Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1992 Jun 11;20(11):2835–2840. doi: 10.1093/nar/20.11.2835

Extensive phosphorothioate substitution yields highly active and nuclease-resistant hairpin ribozymes.

B M Chowrira 1, J M Burke 1
PMCID: PMC336929  PMID: 1377380

Abstract

The catalytic function of the hairpin ribozyme has been investigated by modification-interference analysis of both ribozyme and substrate, using ribonucleoside phosphorothioates. Thiophosphate substitutions in two ribozyme domains were examined by using a novel and highly efficient two-piece ribozyme assembled from two independently synthesized oligoribonucleotides. The catalytic proficiency of the two-piece construct (KM = 48 nM, kcat = 2.3 min-1) is nearly identical to that of the one-piece ribozyme. The two-piece ribozyme is essentially unaffected by substitution with thiophosphates 5' to all guanosines, cytidines, and uridines. In contrast, incorporation of multiple adenosine phosphorothioates in the 5' domain of the ribozyme decreases ribozyme activity by a factor of 25. Modification-interference experiments using ribozymes partially substituted with adenosine phosphorothioate suggest that thiophosphates 5' to A7, A9 and A10 interfere with cleavage to a greater extent than substitutions at other sites within the molecule, but the effect is modest. Within the substrate, phosphorothioate substitution does not directly interfere with cleavage, rather, increasing thiophosphate content decreases the stability of the ribozyme-substrate complex. We describe the construction of a hairpin ribozyme containing dinucleotide extensions at its 5' and 3' ends. Full substitution of this molecule with G and C phosphorothioates results in a ribozyme with greatly enhanced stability against cellular ribonucleases without significant loss of catalytic efficiency.

Full text

PDF
2835

Images in this article

2837Image
on p.2837
2837Image
on p.2837
2838Image
on p.2838
2838Image
on p.2838
2839Image
on p.2839
2839Image
on p.2839

Selected References

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

  1. Burke J. M., Irvine K. D., Kaneko K. J., Kerker B. J., Oettgen A. B., Tierney W. M., Williamson C. L., Zaug A. J., Cech T. R. Role of conserved sequence elements 9L and 2 in self-splicing of the Tetrahymena ribosomal RNA precursor. Cell. 1986 Apr 25;45(2):167–176. doi: 10.1016/0092-8674(86)90380-6. [DOI] [PubMed] [Google Scholar]
  2. Buzayan J. M., Feldstein P. A., Segrelles C., Bruening G. Autolytic processing of a phosphorothioate diester bond. Nucleic Acids Res. 1988 May 11;16(9):4009–4023. doi: 10.1093/nar/16.9.4009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cech T. R. Ribozymes and their medical implications. JAMA. 1988 Nov 25;260(20):3030–3034. [PubMed] [Google Scholar]
  4. Chowrira B. M., Berzal-Herranz A., Burke J. M. Novel guanosine requirement for catalysis by the hairpin ribozyme. Nature. 1991 Nov 28;354(6351):320–322. doi: 10.1038/354320a0. [DOI] [PubMed] [Google Scholar]
  5. Chowrira B. M., Burke J. M. Binding and cleavage of nucleic acids by the "hairpin" ribozyme. Biochemistry. 1991 Sep 3;30(35):8518–8522. doi: 10.1021/bi00099a003. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Eckstein F., Gish G. Phosphorothioates in molecular biology. Trends Biochem Sci. 1989 Mar;14(3):97–100. doi: 10.1016/0968-0004(89)90130-8. [DOI] [PubMed] [Google Scholar]
  8. Eckstein F. Nucleoside phosphorothioates. Annu Rev Biochem. 1985;54:367–402. doi: 10.1146/annurev.bi.54.070185.002055. [DOI] [PubMed] [Google Scholar]
  9. Hampel A., Tritz R. RNA catalytic properties of the minimum (-)sTRSV sequence. Biochemistry. 1989 Jun 13;28(12):4929–4933. doi: 10.1021/bi00438a002. [DOI] [PubMed] [Google Scholar]
  10. Koizumi M., Ohtsuka E. Effects of phosphorothioate and 2-amino groups in hammerhead ribozymes on cleavage rates and Mg2+ binding. Biochemistry. 1991 May 28;30(21):5145–5150. doi: 10.1021/bi00235a005. [DOI] [PubMed] [Google Scholar]
  11. Latham J. A., Cech T. R. Defining the inside and outside of a catalytic RNA molecule. Science. 1989 Jul 21;245(4915):276–282. doi: 10.1126/science.2501870. [DOI] [PubMed] [Google Scholar]
  12. Milligan J. F., Uhlenbeck O. C. Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol. 1989;180:51–62. doi: 10.1016/0076-6879(89)80091-6. [DOI] [PubMed] [Google Scholar]
  13. Ruffner D. E., Uhlenbeck O. C. Thiophosphate interference experiments locate phosphates important for the hammerhead RNA self-cleavage reaction. Nucleic Acids Res. 1990 Oct 25;18(20):6025–6029. doi: 10.1093/nar/18.20.6025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Sarver N., Cantin E. M., Chang P. S., Zaia J. A., Ladne P. A., Stephens D. A., Rossi J. J. Ribozymes as potential anti-HIV-1 therapeutic agents. Science. 1990 Mar 9;247(4947):1222–1225. doi: 10.1126/science.2107573. [DOI] [PubMed] [Google Scholar]
  15. Slim G., Gait M. J. Configurationally defined phosphorothioate-containing oligoribonucleotides in the study of the mechanism of cleavage of hammerhead ribozymes. Nucleic Acids Res. 1991 Mar 25;19(6):1183–1188. doi: 10.1093/nar/19.6.1183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Spitzer S., Eckstein F. Inhibition of deoxyribonucleases by phosphorothioate groups in oligodeoxyribonucleotides. Nucleic Acids Res. 1988 Dec 23;16(24):11691–11704. doi: 10.1093/nar/16.24.11691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Stein C. A., Cohen J. S. Oligodeoxynucleotides as inhibitors of gene expression: a review. Cancer Res. 1988 May 15;48(10):2659–2668. [PubMed] [Google Scholar]
  18. Van Tol H., Buzayan J. M., Bruening G. Evidence for spontaneous circle formation in the replication of the satellite RNA of tobacco ringspot virus. Virology. 1991 Jan;180(1):23–30. doi: 10.1016/0042-6822(91)90005-v. [DOI] [PubMed] [Google Scholar]
  19. Waring R. B. Identification of phosphate groups important to self-splicing of the Tetrahymena rRNA intron as determined by phosphorothioate substitution. Nucleic Acids Res. 1989 Dec 25;17(24):10281–10293. doi: 10.1093/nar/17.24.10281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. van Tol H., Buzayan J. M., Feldstein P. A., Eckstein F., Bruening G. Two autolytic processing reactions of a satellite RNA proceed with inversion of configuration. Nucleic Acids Res. 1990 Apr 25;18(8):1971–1975. doi: 10.1093/nar/18.8.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES