Ribozymes and siRNA protocols

In this completely updated and expanded edition of a classic bench manual, hands-on experts take advantage of the latest advances in ribozyme, DNAzyme, hammerhead ribozymes and derivatives, and RNA interference technologies to describe in detail the exciting and successful methods now available for gene inactivation in vitro and in vivo. Their optimized techniques employ hairpin ribozymes, DNAzymes, hammerhead ribozymes and derivatives, group I intron ribozymes, RNase P ribozymes, and siRNAs, as well as general methods for RNA structure analysis, delivery of oligonucleotides, and gene therapy. Also provided are novel methods for identifying accessible cellular mRNA sites; group I intron and RNAse P ribozyme protocols for effective design, selection, and therapeutic applications; and the latest RNAi methods for sequence-specific gene silencing in a wide variety of organisms. Additional techniques cover the analysis of ribozyme structures and conformational transitions using nucleotide analog interference mapping and fluorescence resonance energy transfer, the use of ribozymes in clinical and gene therapy, and the use of ribozymes and DNAzymes in rodent models of human disease. Each proven protocol includes a background introduction outlining the principle behind the technique, step-by-step instructions, lists of equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.
Comprehensive and up-to-date, Ribozymes and siRNA Protocols details for experienced and novice investigators alike the many exciting advances in our understanding of nucleic acid enzymes, as well as demonstrating how they may be used to analyze gene function and target validation, and to productively develop novel therapeutics for human diseases.

Includes supplementary material: sn.pub/extras

Zusammenfassung
Focusing on the latest technical advances for interfering with gene expression, this text provides both general and specific protocols for using hammerhead ribozymes and deviates, DNAzymes, hairpin ribozymes, group I intron ribozymes, RNase P ribozymes and siRNAs.

Inhalt
Ribozyme- and siRNA-Mediated mRNA Degradation.- Combination of Chemical and Enzymatic RNA Synthesis.- Determination of Kinetic Parameters for Hammerhead and Hairpin Ribozymes.- Real-Time Monitoring of RNA and DNA Reactions by Fluorescence Detection.- Quantification of Ribozyme Target RNA Using Real-Time PCR.- Analysis of Ribozyme Structure and Function by Nucleotide Analog Interference Mapping.- Analysis of Global Conformational Transitions in Ribozymes.- In Vivo Detection of Ribozyme Cleavage Products and RNA Structure by Use of Terminal Transferase-Dependent PCR.- Identification of Efficient Cleavage Sites in Long-Target RNAs.- Selection In Vitro of Allosteric Ribozymes.- Regulation of Ribozyme Cleavage Activity by Oligonucleotides.- Tetracycline-Regulated Expression of Hammerhead Ribozymes In Vivo.- Ribozyme Expression Systems.- Design and Expression of Chimeric U1/Ribozyme Transgenes.- Design and Validation of Therapeutic Hammerhead Ribozymes for Autosomal Dominant Diseases.- Helicase-Attached Novel Hybrid Ribozymes.- Functional Gene Discovery Using Hybrid Ribozyme Libraries.- Maxizyme Technology.- Target-Site Selection for the 1023 DNAzyme.- In Vitro Selected RNA-Cleaving DNA Enzymes From Combinatorial Libraries.- Nucleic Acid Sequence Analysis Using DNAzymes.- Crystallization of the Hairpin Ribozyme.- An Experimental Method for Selecting Effective Target Sites and Designing Hairpin Ribozymes.- Design and Optimization of Sequence-Specific Hairpin Ribozymes.- Design, Targeting, and Initial Screening of sTRSV-Derived Hairpin Ribozymes for Optimum Helix 1 Length and Catalytic Efficiency In Vitro.- Optimization and Application of the Group I Ribozyme Trans-Splicing Reaction.- Repair of Myotonic Dystrophy Protein Kinase (DMPK) Transcripts by Trans-SplicingRibozymes.- General Design and Construction of RNase P Ribozymes for Gene-Targeting Applications.- In Vitro Selection of RNase P Ribozymes That Efficiently Cleave a Target mRNA.- In Vitro Selection of External Guide Sequences for Directing Human RNase P to Cleave a Target mRNA.- RNase P-Mediated Inhibition of Viral Growth by Exogenous Administration of Short Oligonucleotide External Guide Sequence.- RNase P Ribozyme As an Antiviral Agent Against Human Cytomegalovirus.- Inhibition of Gene Expression by Nucleic Acid Enzymes in Rodent Models of Human Disease.- Potential Design Rules and Enzymatic Synthesis of siRNAs.- RNA Interference (RNAi) With RNase III-Prepared siRNAs.- RNAi Expression Vectors in Mammalian Cells.- Gene-Array Analysis of Glioma Cells After Treatment With an Anti-PKC? siRNA.- RNAi in Living Mice.- Construction and Transfection of PCR Products Expressing siRNAs or shRNAs in Mammalian Cells.- Systemic Delivery of Synthetic siRNAs.- Adenovirus-Delivered siRNA.- RNAi Expression Vectors in Plant Cells.- Delivery Agents for Oligonucleotides.- Selection of Peptides for Specific Delivery of Oligonucleotides Into Cancer Cells.- Clinical Gene Therapy Research Utilizing Ribozymes.- Critical Steps in the Implementation of Hematopoietic Progenitor-Cell Gene Therapy Using Ribozyme Vectors.