Upload
others
View
4
Download
0
Embed Size (px)
Citation preview
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
CarvellH. Williams, 1997 72. NeurotransinitterIVlethods,editedbyffic/iar(/C.iiayne, 1997 71. PRINS and In Situ PCR Protocols, edited by John R.
Gosden, 1997 70. Sequence Data Analysis Guidebook, edited by Simon R.
Swindell, 1997 69. cDNA Library Protocols, edited by Ian G. Cowell
and Caroline A. Austin, 1997 68. Gene Isolation and Mapping Protocols, edited by
Jacqueline Boultwood, 1997 67. PCR Cloning Protocols; From Molecular Cloning to
Genetic Engineering, edited by Bruce A. White, 1996 66. Epitope IVIapping Protocols, edited by Glenn E. Morris, 1996 65. PCR Sequencing Protocols, edited by Ralph Rapley, 1996 64. ProteinSequencingProtocols,editedbyftyan/.&i/A 1996 63. Recombinant Proteins: Detection and Isolation Protocols,
edited by Rocfy S. Tuan. 1996 62. Recombinant Gene Expression Protocols, edited by Rocky
S. Tuan, 1996 61. Protein and Peptide Analysis by Mass Spectrometry,
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
and Stephen J Hill, 1995 40. Protein Stability and Folding: Theory and Practice,
edited by Bret A. Shirley, 1995 39. Baculovirus Expression Protocols, edited by Christopher
D. Richardson, 1995 38. Cryoprescrvation and Freeze-Drying Protocols,
edited by John G. Day and Mark R. McLellan, 1995 37. In Vitro Transcription and Translation Protocols,
edited by Martin J. Tymms. 1995 36. Peptide Analysis Protocols, edited by Ben M. Dunn and
Michael W. Pennington, 1994 35. Peptide Synthesis Protocols, edited by Michael W.
Pennington and Ben M. Dunn, 1994 34. Immunocytocheraical Methods and Protocols, edited by
Lorette C. Javois, 1994 33. In Situ Hybridization Protocols, edited by K. H. Andy
Choo. 1994 32. Basic Protein and Peptide Protocols, edited by John M.
Walker, 1994 31. Protocols for Gene Analysis, edited hy Adrian J. Harwood,
1994 30. DNA-Protein Interactions, edited by G. Geoff Kneale, 1994 29. Chromosome Analysis Protocols, edited by John R.
Gosden, 1994 28. Protocols for Nucleic Acid Analysis by Nonradioactive
Probes, edited by Peter G Isaac, 1994 27. Biomembrane Protocols: 11. Architecture and Function.
edited by John M. Graham and Joan A. Higgins, 1994 26. Protocols for Oligonucleotide Conjugates: Synthesis and
Analytical Techniques, edited by Sudhir Agrawal, 1994 25. Computer Analysis of Sequence Data: Part II, edited by
Annette M. Griffin and Hugh G. Griffin, 1994 24. Computer Analysis of Sequence Data: Part I, edited by
Annette M. Griffin and Hugh G. Griffin, 1994 23. DNA Sequencing Protocols, edited by Hugh G. Griffin and
Annette M. Griffin, I99S 22. Microscopy, Optical Spectroscopy, and Macroscopic
Techniques, edited by Qmto;)/!cr Jones, Barbara Mulloy, and Adrian H. Thomas, 1993
21. Protocols in Molecular Parasitology, edited hy John E. Hyde, 1991
20. Protocols for Oligonucleotides and Analogs: Synthesis and Properties, edited by Sudhir Agrawal, 1993
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
All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Biology" is a trademark of The Humana Press Inc.
All authored papers, comments, opinions, conclusions, or recommendations are those of the author(s), and do not necessarily reflect the views of the publisher.
This publication is printed on acid-free paper. <S> ANSI Z39.48-I984 (American Standards Institute) Permanence of Paper for Printed Library Materials.
Cover illustration: Fig. 2 from Chapter 26, "Using Fluorescence Resonance Energy Transfer to Investigate Hammerhead Ribozyme Kinetics," by Thomas A. Perkins and John Goodchild.
Cover design by Patricia F. Cleary.
For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers; Tel: 201-256-1699; Fax: 201-256-8341; E-mail: [email protected] or visit our website at http://www.humanapress.com
Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US S8.00 per copy, plus US $00.25 per page, is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [0-89603-389-9/97 $8.00 + $00.25].
Printedin the United States of America. 1 0 9 8 7 6 5 4 3 2 1
Library of Congress Cataloging in Publication Data
Main entry under title:
Methods in molecular biology'".
Ribozyme protocols/edited by Philip C. Turner p. cm.—(Methods in molecular biologyT"; vol. 74)
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]
QP623.5.C36R53 1997 572' .788-dc21 DNLM/DLC for Library of Congress 97-12062
CIP
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