Abstract
Among the multiple publications that describe the use of ribozymes to knock out gene expression, only a few have directly proven that the ribozymes were catalytically active in vivo (1–3). Most studies rely on indirect evidence, such as a decrease in the level of the target RNA, and the comparison of the ribozyme with a mutant version that has lost cleavage activity. However, demonstration of the in vivo activity of a ribozyme requires detection of its cleavage products. For example, a ribozyme hybridized to its target RNA could promote its degradation indirectly, through cellular, double-strand specific RNases (4). Furthermore, a mutant ribozyme could be inactive for reasons independent of the loss of catalytic activity (e.g., the mutation could induce a change in the 3-D structure of the ribozyme, resulting in less efficient hybridization to the target RNA).
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References
Bertrand, E., Pictet, R., and Grange, G. (1994) Can hammerhead ribozymes be efficient tools for inactivating gene function? Nucleic. Acids. Res. 22, 293–300.
Saxena, S. K. and Ackerman, E. J. (1990) Ribozymes correctly cleave a model substrate and endogenous RNA in vivo. J. Biol. Chem. 265, 17,106–17,109.
Steinecke, P., Herget, T., and Schreler, P. H. (1992) Expression of a chimeric ribozyme gene results in endonucleolytic cleavage of target mRNA and a concomitant reduction of gene expression in vivo. EMBO J. 11, 1525–1530.
Hildebrandt, M. and Nellen, W. (1992) Differential antisense transcription from the Dictyostelium EB4 gene locus. implications on antisense-mediated regulation of mRNA stability. Cell. 69, 197–204.
Symons, R. H. (1992) Small catalytic RNAs. Annu. Rev. Biochem. 61, 641–671.
Bertrand, E., Fromont-Racine, M., Pictet, R., and Grange, T. (1993) Visualization of the in vivo interaction of a regulatory protein with RNA. Proc. Natl. Acad. Sci. USA 90, 3496–3500.
Mueller, P. and Wold, B. (1989) In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science 246, 780–786.
Peattie, D. A. (1979) Direct chemical method for sequencing RNA. Proc. Natl. Acad. Sci. USA 76, 1760–1764.
Frohman, M. A., Dush, M. K., and Martin, G. R. (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85, 8998–9002.
Edwards, J. B., Delort, J., and Mallet, J. (1991) Oligodeoxyribonucleotide ligation to single-stranded cDNAs. a new tool for cloning 5′ ends of mRNAs and for constructing cDNA libraries by in vitro amplification. Nucleic. Acids. Res. 19, 5227–5232.
Troutt, A. B., McHeyzer, W. M., Pulendran, B., and Nossal, G. J. (1992) Ligation-anchored PCR: a sample amplification technique with single-sided specificity [published erratum appears in Proc. Natl. Acad. Sci. USA (1993) 90, 3775] Proc. Natl. Acad. Sci. USA 89, 9823–9825
Fromont-Racine, M., Bertrand, E., Pictet, R., and Grange, T. (1993) A highly sensitive method for mapping the 5′ termini of mRNAs. Nucleic. Acids. Res. 21, 1683, 1684
Sambrook, J., Fritsch, E. F., and Mamatis, T. (1989) Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Milligan, J. F., Groebe, D. R., Witherell, G. W., and Uhlenbeck, O. C. (1987) Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA template. Nucleic. Acids. Res. 15, 8783–8798.
Meinkoth, J. and Wahl, G. (1984) Hybridization of nucleic acids immobilized on solid supports. Anal. Biochem. 138, 267–284.
Auffray, C. and Rougeon, R. (1980) Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur. J. Biochem. 107, 303–314.
Ehresmann, C., Baudin, F., Mougel, M., Romby, P., Ebel, J. P., and Ehresmann, B. (1987) Probing the structure of RNA in solution. Nucleic. Acids. Res. 15, 9109–9128.
Innis, M. A. and Gelfand, D. H. (1990) Optimization of PCRs, In: PCR Protocols: A Guide to Methods and Applications (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. W., eds.), Academic, San Diego, CA, pp. 3–12.
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Bertrand, E., Fromont-Racine, M., Pictet, R., Grange, T. (1997). Detection of Ribozyme Cleavage Products Using Reverse Ligation-Mediated PCR (RL-PCR). In: Turner, P.C. (eds) Ribozyme Protocols. Methods in Molecular Biology™, vol 74. Humana Press. https://doi.org/10.1385/0-89603-389-9:311
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DOI: https://doi.org/10.1385/0-89603-389-9:311
Publisher Name: Humana Press
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