Ribozyme Protocols

ISBN-13: 9781489942852 / Angielski / Miękka / 2013 / 492 str.

Philip C. Turner

Opis

The purpose of Ribozyme Protocols is to provide a helpful compilation of protocols that will be of use- DEGREESnot only to those with some experience of ribozymes- DEGREESbut 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 pre- dict 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 tar- get 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 select- ing those that actually hybridize to and/or cleave the target; these methods are described in Chapters 5 and 6.

Spis treści

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. Sull

Blurb

Ribozyme Protocols brings together a broad spectrum of essential and easily reproducible techniques currently used by leading researchers to manipulate RNA molecules and to inactivate gene expression in vivo. The methods collected here 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 generally to be found in the research literature.

Ribozyme Protocols will help all investigators perform meaningful and successful experiments using ribozymes in both clinical and basic research, as well as launch fresh application initiatives. It is certain to be found indispensable not only by those workers new to the field, but also by experienced researchers who wish to expand their range of expertise.