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.

Ribozyme- and siRNA-Mediated mRNA Degradation: A General Introduction Mouldy Sioud Combination of Chemical and Enzymatic RNA Synthesis Rajesh K. Gaur, Andreas Hanne, and Guido Krupp Determination of Kinetic Parameters for Hammerhead and Hairpin Ribozymes Martha J. Fedor Real-Time Monitoring of RNA and DNA Reactions by Fluorescence Detection Kumud K. Singh, Andreas Hanne, and Guido Krupp Quantification of Ribozyme Target RNA Using Real-Time PCR Dagmar Klein, Camillo Ricordi, and Ricardo L. Pastori Analysis of Ribozyme Structure and Function by Nucleotide Analog Interference Mapping Soumitra Basu, Catherine Pazsint, and Gopeswar Chowdhury Analysis of Global Conformational Transitions in Ribozymes David M. J. Lilley In Vivo Detection of Ribozyme Cleavage Products and RNA Structure by Use of Terminal Transferase-Dependent PCR Hsiu-Hua Chen, Daniela Castanotto, Jeanne LeBon, John J. Rossi, and Arthur D. Riggs Identification of Efficient Cleavage Sites in Long-Target RNAs Wei-Hua Pan and Gary A. Clawson Selection In Vitro of Allosteric Ribozymes Adam Roth and Ronald R. Breaker Regulation of Ribozyme Cleavage Activity by Oligonucleotides Yasuo Komatsu and Eiko Ohtsuka Tetracycline-Regulated Expression of Hammerhead Ribozymes In Vivo Emma T. Bowden and Anna T. Riegel Ribozyme Expression Systems Masayuki Sano and Kazunari Taira Design and Expression of Chimeric U1/Ribozyme Transgenes Roger Abounader, Robert Montgomery, Harry Dietz, and John Laterra Design and Validation of Therapeutic Hammerhead Ribozymes for Autosomal Dominant Diseases Jason J. Fritz, Marina Gorbatyuk, Alfred S. Lewin, and William W. Hauswirth Helicase-Attached Novel Hybrid Ribozymes Hiroaki Kawasaki, Masaki Warashina, Tomoko Kuwabara, and Kazunari Taira Functional Gene Discovery Using Hybrid Ribozyme Libraries Yoshio Kato, Masaru Tsunemi, Makoto Miyagishi, Hiroaki Kawasaki, and Kazunari Taira Maxizyme Technology Mayu Iyo, Hiroaki Kawasaki, and Kazunari Taira Target-Site Selection for the 10-23 DNAzyme Murray J. Cairns and Lun-Quan Sun In Vitro Selected RNA-Cleaving DNA Enzymes From Combinatorial Libraries Samitabh Chakraborti, Bandi Sriram and Akhil C. Banerjea Nucleic Acid Sequence Analysis Using DNAzymes Murray J. Cairns and Lun-Quan Sun Crystallization of the Hairpin Ribozyme: Illustrative Protocols Peter B. Rupert and Adrian R. Ferre-D'Amare An Experimental Method for Selecting Effective Target Sites and Designing Hairpin Ribozymes Alicia Barroso-delJesus, Elena Puerta-Fernandez, Cristina Romero-Lopez, and Alfredo Berzal-Herranz Design and Optimization of Sequence-Specific Hairpin Ribozymes Cristina Romero-Lopez, Alicia Barroso-delJesus, Elena Puerta-Fernandez, and Alfredo Berzal-Herranz Design, Targeting, and Initial Screening of sTRSV-Derived Hairpin Ribozymes for Optimum Helix 1 Length and Catalytic Efficiency In Vitro Max W. Richardson, Linda Hostalek, Michelle Dobson, Jason Hu, Richard Shippy, Andrew Siwkowski, Jonathan D. Marmur, Kamel Khalili, Paul E. Klotman, Arnold Hampel, and Jay Rappaport Optimization and Application of the Group I Ribozyme Trans-Splicing Reaction Christer Einvik, Tonje Fiskaa, Eirik W. Lundblad, and Steinar Johansen Repair of Myotonic Dystrophy Protein Kinase (DMPK) Transcripts by Trans-Splicing Ribozymes Leonidas A. Phylactou General Design and Construction of RNase P Ribozymes for Gene-Targeting Application Hua Zou, Karen Chan, ...