Library Science

In this completely updated and expanded edition of a classic bench manual, hands-on experts take advantage of the latest advances in ribozyme, DNAzyme, 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 hairpain 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 ribozymes protocols for effective design, selection, and therapeutic applications; and the latest RNAi methods for sequencing-specific gene silencing in a wide variety of organisms. Comprehensive and up-to-date, Ribozymes and siRNA Protocols synthesizes for experienced and novice investigators alike the exciting advances in understanding nucleic acid enzymes and demonstrates how they may be used to analyze gene function and target validation, and to productively develop new therapeutics for human diseases.

Ed. Sioud, Mouldy

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Table of Contents

1. Ribozyme- and siRNA-Mediated mRNA Degradation: A General Introduction (pp. 001-008)
   Sioud, Mouldy
2. Combination of Chemical and Enzymatic RNA Synthesis (pp. 009-018)
   Gaur, Rajesh K.; Hanne, Andreas; Krupp, Guido
3. Determination of Kinetic Parameters for Hammerhead and Hairpin Ribozymes (pp. 019-032)
   Fedor, Martha J.
4. Real-Time Monitoring of RNA and DNA Reactions by Fluorescence Detection (pp. 033-048)
   Singh, Kumud K.; Hanne, Andreas; Krupp, Guido
5. Quantification of Ribozyme Target RNA Using Real-Time PCR (pp. 049-056)
   Klein, Dagmar; Ricordi, Camillo; Pastori, Ricardo L.
6. Analysis of Ribozyme Structure and Function by Nucleotide Analog Interference Mapping (pp. 057-076)
   Soumitrasu, Soumitrasu; Pazsint, Catherine; Chowdhury, Gopeswar
7. Analysis of Global Conformational Transitions in Ribozymes (pp. 077-108)
   Lilley, David M. J.
8. In Vivo Detection of Ribozyme Cleavage Products and RNA Structure by Use of Terminal Transferase-Dependent PCR (pp. 109-124)
   Chen, Hsiu-Hua; Castanotto, Daniela; LeBon, Jeanne; Rossi, John J.; Riggs, Arthur D.
9. Identification of Efficient Cleavage Sites in Long-Target RNAs (pp. 125-144)
   Pan, Wei-Hua; Clawson, Gary A.
10. Selection In Vitro of Allosteric Ribozymes (pp. 145-164)
    Roth, Adam; Breaker, Ronald R.
11. Regulation of Ribozyme Cleavage Activity by Oligonucleotides (pp. 165-178)
    Komatsu, Yasuo; Ohtsuka, Eiko
12. Tetracycline-Regulated Expression of Hammerhead Ribozymes In Vivo (pp. 179-194)
    Bowden, Emma T.; Riegel, Anna T.
13. Ribozyme Expression Systems (pp. 195-208)
    Sano, Masayuki; Taira, Kazunari
14. Design and Expression of Chimeric U1/Ribozyme Transgenes (pp. 209-220)
    Abounader, Roger; Montgomery, Robert; Dietz, Harry; Laterra, John
15. Design and Validation of Therapeutic Hammerhead Ribozymes for Autosomal Dominant Diseases (pp. 221-236)
    Fritz, Jason J.; Gorbatyuk, Marina; Lewin, Alfred S.; Hauswirth, William W.
16. Helicase-Attached Novel Hybrid Ribozymes (pp. 237-244)
    Kawasaki, Hiroaki; Warashina, Masaki; Kuwabara, Tomoko; Taira, Kazunari
17. Functional Gene Discovery Using Hybrid Ribozyme Libraries (pp. 245-256)
    Kato, Yoshio; Tsunemi, Masaru; Miyagishi, Makoto; Kawasaki, Hiroaki; Taira, Kazunari
18. Maxizyme Technology (pp. 257-266)
    Iyo, Mayu; Kawasaki, Hiroaki; Taira, Kazunari
19. Target-Site Selection for the 10–23 DNAzyme (pp. 267-278)
    Cairns, Murray J.; Sun, Lun-Quan
20. In Vitro Selected RNA-Cleaving DNA Enzymes From Combinatorial Libraries (pp. 279-290)
    Chakraborti, Samitabh; Sriram, Bandi; Banerjea, Akhil C.
21. Nucleic Acid Sequence Analysis Using DNAzymes (pp. 291-302)
    Cairns, Murray J.; Sun, Lun-Quan
22. Crystallization of the Hairpin Ribozyme: Illustrative Protocols (pp. 303-312)
    Rupert, Peter B.; Ferre-D’Amare, Adrian R.
23. An Experimental Method for Selecting Effective Target Sites and Designing Hairpin Ribozymes (pp. 313-326)
    Barroso-delJesus, Alicia; Puerta-Fernandez, Elena; Romero-Lopez, Cristina; Berzal-Herranz, Alfredo
24. Design and Optimization of Sequence-Specific Hairpin Ribozymes (pp. 327-338)
    Romero-Lopez, Cristina; Aliciarroso-delJesus, Aliciarroso-delJesus; Puerta-Fernandez, Elena; Berzal-Herranz, Alfredo
25. Design, Targeting, and Initial Screening of sTRSV- Derived Hairpin Ribozymes for Optimum Helix 1 Length and Catalytic Efficiency In Vitro (pp. 339-358)
    Richardson, Max W.; Hostalek, Linda; Michellebson, Michellebson; Hu, Jason; Shippy, Richard; Siwkowski, Andrew; Marmur, Jonathan D.; Khalili, Kamel; Klotman, Paul E.; Hampel, Arnold; Rappaport, Jay
26. Optimization and Application of the Group I Ribozyme Trans-Splicing Reaction (pp. 359-372)
    Einvik, Christer; Fiskaa, Tonje; Lundblad, Eirik W.; Johansen, Steinar
27. Repair of Myotonic Dystrophy Protein Kinase (DMPK) Transcripts by Trans-Splicing Ribozymes (pp. 373-384)
    Phylactou, Leonidas A.
28. General Design and Construction of RNase P Ribozymes for Gene-Targeting Applications (pp. 385-398)
    Zou, Hua; Chan, Karen; Trang, Phong; Liu, Fenyong
29. In Vitro Selection of RNase P Ribozymes That Efficiently Cleave a Target mRNA (pp. 399-412)
    Kim, Kihoon; Liu, Fenyong
30. In Vitro Selection of External Guide Sequences for Directing Human RNase P to Cleave a Target mRNA (pp. 413-424)
    Raj, Stephen; Liu, Fenyong
31. RNase P-Mediated Inhibition of Viral Growth by Exogenous Administration of Short Oligonucleotide External Guide Sequence (pp. 425-436)
    Dunn, Walter; Liu, Fenyong
32. RNase P Ribozyme As an Antiviral Agent Against Human Cytomegalovirus (pp. 437-450)
    Trang, Phong; Liu, Fenyong
33. Inhibition of Gene Expression by Nucleic Acid Enzymes in Rodent Models of Human Disease (pp. 451-456)
    Ole Iversen, Per; Sioud, Mouldy
34. Potential Design Rules and Enzymatic Synthesis of siRNAs (pp. 457-470)
    Sioud, Mouldy; Leirdal, Marianne
35. RNA Interference (RNAi) With RNase III-Prepared siRNAs (pp. 471-482)
    Yang, Dun; Goga, Andrei; Bishop, J. Michael
36. RNAi Expression Vectors in Mammalian Cells (pp. 483-492)
    Miyagishi, Makoto; Taira, Kazunari
37. Gene-Array Analysis of Glioma Cells After Treatment With an Anti-PKC? siRNA: A General Protocol (pp. 493-500)
    Leirdal, Marianne; Sioud, Mouldy
38. RNAi in Living Mice (pp. 501-508)
    Hasuwa, Hidetoshi; Okabe, Masaru
39. Construction and Transfection of PCR Products Expressing siRNAs or shRNAs in Mammalian Cells (pp. 509-514)
    Castanotto, Daniela; Rossi, John J.
40. Systemic Delivery of Synthetic siRNAs (pp. 515-522)
    Sioud, Mouldy; Sorensen, Dag R.
41. Adenovirus-Delivered siRNA (pp. 523-532)
    Shen, Changxian; Reske, Sven N.
42. RNAi Expression Vectors in Plant Cells (pp. 533-544)
    Akashi, Hideo; Miyagishi, Makoto; Taira, Kazunari
43. Delivery Agents for Oligonucleotides (pp. 545-568)
    Seksek, Olivier; Bolard, Jacques
44. Selection of Peptides for Specific Delivery of Oligonucleotides Into Cancer Cells (pp. 569-580)
    Shadidi, Mohsen; Sioud, Mouldy
45. Clinical Gene Therapy Research Utilizing Ribozymes: Application to the Treatment of HIV/AIDS (pp. 581-598)
    Ngok, Frances K.; Mitsuyasu, Ronald T.; Macpherson, Janet L.; Boyd, Maureen P.; Symonds, Geoff P.; Amado, Rafael G.
46. Critical Steps in the Implementation of Hematopoietic Progenitor-Cell Gene Therapy Using Ribozyme Vectors: A Laboratory Protocol (pp. 599-616)
    Boyd, Maureen P.; Ngok, Frances K.; Todd, Alison V.; Symonds, Geoff P.; Macpherson, Janet L.; Amado, Rafael G.

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