Ribozymes and siRNA Protocols

Ribozymes and siRNA Protocols: Second Edition......Page 4
Preface......Page 6
Contents......Page 8
Contributors......Page 12
1 Ribozyme- and siRNA-Mediated mRNA Degradation: A General Introduction......Page 17
2 Combination of Chemical and Enzymatic RNA Synthesis......Page 25
3 Determination of Kinetic Parameters for Hammerhead and Hairpin Ribozymes......Page 35
4 Real-Time Monitoring of RNA and DNA Reactions by Fluorescence Detection......Page 49
5 Quantification of Ribozyme Target RNA Using Real-Time PCR......Page 65
6 Analysis of Ribozyme Structure and Function by Nucleotide Analog Interference Mapping......Page 73
7 Analysis of Global Conformational Transitions in Ribozymes......Page 93
8 In Vivo Detection of Ribozyme Cleavage Products and RNA Structure by Use of Terminal Transferase-Dependent PCR......Page 125
9 Identification of Efficient Cleavage Sites in Long-Target RNAs......Page 141
10 Selection In Vitro of Allosteric Ribozymes......Page 161
11 Regulation of Ribozyme Cleavage Activity by Oligonucleotides......Page 181
12 Tetracycline-Regulated Expression of Hammerhead Ribozymes In Vivo......Page 195
13 Ribozyme Expression Systems......Page 211
14 Design and Expression of Chimeric U1/Ribozyme Transgenes......Page 225
15 Design and Validation of Therapeutic Hammerhead Ribozymes for Autosomal Dominant Diseases......Page 237
16 Helicase-Attached Novel Hybrid Ribozymes......Page 253
17 Functional Gene Discovery Using Hybrid Ribozyme Libraries......Page 261
18 Maxizyme Technology......Page 273
19 Target-Site Selection for the 10–23 DNAzyme......Page 283
20 In Vitro Selected RNA-Cleaving DNA Enzymes From Combinatorial Libraries......Page 295
21 Nucleic Acid Sequence Analysis Using DNAzymes......Page 307
22 Crystallization of the Hairpin Ribozyme: Illustrative Protocols......Page 319
23 An Experimental Method for Selecting Effective Target Sites and Designing Hairpin Ribozymes......Page 329
24 Design and Optimization of Sequence-Specific Hairpin Ribozymes......Page 343
25 Design, Targeting, and Initial Screening of sTRSVDerived Hairpin Ribozymes for Optimum Helix 1 Length and Catalytic Efficiency In Vitro......Page 355
26 Optimization and Application of the Group I Ribozyme Trans-Splicing Reaction......Page 375
27 Repair of Myotonic Dystrophy Protein Kinase (DMPK) Transcripts by Trans-Splicing Ribozymes......Page 389
28 General Design and Construction of RNase P Ribozymes for Gene-Targeting Applications......Page 401
29 In Vitro Selection of RNase P Ribozymes That Efficiently Cleave a Target mRNA......Page 415
30 In Vitro Selection of External Guide Sequences for Directing Human RNase P to Cleave a Target mRNA......Page 429
31 RNase P-Mediated Inhibition of Viral Growth by Exogenous Administration of Short Oligonucleotide External Guide Sequence......Page 441
32 RNase P Ribozyme As an Antiviral Agent Against Human Cytomegalovirus......Page 453
33 Inhibition of Gene Expression by Nucleic Acid Enzymes in Rodent Models of Human Disease......Page 467
34 Potential Design Rules and Enzymatic Synthesis of siRNAs......Page 473
35 RNA Interference (RNAi) With RNase III-Prepared siRNAs......Page 487
36 RNAi Expression Vectors in Mammalian Cells......Page 499
37 Gene-Array Analysis of Glioma Cells After Treatment With an Anti-PKC alpha siRNA A: General Protocol......Page 509
38 RNAi in Living Mice......Page 517
39 Construction and Transfection of PCR Products Expressing siRNAs or shRNAs in Mammalian Cells......Page 525
40 Systemic Delivery of Synthetic siRNAs......Page 531
41 Adenovirus-Delivered siRNA......Page 539
42 RNAi Expression Vectors in Plant Cells......Page 549
43 Delivery Agents for Oligonucleotides......Page 561
44 Selection of Peptides for Specific Delivery of Oligonucleotides Into Cancer Cells......Page 585
45 Clinical Gene Therapy Research Utilizing Ribozymes: Application to the Treatment of HIV/AIDS......Page 597
46 Critical Steps in the Implementation of Hematopoietic Progenitor-Cell Gene Therapy Using Ribozyme Vectors: A Laboratory Protocol......Page 615
Index......Page 633