Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1998 Jul 1;26(13):3159–3164. doi: 10.1093/nar/26.13.3159

Chemical and enzymatic properties of bridging 5'-S-phosphorothioester linkages in DNA.

Y Xu 1, E T Kool 1
PMCID: PMC147688  PMID: 9628913

Abstract

We describe physicochemical and enzymatic properties of 5' bridging phosphorothioester linkages at specific sites in DNA oligonucleotides. The susceptibility to hydrolysis at various pH values is examined and no measurable hydrolysis is observed at pH 5-9 after 4 days at 25 degrees C. The abilities of three 3'- and 5'-exonuclease enzymes to hydrolyze the DNA past this linkage are examined and it is found that the linkage causes significant pauses at the sulfur linkage for T4 DNA polymerase and calf spleen phosphodiesterase, but not for snake venom phosphodiesterase. Restriction endonuclease (Nsi I) cleavage is also attempted at a 5'-thioester junction and strong resistance to cleavage is observed. Also tested is the ability of polymerase enzymes to utilize templates containing single 5'-S-thioester linkages; both Klenow DNA polymerase and T7 RNA polymerase are found to synthesize complementary strands successfully without any apparent pause at the sulfur linkage. Finally, the thermal stabilities of duplexes containing such linkages are measured; results show that T m values are lowered by a small amount (2 degrees C) when one or two thioester linkages are present in an otherwise unmodified duplex. The chemical stability and surprisingly small perturbation by the 5' bridging sulfur make it a good candidate as a physical and mechanistic probe for specific protein or metal interactions involving this position in DNA.

Full Text

The Full Text of this article is available as a PDF (548.1 KB).

Selected References

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

  1. Beaudry A. A., Joyce G. F. Directed evolution of an RNA enzyme. Science. 1992 Jul 31;257(5070):635–641. doi: 10.1126/science.1496376. [DOI] [PubMed] [Google Scholar]
  2. Carmi N., Balkhi S. R., Breaker R. R. Cleaving DNA with DNA. Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2233–2237. doi: 10.1073/pnas.95.5.2233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chladek S., Nagyvary J. Nucleophilic reactions of some nucleoside phosphorothioates. J Am Chem Soc. 1972 Mar 22;94(6):2079–2085. doi: 10.1021/ja00761a047. [DOI] [PubMed] [Google Scholar]
  4. Cosstick R., Vyle J. S. Synthesis and properties of dithymidine phosphate analogues containing 3'-thiothymidine. Nucleic Acids Res. 1990 Feb 25;18(4):829–835. doi: 10.1093/nar/18.4.829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Eckstein F., Thomson J. B. Phosphate analogs for study of DNA polymerases. Methods Enzymol. 1995;262:189–202. doi: 10.1016/0076-6879(95)62018-4. [DOI] [PubMed] [Google Scholar]
  6. HILMOE R. J. Purification and properties of spleen phosphodiesterase. J Biol Chem. 1960 Jul;235:2117–2121. [PubMed] [Google Scholar]
  7. Heidenreich O., Pieken W., Eckstein F. Chemically modified RNA: approaches and applications. FASEB J. 1993 Jan;7(1):90–96. doi: 10.1096/fasebj.7.1.7678566. [DOI] [PubMed] [Google Scholar]
  8. Ho N. W., Gilham P. T. The analysis of polydeoxyribonucleotides by digestion with phosphatase and phosphodiesterases. Biochim Biophys Acta. 1973 Apr 21;308(7):53–58. doi: 10.1016/0005-2787(73)90121-4. [DOI] [PubMed] [Google Scholar]
  9. Huang W. M., Lehman I. R. On the exonuclease activity of phage T4 deoxyribonucleic acid polymerase. J Biol Chem. 1972 May 25;247(10):3139–3146. [PubMed] [Google Scholar]
  10. Knight W. B., Sem D. S., Smith K., Miziorko H. M., Rendina A. R., Cleland W. W. Phosphorylated thiosugars: synthesis, properties, and reactivity in enzymatic reactions. Biochemistry. 1991 May 21;30(20):4970–4977. doi: 10.1021/bi00234a019. [DOI] [PubMed] [Google Scholar]
  11. Komiyama M. Sequence-specific and hydrolytic scission of DNA and RNA by lanthanide complex-oligoDNA hybrids. J Biochem. 1995 Oct;118(4):665–670. doi: 10.1093/oxfordjournals.jbchem.a124961. [DOI] [PubMed] [Google Scholar]
  12. Kuimelis R. G., McLaughlin L. W. Cleavage properties of an oligonucleotide containing a bridged internucleotide 5'-phosphorothioate RNA linkage. Nucleic Acids Res. 1995 Dec 11;23(23):4753–4760. doi: 10.1093/nar/23.23.4753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kuimelis R. G., McLaughlin L. W. Ribozyme-mediated cleavage of a substrate analogue containing an internucleotide-bridging 5'-phosphorothioate: evidence for the single-metal model. Biochemistry. 1996 Apr 23;35(16):5308–5317. doi: 10.1021/bi952994p. [DOI] [PubMed] [Google Scholar]
  14. Mag M., Lüking S., Engels J. W. Synthesis and selective cleavage of an oligodeoxynucleotide containing a bridged internucleotide 5'-phosphorothioate linkage. Nucleic Acids Res. 1991 Apr 11;19(7):1437–1441. doi: 10.1093/nar/19.7.1437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Olsen D. B., Kotzorek G., Sayers J. R., Eckstein F. Inhibition of the restriction endonuclease BanII using modified DNA substrates. Determination of phosphate residues critical for the formation of an active enzyme-DNA complex. J Biol Chem. 1990 Aug 25;265(24):14389–14394. [PubMed] [Google Scholar]
  16. Rybakov V. N., Rivkin M. I., Kumarev V. P. Some substrate properties of analogues of oligothymidylates with p-s-C5' bonds. Nucleic Acids Res. 1981 Jan 10;9(1):189–201. doi: 10.1093/nar/9.1.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Suh E., Waring R. B. A phosphorothioate at the 3' splice-site inhibits the second splicing step in a group I intron. Nucleic Acids Res. 1992 Dec 11;20(23):6303–6309. doi: 10.1093/nar/20.23.6303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tsang J., Joyce G. F. Evolutionary optimization of the catalytic properties of a DNA-cleaving ribozyme. Biochemistry. 1994 May 17;33(19):5966–5973. doi: 10.1021/bi00185a038. [DOI] [PubMed] [Google Scholar]
  20. Vyle J. S., Connolly B. A., Kemp D., Cosstick R. Sequence- and strand-specific cleavage in oligodeoxyribonucleotides and DNA containing 3'-thiothymidine. Biochemistry. 1992 Mar 24;31(11):3012–3018. doi: 10.1021/bi00126a024. [DOI] [PubMed] [Google Scholar]
  21. 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