regulation of translation 121 tetrameric aptamer 154 theophylline aptamer 113 – allosteric hammerhead ribozyme 106 – iterative computational deletion 106 – molecular beacon 106 – NMR 105 – ribozyme ligase 106 – structure 105 – theophylline-responsive riboswitch 106 – to regulate gene expression 106 thermodynamic equilibrium 195 thermodynamic stability 13 thermodynamics 33 thioaptamers 379 threose nucleic acid (TNA) 17, 20 thrombin 389 thrombin aptamer (ARC 183) 330, 373, 383, 389, 464 – anticoagulant 400 – biotinylated aptamer 378 – CABG 400 – clinical development 463 – clinical trials 401 – ligand in affinity capillary electrophoresis 336-339 – NMR studies 400 – non-nucleotide modifications 378 – radioiodinated (123I) aptamer 359 thrombocytopenia 152 thrombosis 349 thymine dimer 253 tissue penetration 345 tissue remodeling 157 TNA; see threose nucleic acid (TNA) toxicity 397, 433 toxicology 388, 392 transfection 281 transfer RNA (tRNA) 45, 47, 71-72, 83-84, 116, 125, 131, 172, 219, 223, 229, 282, 299 transition state analog (TSAs) 212, 232 Transmissible spongiform encephalopathies (TSEs) 158 triple helix 168, 185 triplex 248 triplex-forming oligonucleotides 167

TSA; see transition state analog (TSA) TTA1; see tenascin aptamer TTA1 tumor diagnosis 350 tumor immunotherapy 154 tumor model 144 tumor uptake 359, 366 tumor-to-blood ratio 366 tumors 273-274, 356

u UDPGT (uridine diphosphate glucoronyl transferase) 390 urinary excretion 345 5l-UTR (5l untranslated region) 191, 192, 196, 201, 203 5l-UTR intron 193 UV crosslinking 121 UV detection 331 UV irradiation 328

v vaccination 154 VEGF aptamer; see Macugen (pegaptanib sodium) VEGF and human disease – age-related macular degeneration (AMD) 446 – anti-VEGF therapy 456-457 – cancer 445 – diabetic retinopathy 448 – neovascular AMD 456 – neovascularization 451-452 – pathologic neovascularization 451 – physiologic neovascularization 451 – regression of existing tumor vessels 450 – VEGF and human physiology 449 – VEGF inhibition 450 – VEGF isoforms 449-452 – VEGF pathway 464 – VEGF therapeutic dilemma 449 – VEGF-TRAP 450 – wound-healing 449 viral entry 366 viral vectors 281 viral–cell adhesion 366 vitraveneTM (fomivirsen) 387 volume of distribution 388, 396

w Warburg effect 350 Watson–Crick base pairing 167, 169, 199, 239, 241, 245, 306, 378 Wilms’ tumor 274

489

490

Index

x

y

X-ray crystallography 167, 196, 200 – bacteriophage MS2 coat protein 312 – thrombin 312 – transcription factor NFkB 312 xenograft model 275

yeast three-hybrid system 282

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