Abstract
The chemical synthesis is described of oligoribonucleotides containing a single phosphorothioate linkage of defined Rp and Sp configuration. The oligoribonucleotides were used as substrates in the study of the mechanism of cleavage of an RNA hammerhead domain having the phosphorothioate group at the cleavage site. Whereas the Rp isomer was cleaved only very slowly in the presence of magnesium ion, the rate of cleavage of the Sp isomer was only slightly reduced from that of the unmodified phosphodiester. This finding gives further evidence for the hypothesis that the magnesium ion is bound to the pro-R oxygen in the transition state of the hammerhead cleavage reaction. Also, inversion of configuration at phosphorus is confirmed for a two-stranded hammerhead.
Full text
PDF





Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Burgers P. M., Eckstein F. Absolute configuration of the diastereomers of adenosine 5'-O-(1-thiotriphosphate): consequences for the stereochemistry of polymerization by DNA-dependent RNA polymerase from Escherichia coli. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4798–4800. doi: 10.1073/pnas.75.10.4798. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Cathala G., Brunel C. Use of n-butanol for efficient recovery of minute amounts of small RNA fragments and branched nucleotides from dilute solutions. Nucleic Acids Res. 1990 Jan 11;18(1):201–201. doi: 10.1093/nar/18.1.201. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eckstein F. Nucleoside phosphorothioates. Annu Rev Biochem. 1985;54:367–402. doi: 10.1146/annurev.bi.54.070185.002055. [DOI] [PubMed] [Google Scholar]
- Fedor M. J., Uhlenbeck O. C. Substrate sequence effects on "hammerhead" RNA catalytic efficiency. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1668–1672. doi: 10.1073/pnas.87.5.1668. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Forster A. C., Jeffries A. C., Sheldon C. C., Symons R. H. Structural and ionic requirements for self-cleavage of virusoid RNAs and trans self-cleavage of viroid RNA. Cold Spring Harb Symp Quant Biol. 1987;52:249–259. doi: 10.1101/sqb.1987.052.01.030. [DOI] [PubMed] [Google Scholar]
- Forster A. C., Symons R. H. Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites. Cell. 1987 Apr 24;49(2):211–220. doi: 10.1016/0092-8674(87)90562-9. [DOI] [PubMed] [Google Scholar]
- Green M. R. Mobile RNA catalysts. Nature. 1988 Dec 22;336(6201):716–718. doi: 10.1038/336716a0. [DOI] [PubMed] [Google Scholar]
- Heus H. A., Pardi A. Nuclear magnetic resonance studies of the hammerhead ribozyme domain. Secondary structure formation and magnesium ion dependence. J Mol Biol. 1991 Jan 5;217(1):113–124. doi: 10.1016/0022-2836(91)90615-d. [DOI] [PubMed] [Google Scholar]
- Jeffries A. C., Symons R. H. A catalytic 13-mer ribozyme. Nucleic Acids Res. 1989 Feb 25;17(4):1371–1377. doi: 10.1093/nar/17.4.1371. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Koizumi M., Hayase Y., Iwai S., Kamiya H., Inoue H., Ohtsuka E. Design of RNA enzymes distinguishing a single base mutation in RNA. Nucleic Acids Res. 1989 Sep 12;17(17):7059–7071. doi: 10.1093/nar/17.17.7059. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Koizumi M., Iwai S., Ohtsuka E. Cleavage of specific sites of RNA by designed ribozymes. FEBS Lett. 1988 Nov 7;239(2):285–288. doi: 10.1016/0014-5793(88)80935-9. [DOI] [PubMed] [Google Scholar]
- Koizumi M., Iwai S., Ohtsuka E. Construction of a series of several self-cleaving RNA duplexes using synthetic 21-mers. FEBS Lett. 1988 Feb 15;228(2):228–230. doi: 10.1016/0014-5793(88)80004-8. [DOI] [PubMed] [Google Scholar]
- Kruger K., Grabowski P. J., Zaug A. J., Sands J., Gottschling D. E., Cech T. R. Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena. Cell. 1982 Nov;31(1):147–157. doi: 10.1016/0092-8674(82)90414-7. [DOI] [PubMed] [Google Scholar]
- Mei H. Y., Kaaret T. W., Bruice T. C. A computational approach to the mechanism of self-cleavage of hammerhead RNA. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9727–9731. doi: 10.1073/pnas.86.24.9727. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Potter B. V., Connolly B. A., Eckstein F. Synthesis and configurational analysis of a dinucleoside phosphate isotopically chiral at phosphorus. Stereochemical course of Penicillium citrum nuclease P1 reaction. Biochemistry. 1983 Mar 15;22(6):1369–1377. doi: 10.1021/bi00275a008. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Sheldon C. C., Symons R. H. Mutagenesis analysis of a self-cleaving RNA. Nucleic Acids Res. 1989 Jul 25;17(14):5679–5685. doi: 10.1093/nar/17.14.5679. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Taira K., Uebayasi M., Maeda H., Furukawa K. Energetics of RNA cleavage: implications for the mechanism of action of ribozymes. Protein Eng. 1990 Aug;3(8):691–701. doi: 10.1093/protein/3.8.691. [DOI] [PubMed] [Google Scholar]
- Tuerk C., Gauss P., Thermes C., Groebe D. R., Gayle M., Guild N., Stormo G., d'Aubenton-Carafa Y., Uhlenbeck O. C., Tinoco I., Jr CUUCGG hairpins: extraordinarily stable RNA secondary structures associated with various biochemical processes. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1364–1368. doi: 10.1073/pnas.85.5.1364. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Uhlenbeck O. C. A small catalytic oligoribonucleotide. Nature. 1987 Aug 13;328(6131):596–600. doi: 10.1038/328596a0. [DOI] [PubMed] [Google Scholar]
- Usher D. A., Erenrich E. S., Eckstein F. Geometry of the first step in the action of ribonuclease-A (in-line geometry-uridine2',3'-cyclic thiophosphate- 31 P NMR). Proc Natl Acad Sci U S A. 1972 Jan;69(1):115–118. doi: 10.1073/pnas.69.1.115. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]