Abstract
Oligodeoxynucleotides containing 3′-S-phosphorothiolate (3′-PS) linkages have become useful tools for probing enzyme-catalyzed cleavage processes in DNA. This protocol describes the synthesis of the phosphorothioamidite monomers derived from thymidine and 2′-deoxycytidine, and their application to a fully automated procedure for synthesising oligodeoxynucleotides containing 3′-PS linkages. The synthesis of the 5′-protected-3′-amidites is achievable in 2 weeks with the DNA synthesis and purification taking another 1 week.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Vyle, J.S., Connolly, B.A., Kemp, D. & Cosstick, R. Sequence-specific and strand-specific cleavage in oligodeoxyribonucleotides and DNA containing 3′-thiothymidine. Biochemistry 31, 3012–3018 (1992).
Piccirilli, J.A., Vyle, J.S., Caruthers, M.H. & Cech, T.R. Metal-ion catalysis in the Tetrahymena ribozyme reaction. Nature 361, 85–88 (1993).
Szczelkun, M.D. & Connolly, B.A. Sequence-specific binding of DNA by the EcoRV restriction and modification enzymes with nucleic-acid and cofactor analogues. Biochemistry 34, 10724–10733 (1995).
Horton, N.C., Connolly, B.A. & Perona, J.J. Inhibition of EcoRV endonuclease by deoxyribo-3′-S-phosphorothiolates: a high-resolution X-ray crystallographic study. J. Am. Chem. Soc. 122, 3314–3324 (2000).
Liu, X.H. & Reese, C.B. 3′-Thiouridylyl-(3′-5′)-uridine. Tetrahedron Lett. 37, 925–928 (1996).
Weinstein, L.B., Earnshaw, D.J., Cosstick, R. & Cech, T.R. Synthesis and characterization of an RNA dinucleotide containing a 3′-S-phosphorothiolate linkage. J. Am. Chem. Soc. 118, 10341–10350 (1996).
Sun, S.G., Yoshida, A. & Piccirilli, J.A. Synthesis of 3′-thioribonucleosides and their incorporation into oligoribonucleotides via phosphoramidite chemistry. RNA 3, 1352–1363 (1997).
Sontheimer, E.J. Bridging sulfur substitutions in the analysis of pre-mRNA splicing. Methods 18, 29–37 (1999).
Curley, J.F., Joyce, C.M. & Piccirilli, J.A. Functional evidence that the 3′-5′ exonuclease domain of Escherichia coli DNA polymerase I employs a divalent metal ion in leaving group stabilization. J. Am. Chem. Soc. 119, 12691–12692 (1997).
Brautigam, C.A., Sun, S., Piccirilli, J.A. & Steitz, T.A. Structures of normal single-stranded DNA and deoxyribo-3′-S-phosphorothiolates bound to the 3′-5′ exonucleolytic active site of DNA polymerase I from Escherichia coli. Biochemistry 38, 696–704 (1999).
Shah, R., Cosstick, R. & West, S.C. The RuvC protein dimer resolves Holliday junctions by a dual incision mechanism that involves base-specific contacts. EMBO J. 16, 1464–1472 (1997).
Elliott, S.L., Brazier, J., Cosstick, R. & Connolly, B.A. Mechanism of the Escherichia coli DNA T: G-mismatch endonuclease (Vsr protein) thiophosphate-containing probed with oligodeoxynucleotides. J. Mol. Biol. 353, 692–703 (2005).
Beevers, A.P. et al. Conformational analysis of 3′-S-PO3-linked ribo- and deoxyribodinucleoside monophosphates. Magn. Reson. Chem. 37, 814–820 (1999).
Beevers, A.P. et al. Probing the effect of a 3′-S-phosphorothiolate link on the conformation of a DNA: RNA hybrid; implications for antisense drug design. Chem. Commun. (Camb.), 1458–1459 (2002).
Brazier, J.A., Fisher, J. & Cosstick, R. Stabilization of the DNA I-motif structure by incorporation of 3′-S-phosphorothiolate linkages. Angew. Chem. Int. Ed. Engl. 45, 114–117 (2006).
Vyle, J.S., Li, X. & Cosstick, R. New methods for the synthesis of 3′-S-phosphorothiolate internucleoside linkages. Tetrahedron Lett. 33, 3017–3020 (1992).
Li, X. et al. Application of the Michaelis-Arbuzov reaction to the synthesis of internucleoside 3′-S-phosphorothiolate linkages. J. Chem. Soc., Perkin Trans. 1, 2123–2129 (1994).
Cosstick, R. & Vyle, J.S. Solid-phase synthesis of oligonucleotides containing 3′-thiothymidine. Tetrahedron Lett. 30, 4693–4696 (1989).
Cosstick, R. & Vyle, J.S. Synthesis and properties of dithymidine phosphate analogs containing 3′-thiothymidine. Nucleic Acids Res. 18, 829–835 (1990).
Sun, S.G. & Piccirilli, J.A. Synthesis of 3′-thioribouridine, 3′-thioribocytidine, and their phosphoramidites. Nucleosides Nucleotides 16, 1543–1545 (1997).
Sabbagh, G., Fettes, K.J., Gosain, R., O'Neil, I.A. & Cosstick, R. Synthesis of phosphorothioamidites derived from 3′-thio-3′- deoxythymidine and 3′-thio-2′,3′-dideoxycytidine and the automated synthesis of oligodeoxynucleotides containing a 3′- S-phosphorothiolate linkage. Nucleic Acids Res. 32, 495–501 (2004).
Li, X., Andrews, D.M. & Cosstick, R. Synthesis of a dinucleoside 3′-S-phosphorothiolate containing 2′-deoxy-3′-thioadenosine. Tetrahedron 48, 2729–2738 (1992).
Meena, Sam, M., Pierce, K., Szostak, J.W. & McLaughlin, L.W. 2′,3′-Dideoxy-3′-thionucleoside triphosphates: syntheses and polymerase substrate activities. Org. Lett. 9, 1161–1163 (2007).
Bdour, H.M., Kao, J.L.F. & Taylor, J.S. Synthesis and characterization of a [3-N-15]-labeled cis-syn thymine dimer-containing DNA duplex. J. Org. Chem. 71, 1640–1646 (2006).
Narang, S.A. DNA synthesis. Tetrahedron 39, 3–22 (1983).
Schaller, H., Weimann, G., Khorana, H.G. & Lerch, B. Studies on polynucleotides .24. Stepwise synthesis of specific deoxyribopolynucleotides (4)—protected derivatives of deoxyribonucleosides and new syntheses of deoxyribonucleoside-3-phosphates. J. Am. Chem. Soc. 85, 3821–3827 (1963).
Sonveaux, E. The organic chemistry underlying DNA synthesis. Bioorg. Chem. 14, 274–325 (1986).
Radi, M., Spinosa, R., Parlato, M.C., Corelli, F. & Botta, M. Exploring the synthesis of masked phosphoramido 6-vinylcytidine derivatives as building blocks for cross-linking oligonucleotides. Heterocycles 69, 151–166 (2006).
Ti, G.S., Gaffney, B.L. & Jones, R.A. Transient protection—efficient one-flask syntheses of protected deoxynucleosides. J. Am. Chem. Soc. 104, 1316–1319 (1982).
Yang, J.W., Stadler, M. & List, B. Practical Proline-catalyzed asymmetric Mannich reaction of aldehydes with N-Boc-imines. Nat. Protoc. 2, 1937–1942 (2007).
Bentley, J., Brazier, J.A., Fisher, J. & Cosstick, R. Duplex stability of DNA.DNA and DNA.RNA duplexes containing 3′-S-phosphorothiolate linkages. Org. Biomol. Chem. 5, 3698–3702 (2007).
Acknowledgements
The authors thank Alan Mills for obtaining mass spectra.
Author information
Authors and Affiliations
Corresponding authors
Rights and permissions
About this article
Cite this article
Gaynor, J., Bentley, J. & Cosstick, R. Synthesis of the 3′-thio-nucleosides and subsequent automated synthesis of oligodeoxynucleotides containing a 3′-S-phosphorothiolate linkage. Nat Protoc 2, 3122–3135 (2007). https://doi.org/10.1038/nprot.2007.451
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2007.451
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.