Ribozyme Protocols

M E T H O D S I N M O L E C U L A R B I O L O G Y '

John M. Walker, SERIES EDITOR

74. Ribozyme Protocols, edited by Philip C. Turner, 1997 73. Neuropeptide Protocols, edited by G. Brent Irvine and

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Swindell, 1997 69. cDNA Library Protocols, edited by Ian G. Cowell

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edited by John R. Chapman, 1996 60. Protein NMR Protocols, edited hy David G. Reid, 1996 59. Protein Purification Protocols, edited by Shawn Doonan,

1996 58. Basic DNA and RNA Protocols, edited by Adrian J.

Harwood. 1996 57. In Vitro Mutagenesis Protocols, edited by Michael K.

Trower, 1996 56. Crystallographic Methods and Protocols, edited by Chris­

topher Jones, Barbara Mulloy, and Mark Sanderson, 1996 55 Plant Cell Electroporation and Electrofusion Protocols,

edited by Jac A. Nickoloff, 1995 54. YAC Protocols, edited by David Marfe, 1995 53. Yeast Protocols: Methods in Cell and Molecular Biology,

edited by Ivor H. Evans, 1996 52. Capillary Electrophoresis: Principles, Instrumentation,

and Applications, edited hy Kevin D. Altria, 1996 51. Antibody Engineering Protocols, edited by Sudhir

Paul, 1995 50. Species Diagnostics Protocols: PCR and Other Nucleic

Acid Methods, edited by Justin P. Clapp, 1996 49. Plant Gene Transfer and Expression Protocols, edited by

Heddwyn Jones, 1995 48. Animal Cell Electroporation and Electrofusion Proto­

cols, edited by Jac A. Nickoloff, 1995 47. Electroporation Protocols for Microorganisms, edited by

Jac A. Nickoloff 1995 46. Diagnostic Bacteriology Protocols, edited by Jenny

Howard and David M. Whitcombe, 1995

45. Monoclonal Antibody Protocols, edited by William C. Davis, 1995

44. Agrobacterium Protocols, edited by Kevan M. A. Gartland and Michael R. Davey, 1995

43. In Vitro Toxicity Testing Protocols, edited by Sheila O'Hare and Chris K. Atletvill, 1995

42. ELISA: Theory and Practice, by John R. Crowther. 1995 41. Signal Transduction Protocols, edited by David A. Kendall

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Lorette C. Javois, 1994 33. In Situ Hybridization Protocols, edited by K. H. Andy

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21. Protocols in Molecular Parasitology, edited hy John E. Hyde, 1991

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M E T H O D S I N M O L E C U L A R B I O L O G Y '

Ribozyme Protocols

Edited by

Philip C. Turner University of Liverpool, UK

Humana Press ^ ( ^ Totowa, New Jersey

© 1997 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512

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Methods in molecular biology'".

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Includes index. ISBN 0-89603-389-9 (alk. paper) 1. Catalytic RNA—^Laboratory manuals. I. Turner, Philip C. 11. Series: Methods in molecular biology (Totowa, NJ); 74. [DNLM: 1. RNA, Catalytic. 2. Genetic Techniques. Wl ME9616J v. 74 1997/QU 58.7 R4865 1997]

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Preface

The purpose of Ribozyme Protocols is to provide a helpful compilation of protocols that will be of use—^not only to those with some experience of ribozymes—^but also to those wishing to use ribozymes for the first time. Although it is usually impossible to cover every aspect of a scientific field, I believe this book approaches that ideal and should help all readers perform meaningful experiments using ribozymes.

To design ribozymes, one must consider whether the target site will be accessible; this task can be facilitated by using computer programs that pre­dict the folding of the target RNA. Such programs are detailed in Chapters 2 and 3. If the chosen target is an RNA virus that can mutate rapidly, it makes sense to consider those parts of the genome that are least likely to change during viral replication. An example of how this can be done is described in Chapter 4.

Although computer analysis may be a useful starting point to select tar­get sites, there seems, at the moment, to be no guarantee that any particular chosen site will be efficiently cleaved. Some workers have deliberately bypassed this problem by using libraries of ribozyme sequences and by select­ing those that actually hybridize to and/or cleave the target; these methods are described in Chapters 5 and 6.

The production of ribozymes is then considered; first, by using chemi­cal synthesis methods (Chapters 7 and 8), and then by constructing DNA tem­plates for in vitro transcription (Chapters 9—11), which includes protocols using cloned templates, PCR-generated templates, and chemically synthesized tem­plates, as well as how to check the sequence of short RNA transcripts. The introduction of modifications into ribozymes and the uses of modified ribozymes are covered in Chapters 12 and 13.

Chapters 14—17 describe cloning strategies for producing catalytic antisense RNAs (ribozymes with long regions of complementarity to their tar­gets) as well as design rules for minimized hammerhead ribozymes. Design rules and methodology for constructing hairpin ribozymes and RNase P ribozymes follow in Chapters 18—20.

A block of six chapters (Chapters 21—26) covers the theoretical and prac­tical aspects of measuring precise catalytic parameters for ribozyme-mediated

vi Preface

reactions. These are followed by protocols for the optimization of ribozyme reactions (Chapters 27—30) that consider hybridizing arm length, facilitators, and protein enhancement, as well as selection for rapid hybridization.

Three methods of determining cleavage efficiency are detailed in Chap­ters 32—34, but it should be borne in mind that direct detection of the cleavage products is notoriously difficult, presumably owing to their instability. These are followed by two chapters (35 and 36) detailing other ribozyme-mediated reactions, those of transsplicing and ligation. It is worth noting that, if transsplic-ing ribozymes could produce corrected mRNAs in vivo, even at low efficiency, then enough functional protein could be produced to have a clinically benefi­cial effect (e.g., in certain thalassemias).

A group of five chapters concerned with ribozyme structure (Chapters 37-41) describe, first, mutagenesis to confirm ribozyme secondary structure, and, second, methods for crystallizing ribozymes with and without substrate for three-dimensional structure determination.

Delivery systems and methods of delivery of ribozymes to cells are described in the subsequent five chapters (42-46), and the use of both ham­merhead and hairpin ribozymes against AIDS is given in Chapters 47 and 48, respectively. The book ends with a review of the prospects of the clinical use of ribozymes in gene therapy.

I would like to thank the authors for their contributions and all those involved in the production of the book. I hope that readers benefit from using this compilation of protocols and that they ultimately develop some useful applications of ribozymes, which are for the good of all.

Philip C. Turner

Contents

Preface v

Contributors x/

1 Ribozymes: An Introduction Helen A. James and Philip C. Turner /

2 Computer-Aided Calculation of the Local Folding Potential of Target RNA and Its Use for Ribozyme Design

Georg Sczaliiel and Martin Tabler 11

3 Computational Approaches to the Identification of Ribozyme Target Sites

William James and Elizabetti Cowe 17

4 Computer Analysis of the Conservation and Uniqueness of Ribozyme-Targeted HIV Sequences

Mary Beth DeYoung and Arnold Hampel 27

5 Selection of Accessible Sites for Ribozymes on Large RNA Transcripts

Bruce L. Frank and John Goodchild 37

6 Selection of Efficient Ribozyme Cleavage Sites in Target RNAs Andre Lieber, Mikkel Rohde, and Michael Strauss 45

7 Chemical Synthesis, Analysis, and Purification of Ribozymes Ravi Vinayak 51

8 A Practical Method for the Production of RNA and Ribozymes Francine E. Wincott and Nassim Usman 59

9 Preparation of Templates for Production of Ribozymes and Substrates

Rajesh K. Gaur and Guido Krupp 69 10 Enzymatic Synthesis and Characterization of Unmodified Ribozymes

and Substrates Guido Krupp 79

11 T7 Transcript Length Determination Using Enzymatic RNA Sequencing

Andrew Siwkowski 91

VII

via Contents

12 Chemical and Enzymatic Approaches to Construct JVIodified RNAs Rajesh K. Gaur and Guido Krupp 99

13 Applications of Modified Transcripts Rajesh K. Gaur, Frank Conrad, and Guido Krupp 111

14 Cloning Strategies for Catalytic Antisense RNAs Martin Tabler and Mina Tsagris 121

15 PCR-Based Construction of Long Hammerhead Ribozymes Martin Zillman and Gregory Robinson 131

16 Design and Production of Asymmetric Hammerhead Ribozymes Martin Tabier and Georg Sczaldel 141

17 Minimized Hammerhead Ribozymes Maxine J. McCaii, Philip Hendry, and Trevor J. Locliett 151

18 Design of Hairpin Ribozymes for In Vitro and Cellular Applications Qiao Yu and John M. Buriie 161

19 Design of the Hairpin Ribozyme for Targeting Specific RNA Sequences

Arnold Hampel, Mary Beth DeYoung, Scott Galasinski, and Andrew Siwkowski 171

20 Design and Preparation of Sequence-Specific RNase P Ribozymes

Denis Drainas and Guido Krupp 179

21 Theoretical Considerations in Measuring Reaction Parameters Timothy S. McConnell 187

22 Experimental Approaches for Measuring Reaction Parameters Timothy S. McConneli 199

23 Determination of Catalytic Parameters for Hairpin Ribozymes Mary Beth DeYoung, Andrew Siwkowski,

and Arnold Hampel 209

24 Characterizing Ribozyme Cleavage Reactions Philip Hendry, Maxine J. McCall, and Trevor J. Lockett 221

25 Defining Optimum Reaction Conditions for Hammerhead Ribozymes

Philip Hendry, Maxine J. McCall, and Trevor J. Lockett 231

26 Using Fluorescence Resonance Energy Transfer to Investigate Hammerhead Ribozyme Kinetics

Thomas A. Perkins and John Goodchild 241

27 Design of Hybridizing Arms in Hammerhead Ribozymes Philip Hendry, Maxine J. McCall, and Trevor J. Lockett 253

Contents ix

28 Optimization of Hammerhead Flanking Sequences Using Oligonucleotide Facilitators

John Goodchild 265 29 Enhancement of Ribozyme Function by RNA Binding Proteins

Nan Sook Lee, Edouard Bertrand, and John J. Rossi 275

30 Selection of Fast-Hybridizing Complementary RNA Species In Vitro Ralf Kronenwett and Georg Sczakiel 281

31 In Vitro Selection of Hairpin Ribozymes Bruno Sargueil and John M. Burke 289

32 The Detection of Hammerhead Ribozyme Cleavage by RT-PCR Methods

Rhonda Perriman 301

33 Detection of Ribozyme Cleavage Products Using Reverse Ligation-Mediated PCR (RL-PCR)

Edouard Bertrand, Micheline Fromont-Racine, Raymond Pictet, and Thierry Grange 311

34 Quantitation of Ribozyme Target Abundance by QCPCR Amber A. Beaudry and James A. McSwiggen 325

35 Trans-Splicing Reactions by Ribozymes Joshua T. Jones, Seong-Wook Lee, and Bruce A. Sullenger 341

36 Ligation of RNA Molecules by the Hairpin Ribozyme Alfredo Berzal-Herranz and John M. Burke 349

37 Mutagenesis and Modeling of the Hairpin Ribozyme Family Andrew Siwkowski, Mary Beth DeYoung, Pamela Anderson,

and Arnold Hampel 357

38 Preparation of Homogeneous Ribozyme RNA for Crystallization Jennifer A. Doudna 365

39 Establishing Suitability of RNA Preparations for Crystallization: Determination of Polydispersity

Adrian R. Ferre-d'Amare and Jennifer A. Doudna 371

40 A Sparse Matrix Approach to Crystallizing Ribozymes and RNA Motifs

Jamie H. Cate and Jennifer A. Doudna 379 41 Crystaliographic Analyses of Chemically Synthesized Modified

Hammerhead RNA Sequences as a General Approach Toward Understanding Ribozyme Structure and Function

William G. Scott 387

42 tRNA Delivery Systems for Ribozymes Rhonda Perriman and Rob de Feyter 393

X Contents

43 Expressing Ribozymes in Plants Rob de Feyter and Judith Gaudron 403

44 Using IVIicroinjection of Xenopus Oocytes to Express and Optimize Ribozymes In Vivo

Philip C. Turner 417

45 Exogenous Cellular Delivery of Ribozymes and Ribozyme Encoding DNAs

Daniela Castanotto, Edouard Bertrand, and John J. Rossi 429 46 Optimization of Lipid-Mediated Ribozyme Delivery to Cells

in Culture SuzyA. Brown and Thale C. Jarvis 441

47 Retroviral Delivery and Anti-HIV Testing of Hammerhead Ribozymes

Laurence Cagnon and John Rossi 457

48 Hairpin Ribozyme Gene Therapy for AIDS Elizabeth A. Duarte, Mark C. Leavitt, Osamu Yamada,

and Mang Yu 459

49 Clinical Aspects of Ribozymes as Therapeutics in Gene Therapy David Looney and Mang Yu 469

index 487

Contributors

PAMELA ANDERSON • Department of Biological Sciences, Northern Illinois University, DeKalb, IL

AMBER A. BEAUDRY • Ribozyme Pharmaceuticals, Boulder, CO EDOUARD BERTRAND • Institut Jacques Monod du CNRS, Universite Paris,

France ALFREDO BERZAL-HERRANZ • Instituto de Parasitologia y Biomedicina

(CSIC), Granada, Spain SuzY A. BROWN • Ribozyme Pharmaceuticals, Boulder, CO JOHN M . BURKE • Markey Center for Molecular Genetics, Department

of Microbiology and Molecular Genetics, The University of Vermont, Burlington, VT

LAURENCE CAGNON • Department of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA

DANIELA CASTANOTTO • Department of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA

JAMIE H . CATE • Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT

FRANK CONRAD • Institut fiir Allgemeine Mikrobiologie, der Universitdt Kiel, Kiel, Germany

ELIZABETH COWE • Oxford University Molecular Biology Data Centre, University of Oxford, UK

JENNIFER A. DOUDNA • Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT

DENIS DRAINAS • University ofPatras, Rio-Patras, Patras, Greece ELIZABETH A. DUARTE • Immusol, San Diego, CA ADRIAN R. FERRE-D'AMARE • Department of Molecular Biophysics

and Biochemistry, Yale University, New Haven, CT ROB DE FEYTER • CSIRO Division of Plant Industry, Canberra, Australia BRUCE L . FRANK • Hybridan, Worcester, MA

SCOTT GALASINSKI • Department of Biological Sciences, Northern Illinois

University, DeKalb, IL JUDITH GAUDRON • CSIRO Division of Plant Industry, Canberra, Australia

XI

xii Contributors

RAJESH K . GAUR • Howard Hughes Medical Institute, Program in Molecular Medicine, University of Massachusetts, Worcester, MA

JOHN GOODCHILD • Hybridon, Worcester, MA THIERRY GRANGE • Institut Jacques Monad du CNRS, Universite

Paris, France ARNOLD HAMPEL • Department of Biological Sciences, Northern Illinois

University, DeKalb, IL PHILIP HENDRY • CSIRO Division of Biomolecular Engineering,

North Ryde, Australia HELEN A. JAMES • Department of Biological Sciences, University of East

Anglia, Norwich, Norfolk, UK WILLIAM JAMES • Sir William Dunn School of Pathology, University

of Oxford, UK THALE C . JARVIS • Ribozyme Pharmaceuticals, Boulder, CO JOSHUA T . JONES • Program in Molecular Therapeutics, Departments of

Experimental Surgery and Genetics, Duke University Medical Center, Durham, NC

RALE KRONENWETT • Klinische Kooperationseinheit Molekulare Hdmatologie und Onkologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany

GuiDO KRUPP • Institut fiir Allgemeine Mikrobiologie, der Universitdt Kiel, Kiel, Germany

MARK C . LEAVITT • Immusol, San Diego, CA

NAN SOOK LEE • Department ofMolcular Biology, City of Hope Beckman Research Institute, Duarte, CA

SEONG-WOOK LEE • Program in Molecular Therapeutics, Departments of Experimental Surgery and Genetics, Duke University Medical Center, Durham, NC

ANDRE LIEBER • Markey Molecular Medicine Center. Division of Medical Genetics, University of Washington, Seattle, WA

TREVOR J. LOCKETT • CSIRO Division of Biomolecular Engineering, North Ryde, Australia

DAVID LOONEY • San Diego Veterans Administration Medical Center, San Diego, CA

MAXINE J. MCCALL • CSIRO Division of Biomolecular Engineering, North Ryde, Australia

TIMOTHY S. MCCONNELL • Department of Molecular Biophysics and Biochemistry, Boyer Center for Molecular Medicine/HHMI, Yale University School of Medicine, New Haven, CT

Contributors xiii

JAMES A. MCSWIGGEN • Ribozyme Pharmaceuticals, Boulder, CO THOMAS A. PERKINS • Hybridon, Worcester, MA RHONDA PERRIMAN • Sinsheimer Laboratories, University of California,

Santa Cruz, CA RAYMOND PICTET • Institut Jacques Monod du CNRS, Universite

Paris, France MiCHELiNE FROMONT-RACINE • Institut Jacques Monod du CNRS,

Universite Paris, France GREG ROBINSON • Hybridon, Worcester, MA MIKKEL ROHDE • Danish Cancer Society, Division of Cancer Biology,

Department of Cell Cycle and Cancer, Copenhagen, Denmark JOHN J. Rossi • Department of Molecular Biology, Beckman Research

Institute of the City of Hope, Duarte, CA BRUNO SARGUEIL • Markey Center for Molecular Genetics, Department

of Microbiology and Molecular Genetics, The University of Vermont, Burlington, VT

WILLIAM G . SCOTT • MRC Laboratory of Molecular Biology, Cambridge, UK; Current address: Department of Chemistry, Indiana University, Bloomington, IN

GEORG SCZAKIEL • Forschungsschwerpunkt Angewandte Tiimorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany

ANDREW SIWKOWSKI • Northern Illinois University, DeKalb, IL MICHAEL STRAUSS • MPG Group, Max Delbriick Center of Molecular

Medicine, Humboldt University, Berlin-Buch, Germany BRUCE A. SULLENGER • Program in Molecular Therapeutics, Departments of

Experimental Surgery and Genetics, Duke University Medical Center, Durham, NC

MARTIN TABLER • Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology, Crete, Greece

MINA TSAGRIS • Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology, Crete, Greece

PHILIP C . TURNER • School of Biological Sciences, University of Liverpool, UK NASSIM USMAN • Ribozyme Pharmaceuticals, Boulder, CO RAVI VINA YAK • Perkin Elmer Corporation, Applied Biosystems Division,

Foster City, CA FRANCINE E . WINCOTT • Ribozyme Pharmaceuticals, Boulder, CO OSAMU YAMADA • Immusol, San Diego, CA

MARY BETH D E YOUNG • Gladstone Institute of Cardiovascular Biology,

San Francisco, CA

xiv Contributors

MANG YU • Immusol, San Diego, CA QIAO YU • Marlcey Center for Molecular Genetics, Department

of Microbiology and Molecular Genetics, The University of Vermont, Burlington, VT

MARTIN ZILLMANN • Hybridon, Worcester, MA