Ribozyme Protocols: (74 Methods in Molecular Biology)

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 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.

Table of Contents:
Ribozymes.- Computer-Aided Calculation of the Local Folding Potential of Target RNA and Its Use for Ribozyme Design.- Computational Approaches to the Identification of Ribozyme Target Sites.- Computer Analysis of the Conservation and Uniqueness of Ribozyme-Targeted HIV Sequences.- Selection of Accessible Sites for Ribozymes on Large RNA Transcripts.- Selection of Efficient Ribozyme Cleavage Sites in Target RNAs.- Chemical Synthesis, Analysis, and Purification of Ribozymes.- A Practical Method for the Production of RNA and Ribozymes.- Preparation of Templates for Production of Ribozymes and Substrates.- Enzymatic Synthesis and Characterization of Unmodified Ribozymes and Substrates.- T7 Transcript Length Determination Using Enzymatic RNA Sequencing.- Chemical and Enzymatic Approaches to Construct Modified RNAs.- Applications of Modified Transcripts.- Cloning Strategies for Catalytic Antisense RNAs.- PCR-Based Construction of Long Hammerhead Ribozymes.- Design and Production of Asymmetric Hammerhead Ribozymes.- Minimized Hammerhead Ribozymes.- Design of Hairpin Ribozymes for In Vitro and Cellular Applications.- Design of the Hairpin Ribozyme for Targeting Specific RNA Sequences.- Design and Preparation of Sequence-Specific RNase P Ribozymes.- Theoretical Considerations in Measuring Reaction Parameters.- Experimental Approaches for Measuring Reaction Parameters.- Determination of Catalytic Parameters for Hairpin Ribozymes.- Characterizing Ribozyme Cleavage Reactions.- Defining Optimum Reaction Conditions for Hammerhead Ribozymes.- Using Fluorescence Resonance Energy Transfer to Investigate Hammerhead Ribozyme Kinetics.- Design of Hybridizing Arms in Hammerhead Ribozymes.- Optimization of Hammerhead Flanking Sequences Using Oligonucleotide Facilitators.- Enhancement of Ribozyme Function by RNA Binding Proteins.- Selection of Fast-Hybridizing Complementary RNA Species In Vitro.- In Vitro Selection of Hairpin Ribozymes.- The Detection of Hammerhead Ribozyme Cleavage by RT-PCR Methods.- Detection of Ribozyme Cleavage Products Using Reverse Ligation-Mediated PCR (RL-PCR).- Quantitation of Ribozyme Target Abundance by QCPCR.- Trans-Splicing Reactions by Ribozymes.- Ligation of RNA Molecules by the Hairpin Ribozyme.- Mutagenesis and Modeling of the Hairpin Ribozyme Family.- Preparation of Homogeneous Ribozyme RNA for Crystallization.- Establishing Suitability of RNA Preparations for Crystallization.- A Sparse Matrix Approach to Crystallizing Ribozymes and RNA Motifs.- Crystallographic Analyses of Chemically Synthesized Modified Hammerhead RNA Sequences as a General Approach Toward Understanding Ribozyme Structure and Function.- tRNA Delivery Systems for Ribozymes.- Expressing Ribozymes in Plants.- Using Microinjection of Xenopus Oocytes to Express and Optimize Ribozymes In Vivo.- Exogenous Cellular Delivery of Ribozymes and Ribozyme Encoding DNAs.- Optimization of Lipid-Mediated Ribozyme Delivery to Cells in Culture.- Retroviral Delivery and Anti-HIV Testing of Hammerhead Ribozymes.- Hairpin Ribozyme Gene Therapy for AIDS.- Clinical Aspects of Ribozymes as Therapeutics in Gene Therapy.