Ribozyme Protocols

About this book

A comprehensive collection of essential and easily reproducible techniques currently used by leading researchers to manipulate RNA molecules and to inactivate gene expression in vivo. The methods range from theoretical design to clinical application and include the synthesis and assay of ribozymes, as well as their use both in vitro and in vivo. Each method is presented by an expert with substantial hands-on experience and in far greater detail than is to be generally found in the research literature. The book will be indispensable not only to those workers new to the field, but also to experienced researchers who want to expand their range of expertise.

Contents

Ribozymes: An Introduction Helen A. James and Philip C. Turner. Computer-Aided Calculation of the Local Folding Potential of Target RNA and its Use for Ribozyme Design Georg Sczakiel and Martin Tabler. Computational Approaches to the Identification of Ribozyme Target Sites William James and Elizabeth Cowe. Computer Analysis of the Conservation and Uniqueness of Ribozyme-Targeted HIV Sequences Mary Beth DeYoung and Arnold Hampel. Selection of Accessible Sites for Ribozymes on Large RNA Transcripts Bruce L. Frank and John Goodchild. Selection of Efficient Ribozyme Cleavage Sites in Target RNAs Andre Lieber Mikkel Rohde and Michael Strauss. Chemical Synthesis Analysis and Purification of Ribozymes Ravi Vinayak. A Practical Method for the Production of RNA and Ribozymes Francine E. Wincott and Nassim Usman. Preparation of Templates for Production of Ribozymes and Substrates Rajesh K. Gaur and Guido Krupp. Enzymatic Synthesis and Characterization of Unmodified Ribozymes and Substrates Guido Krupp. T7 Transcript Length Determination Using Enzymatic RNA Sequencing Andrew Siwkowski. Chemical and Enzymatic Approaches to Construct Modified RNAs Rajesh K. Gaur and Guido Krupp. Applications of Modified Transcripts Rajesh K. Gaur Frank Conrad and Guido Krupp. Cloning Strategies for Catalytic Antisense RNAs Martin Tabler and Mina Tsagris. PCR-Based Construction of Long Hammerhead Ribozymes Martin Zillman and Gregory Robinson. Design and Production of Asymmetric Hammerhead Ribozymes Martin Tabler and Georg Sczakiel. Minimized Hammerhead Ribozymes Maxine J. McCall Philip Hendry and Trevor J. Lockett. Design of Hairpin Ribozymes for In Vitro and Cellular Applications Qiao Yu and John M. Burke. Design of the Hairpin Ribozyme for Targeting Specific RNA Sequences Arnold Hampel Mary Beth DeYoung Scott Galasinski and Andrew Siwkowski. Design and Preparation of Sequence Specific RNase P Ribozymes Denis Drainas and Guido Krupp. Theoretical Considerations in Measuring Reaction Parameters Timothy S. McConnell. Experimental Approaches for Measuring Reaction Parameters Timothy S. McConnell. Determination of Catalytic Parameters for Hairpin Ribozymes Mary Beth DeYoung Andrew Siwkowski and Arnold Hampel. Characterizing Ribozyme Cleavage Reactions Philip Hendry Maxine J. McCall and Trevor J. Lockett. Defining Optimum Reaction Conditions for Hammerhead Ribozymes Philip Hendry Maxine J. McCall and Trevor J. Lockett. Using Fluorescence Resonance Energy Transfer to Investigate Hammerhead Ribozyme Kinetics Thomas A. Perkins and John Goodchild. Design of Hybridizing Arms in Hammerhead Ribozymes Philip Hendry Maxine J. McCall and Trevor J. Lockett. Optimization of Hammerhead Flanking Sequences Using Oligonucleotide Facilitators John Goodchild. Enhancement of Ribozyme Function by RNA Binding Proteins Nan Sook Lee Edouard Bertrand and John J. Rossi. Selection of Fast-Hybridizing Complementary RNA Species In Vitro Ralf Kronenwett and Georg Sczakiel. In Vitro Selection of Hairpin Ribozymes Bruno Sargueil and John M. Burke. The Detection of Hammerhead Ribozyme Cleavage by RT-PCR Methods Rhonda Perriman. Detection of Ribozyme Cleavage Products Using Reverse Ligation Mediated PCR (RL-PCR) Edouard Bertrand Micheline Fromont-Racine Raymond Pictet and Thierry Grange. Quantitation of Ribozyme Target Abundance by QCPCR Amber A. Beaudry and James A. McSwiggen. Trans-splicing Reactions by Ribozymes Joshua T. Jones Seong- Wook Lee and Bruce A. Sullenger. Ligation of RNA Molecules by the Hairpin Ribozyme Alfredo Berzal-Herranz and John M. Burke. Mutagenesis and Modeling of the Hairpin Ribozyme Family Andrew Siwkowski Mary Beth DeYoung Pamela Anderson and Arnold Hampel. Preparation of Homogeneous Ribozyme RNA for Crystallization Jennifer A. Doudna. Establishing Suitability of RNA Preparations for Crystallization: Determination of Polydispersity Adrian R. Ferr'-d'Amar' and Jennifer A. Doudna. A Sparse Matrix Approach to Crystallizing Ribozymes and RNA Motifs Jamie H. Cate and Jennifer A. Doudna. Crystallographic Analyses of Chemically Synthesized Modified Hammerhead RNA Sequences as a General Approach Toward Understanding Ribozyme Structure and Function William G. Scott. tRNA Delivery Systems for Ribozymes Rhonda Perriman and Rob de Feyter. Expressing Ribozymes in Plants Rob de Feyter and Judith Gaudron. Using Microinjection of Xenopus Oocytes to Express and Optimize Ribozymes In Vivo Philip C. Turner. Exogenous Cellular Delivery of Ribozymes and Ribozyme Encoding DNAs Daniela Castanotto Edouard Bertrand and John J. Rossi. Optimization of Lipid Mediated Ribozyme Delivery to Cells in Culture Suzy A. Brown and Thale C. Jarvis. Retroviral Delivery and Anti-HIV Testing of Hammerhead Ribozymes Laurence Cagnon and John Rossi. Hairpin Ribozyme Gene Therapy for AIDS Elizabeth A. Duarte Mark C. Leavitt Osamu Yamada and Mang Yu. Clinical Aspects of Ribozymes as Therapeutics in Gene Therapy David Looney and Mang Yu. Index.

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