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. 1993 Sep 25;21(19):4444–4450. doi: 10.1093/nar/21.19.4444

The synthesis of oligoribonucleotides containing O6-methylguanosine: the role of conserved guanosine residues in hammerhead ribozyme cleavage.

J A Grasby 1, P Jonathan 1, G Butler 1, M J Gait 1
PMCID: PMC311174  PMID: 8233777

Abstract

The synthesis is described of oligoribonucleotides containing the modified nucleoside O6-methylguanosine. Solid-phase oligoribonucleotide assembly was carried out by use of 2'-silyl-protected nucleoside phosphoramidites, a new O6-methylguanosine-containing synthon and a mild deprotection method. The O6-methylguanosine-modified oligonucleotides were used in the study of the role of conserved residues G5, G8 and G12 in hammerhead ribozyme cleavage. Hammerheads thus substituted at any of these positions showed an approximately 75-fold reduction in kcat whereas Km was unaffected. Hammerheads with modifications at G5 or G8 showed a significant reduction in magnesium binding affinity whereas modification at G12 had no effect. The results show that the three conserved G residues play crucial but different role sin hammerhead cleavage.

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Selected References

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

  1. Abbott P. J., Mehta J. R., Ludlum D. B. Synthesis of 8-14C-labeled O6-methyldeoxyguanosine and its deoxynucleotide copolymers. Biochemistry. 1980 Feb 19;19(4):643–647. doi: 10.1021/bi00545a006. [DOI] [PubMed] [Google Scholar]
  2. Brown R. S., Dewan J. C., Klug A. Crystallographic and biochemical investigation of the lead(II)-catalyzed hydrolysis of yeast phenylalanine tRNA. Biochemistry. 1985 Aug 27;24(18):4785–4801. doi: 10.1021/bi00339a012. [DOI] [PubMed] [Google Scholar]
  3. Brown R. S., Hingerty B. E., Dewan J. C., Klug A. Pb(II)-catalysed cleavage of the sugar-phosphate backbone of yeast tRNAPhe--implications for lead toxicity and self-splicing RNA. Nature. 1983 Jun 9;303(5917):543–546. doi: 10.1038/303543a0. [DOI] [PubMed] [Google Scholar]
  4. DEVOE H., TINOCO I., Jr The stability of helical polynucleotides: base contributions. J Mol Biol. 1962 Jun;4:500–517. doi: 10.1016/s0022-2836(62)80105-3. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. 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]
  8. Fersht A. R. Relationships between apparent binding energies measured in site-directed mutagenesis experiments and energetics of binding and catalysis. Biochemistry. 1988 Mar 8;27(5):1577–1580. doi: 10.1021/bi00405a027. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Fu D. J., McLaughlin L. W. Importance of specific adenosine N7-nitrogens for efficient cleavage by a hammerhead ribozyme. A model for magnesium binding. Biochemistry. 1992 Nov 17;31(45):10941–10949. doi: 10.1021/bi00160a001. [DOI] [PubMed] [Google Scholar]
  11. Fu D. J., McLaughlin L. W. Importance of specific purine amino and hydroxyl groups for efficient cleavage by a hammerhead ribozyme. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3985–3989. doi: 10.1073/pnas.89.9.3985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gaffney B. L., Marky L. A., Jones R. A. Synthesis and characterization of a set of four dodecadeoxyribonucleoside undecaphosphates containing O6-methylguanine opposite adenine, cytosine, guanine, and thymine. Biochemistry. 1984 Nov 20;23(24):5686–5691. doi: 10.1021/bi00319a004. [DOI] [PubMed] [Google Scholar]
  13. Gerchman L. L., Dombrowski J., Ludlum D. B. Synthesis and polymerization of O 6 -methylguanosine 5'-diphosphate. Biochim Biophys Acta. 1972 Jul 31;272(4):672–675. doi: 10.1016/0005-2787(72)90527-8. [DOI] [PubMed] [Google Scholar]
  14. Ginell S. L., Kuzmich S., Jones R. A., Berman H. M. Crystal and molecular structure of a DNA duplex containing the carcinogenic lesion O6-methylguanine. Biochemistry. 1990 Nov 20;29(46):10461–10465. doi: 10.1021/bi00498a005. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Leonard G. A., Thomson J., Watson W. P., Brown T. High-resolution structure of a mutagenic lesion in DNA. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9573–9576. doi: 10.1073/pnas.87.24.9573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Newman P. C., Williams D. M., Cosstick R., Seela F., Connolly B. A. Interaction of the EcoRV restriction endonuclease with the deoxyadenosine and thymidine bases in its recognition hexamer d(GATATC). Biochemistry. 1990 Oct 23;29(42):9902–9910. doi: 10.1021/bi00494a021. [DOI] [PubMed] [Google Scholar]
  19. Odai O., Hiroaki H., Sakata T., Tanaka T., Uesugi S. The role of a conserved guanosine residue in the hammerhead-type RNA enzyme. FEBS Lett. 1990 Jul 2;267(1):150–152. doi: 10.1016/0014-5793(90)80311-6. [DOI] [PubMed] [Google Scholar]
  20. Olsen D. B., Benseler F., Aurup H., Pieken W. A., Eckstein F. Study of a hammerhead ribozyme containing 2'-modified adenosine residues. Biochemistry. 1991 Oct 8;30(40):9735–9741. doi: 10.1021/bi00104a024. [DOI] [PubMed] [Google Scholar]
  21. Parthasarathy R., Fridey S. M. Conformation of O6-alkylguanosines: molecular mechanism of mutagenesis. Carcinogenesis. 1986 Feb;7(2):221–227. doi: 10.1093/carcin/7.2.221. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Perreault J. P., Wu T. F., Cousineau B., Ogilvie K. K., Cedergren R. Mixed deoxyribo- and ribo-oligonucleotides with catalytic activity. Nature. 1990 Apr 5;344(6266):565–567. doi: 10.1038/344565a0. [DOI] [PubMed] [Google Scholar]
  24. Pieken W. A., Olsen D. B., Benseler F., Aurup H., Eckstein F. Kinetic characterization of ribonuclease-resistant 2'-modified hammerhead ribozymes. Science. 1991 Jul 19;253(5017):314–317. doi: 10.1126/science.1857967. [DOI] [PubMed] [Google Scholar]
  25. Pullman B., Claverie P., Caillet J. Van der Waals-London interactions and the configuration of hydrogen-bonded purine and pyrimidine pairs. Proc Natl Acad Sci U S A. 1966 Apr;55(4):904–912. doi: 10.1073/pnas.55.4.904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Slim G., Gait M. J. The role of the exocyclic amino groups of conserved purines in hammerhead ribozyme cleavage. Biochem Biophys Res Commun. 1992 Mar 16;183(2):605–609. doi: 10.1016/0006-291x(92)90525-p. [DOI] [PubMed] [Google Scholar]
  28. Smith C. A., Xu Y. Z., Swann P. F. Solid-phase synthesis of oligodeoxynucleotides containing O6-alkylguanine. Carcinogenesis. 1990 May;11(5):811–816. doi: 10.1093/carcin/11.5.811. [DOI] [PubMed] [Google Scholar]
  29. Smith J. M., Thomas D. J. Quantitative analysis of one-dimensional gel electrophoresis profiles. Comput Appl Biosci. 1990 Apr;6(2):93–99. doi: 10.1093/bioinformatics/6.2.93. [DOI] [PubMed] [Google Scholar]
  30. Sriram M., van der Marel G. A., Roelen H. L., van Boom J. H., Wang A. H. Conformation of B-DNA containing O6-ethyl-G-C base pairs stabilized by minor groove binding drugs: molecular structure of d(CGC[e6G]AATTCGCG complexed with Hoechst 33258 or Hoechst 33342. EMBO J. 1992 Jan;11(1):225–232. doi: 10.1002/j.1460-2075.1992.tb05045.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Symons R. H. Small catalytic RNAs. Annu Rev Biochem. 1992;61:641–671. doi: 10.1146/annurev.bi.61.070192.003233. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Uhlenbeck O. C. A small catalytic oligoribonucleotide. Nature. 1987 Aug 13;328(6131):596–600. doi: 10.1038/328596a0. [DOI] [PubMed] [Google Scholar]
  34. Williams D. M., Pieken W. A., Eckstein F. Function of specific 2'-hydroxyl groups of guanosines in a hammerhead ribozyme probed by 2' modifications. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):918–921. doi: 10.1073/pnas.89.3.918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]

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