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. 1995 Oct 11;23(19):3922–3927. doi: 10.1093/nar/23.19.3922

In vitro activity of minimised hammerhead ribozymes.

P Hendry 1, M J McCall 1, F S Santiago 1, P A Jennings 1
PMCID: PMC307311  PMID: 7479037

Abstract

A number of minimised hammerhead ribozymes (minizymes) which lack stem II have been kinetically characterised. These minizymes display optimal cleavage activity at temperatures around 37 degrees C. The cleavage reactions of the minizymes are first order in hydroxide ion concentration up to around pH 9.3 above which the cleavage rate constants decline rapidly. The reactions show a biphasic dependence on magnesium-ion concentration; one of the interactions has an apparent dissociation constant of around 20 mM while the other appears to be very weak, showing no sign of saturation at 200 mM MgCl2. The minizymes are significantly less active than comparable, full-size ribozymes when cleaving short substrates. However, at a particular site in a transcribed TAT gene from HIV-1, minizymes are more effective than ribozymes.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Cameron F. H., Jennings P. A. Specific gene suppression by engineered ribozymes in monkey cells. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9139–9143. doi: 10.1073/pnas.86.23.9139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cotten M., Birnstiel M. L. Ribozyme mediated destruction of RNA in vivo. EMBO J. 1989 Dec 1;8(12):3861–3866. doi: 10.1002/j.1460-2075.1989.tb08564.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dahm S. C., Derrick W. B., Uhlenbeck O. C. Evidence for the role of solvated metal hydroxide in the hammerhead cleavage mechanism. Biochemistry. 1993 Dec 7;32(48):13040–13045. doi: 10.1021/bi00211a013. [DOI] [PubMed] [Google Scholar]
  4. Dahm S. C., Uhlenbeck O. C. Role of divalent metal ions in the hammerhead RNA cleavage reaction. Biochemistry. 1991 Oct 1;30(39):9464–9469. doi: 10.1021/bi00103a011. [DOI] [PubMed] [Google Scholar]
  5. Fedor M. J., Uhlenbeck O. C. Kinetics of intermolecular cleavage by hammerhead ribozymes. Biochemistry. 1992 Dec 8;31(48):12042–12054. doi: 10.1021/bi00163a012. [DOI] [PubMed] [Google Scholar]
  6. Good N. E., Winget G. D., Winter W., Connolly T. N., Izawa S., Singh R. M. Hydrogen ion buffers for biological research. Biochemistry. 1966 Feb;5(2):467–477. doi: 10.1021/bi00866a011. [DOI] [PubMed] [Google Scholar]
  7. Hendry P., McCall M. J. A comparison of the in vitro activity of DNA-armed and all-RNA hammerhead ribozymes. Nucleic Acids Res. 1995 Oct 11;23(19):3928–3936. doi: 10.1093/nar/23.19.3928. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hendry P., McCall M. J., Santiago F. S., Jennings P. A. A ribozyme with DNA in the hybridising arms displays enhanced cleavage ability. Nucleic Acids Res. 1992 Nov 11;20(21):5737–5741. doi: 10.1093/nar/20.21.5737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hendry P., Moghaddam M. J., McCall M. J., Jennings P. A., Ebel S., Brown T. Using linkers to investigate the spatial separation of the conserved nucleotides A9 and G12 in the hammerhead ribozyme. Biochim Biophys Acta. 1994 Oct 18;1219(2):405–412. doi: 10.1016/0167-4781(94)90065-5. [DOI] [PubMed] [Google Scholar]
  10. Hertel K. J., Pardi A., Uhlenbeck O. C., Koizumi M., Ohtsuka E., Uesugi S., Cedergren R., Eckstein F., Gerlach W. L., Hodgson R. Numbering system for the hammerhead. Nucleic Acids Res. 1992 Jun 25;20(12):3252–3252. doi: 10.1093/nar/20.12.3252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Long D. M., Uhlenbeck O. C. Kinetic characterization of intramolecular and intermolecular hammerhead RNAs with stem II deletions. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):6977–6981. doi: 10.1073/pnas.91.15.6977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. McCall M. J., Hendry P., Jennings P. A. Minimal sequence requirements for ribozyme activity. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5710–5714. doi: 10.1073/pnas.89.13.5710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Nesbitt S., Goodchild J. Further studies on the use of oligonucleotide facilitators to increase ribozyme turnover. Antisense Res Dev. 1994 Winter;4(4):243–249. doi: 10.1089/ard.1994.4.243. [DOI] [PubMed] [Google Scholar]
  14. Perreault J. P., Labuda D., Usman N., Yang J. H., Cedergren R. Relationship between 2'-hydroxyls and magnesium binding in the hammerhead RNA domain: a model for ribozyme catalysis. Biochemistry. 1991 Apr 23;30(16):4020–4025. doi: 10.1021/bi00230a029. [DOI] [PubMed] [Google Scholar]
  15. Pley H. W., Flaherty K. M., McKay D. B. Three-dimensional structure of a hammerhead ribozyme. Nature. 1994 Nov 3;372(6501):68–74. doi: 10.1038/372068a0. [DOI] [PubMed] [Google Scholar]
  16. Ruffner D. E., Stormo G. D., Uhlenbeck O. C. Sequence requirements of the hammerhead RNA self-cleavage reaction. Biochemistry. 1990 Nov 27;29(47):10695–10702. doi: 10.1021/bi00499a018. [DOI] [PubMed] [Google Scholar]
  17. Sanchez-Pescador R., Power M. D., Barr P. J., Steimer K. S., Stempien M. M., Brown-Shimer S. L., Gee W. W., Renard A., Randolph A., Levy J. A. Nucleotide sequence and expression of an AIDS-associated retrovirus (ARV-2). Science. 1985 Feb 1;227(4686):484–492. doi: 10.1126/science.2578227. [DOI] [PubMed] [Google Scholar]
  18. Sawata S., Shimayama T., Komiyama M., Kumar P. K., Nishikawa S., Taira K. Enhancement of the cleavage rates of DNA-armed hammerhead ribozymes by various divalent metal ions. Nucleic Acids Res. 1993 Dec 11;21(24):5656–5660. doi: 10.1093/nar/21.24.5656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Shimayama T., Nishikawa F., Nishikawa S., Taira K. Nuclease-resistant chimeric ribozymes containing deoxyribonucleotides and phosphorothioate linkages. Nucleic Acids Res. 1993 Jun 11;21(11):2605–2611. doi: 10.1093/nar/21.11.2605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sioud M., Natvig J. B., Førre O. Preformed ribozyme destroys tumour necrosis factor mRNA in human cells. J Mol Biol. 1992 Feb 20;223(4):831–835. doi: 10.1016/0022-2836(92)90244-e. [DOI] [PubMed] [Google Scholar]
  21. Snyder D. S., Wu Y., Wang J. L., Rossi J. J., Swiderski P., Kaplan B. E., Forman S. J. Ribozyme-mediated inhibition of bcr-abl gene expression in a Philadelphia chromosome-positive cell line. Blood. 1993 Jul 15;82(2):600–605. [PubMed] [Google Scholar]
  22. Takagi Y., Taira K. Temperature-dependent change in the rate-determining step in a reaction catalyzed by a hammerhead ribozyme. FEBS Lett. 1995 Mar 20;361(2-3):273–276. doi: 10.1016/0014-5793(95)00192-c. [DOI] [PubMed] [Google Scholar]
  23. Tuschl T., Eckstein F. Hammerhead ribozymes: importance of stem-loop II for activity. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):6991–6994. doi: 10.1073/pnas.90.15.6991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Uhlenbeck O. C. A small catalytic oligoribonucleotide. Nature. 1987 Aug 13;328(6131):596–600. doi: 10.1038/328596a0. [DOI] [PubMed] [Google Scholar]
  25. Vogel J., Hinrichs S. H., Reynolds R. K., Luciw P. A., Jay G. The HIV tat gene induces dermal lesions resembling Kaposi's sarcoma in transgenic mice. Nature. 1988 Oct 13;335(6191):606–611. doi: 10.1038/335606a0. [DOI] [PubMed] [Google Scholar]
  26. Yang J. H., Usman N., Chartrand P., Cedergren R. Minimum ribonucleotide requirement for catalysis by the RNA hammerhead domain. Biochemistry. 1992 Jun 2;31(21):5005–5009. doi: 10.1021/bi00136a013. [DOI] [PubMed] [Google Scholar]

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