Abstract
Influence of stereochemistry of the 3′-terminal phosphorothioate (PS)-modified primers was studied in a single base extension (SBE) assay to evaluate any improvements in specificity. SBE reactions were catalyzed by members of the high fidelity Pfu family of DNA polymerases with (exo+) or without (exo−) 3′ → 5′ exonucleolytic activity. The diastereomerically pure PS-labeled primers used in these studies were obtained either by the stereospecific chemical synthesis invented in our laboratory or by the more conventional ion-exchange chromatographic method for separation of a mixture of diastereomers (RP and SP). When the SBE reaction was performed in the presence of mispaired 2′-deoxyribonucleoside triphosphates (dNTPs), the “racemic” 3′-phosphorothioate primer mixture resulted in a lower level of 3′ → 5′ exonuclease-mediated cleavage products in comparison to the SBE reactions carried out with the corresponding unmodified primer. When the diastereomerically pure RP 3′-phosphorothioate primer was examined, the results were largely the same as for the racemic 3′-phosphorothioate primer mixture. In contrast, a 3′-PS primer of SP configuration displayed significantly improved performance in the SBE reaction. This included the lack of 3′ → 5′ proofreading products, less mispriming, and improved yield of incorporation of the correct nucleotide.
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References
Leeds, J. M., Henry, S. P., Truong, L., Zutshi, A., Levin, A. A., & Kornbrust, D. (1997). Pharmacokinetics of a potential human cytomegalovirus therapeutic, a phosphorothioate oligonucleotide, after intravitreal injection in the rabbit. Drug Metabolism and Disposition, 25, 921–926.
Shimayama, T., Nishikawa, F., Nishikawa, S., & Taira, K. (1993). Nuclease-resistant chimeric ribozymes containing deoxyribonucleotides and phosphorothioate linkages. Nucleic Acids Research, 21, 2605–2611.
Cieslak, M., Niewiarowska, J., Nawrot, M., Koziolkiewicz, M., Stec, W. J., & Cierniewski, C. S. (2002). DNAzymes to beta 1 and beta 3 mRNA down-regulate expression of the targeted integrins and inhibit endothelial cell capillary tube formation in fibrin and matrigel. Journal of Biological Chemistry, 277, 6779–6787.
Nawrot, B., & Sipa, K. (2006). Chemical and structural diversity of siRNA molecules. Current Topics in Medicinal Chemistry, 6, 913–925.
Stec, W. J., Zon, G., & Uznanski, B. J. (1985). Reversed-phase high-performance liquid chromatographic separation of diastereomeric phosphorothioate analogues of oligodeoxyribonucleotides and other back-bone-modified congeners of DNA. Journal of Chromatography, 326, 263–280.
Stec, W. J., & Wilk, A. (1994). Stereocontrolled synthesis of oligo(nucleoside phosphorothioate)s. Angewandte Chemie-International Edition in English, 33, 709–722.
Wada, T. (2005). Stereocontrolled synthesis of phosphorothioate DNA by the oxazaphospholidine approach. Frontiers in Organic Chemistry, 1, 41–61.
Burgers, P. M., & Eckstein, F. (1979). A study of the mechanism of DNA polymerase I from Escherichia coli with diastereomeric phosphorothioate analogs of deoxyadenosine triphosphate. Journal of Biological Chemistry, 254, 6889–6893.
Lackey, D. B., & Patel, J. (1997). Biochemical synthesis of chirally pure RP oligonucleotide phosphorothioates. Biotechnological Letters, 19, 475–478.
Matamoros, T., Deval, J., Guerreiro, C., Mulard, L., Canard, B., & Menéndez-Arias, L. (2005). Suppression of multidrug-resistant HIV-1 reverse transcriptase primer unblocking activity by alpha-phosphate-modified thymidine analogues. Journal of Molecular Biology, 349, 451–463.
Wang, L., Chen, S., Xu, T., Taghizadeh, K., Wishnok, J. S., Zhou, X., et al. (2007). Phosphorothioation of DNA in bacteria by dnd genes. Nature Chemical Biology, 3, 709–710.
Potter, B. V., & Eckstein, F. (1984). Cleavage of phosphorothioate-substituted DNA by restriction endonucleases. Journal of Biological Chemistry, 259, 14243–14248.
Brautigam, C., & Steitz, T. A. (1998). Structural principles for the inhibition of the 3′ → 5′ exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates. Journal of Molecular Biology, 277, 363–377.
Burgers, P. M., & Eckstein, F. (1979). Diastereomers of 5′-O-adenosyl 3′-O-uridyl phosphorothioate: Chemical synthesis and enzymatic properties. Biochemistry, 18, 592–596.
Koziołkiewicz, M., Owczarek, A., & Gendaszewska, E. (1999). Enzymatic assignment of diastereomeric purity of stereodefined phosphorothioate oligonucleotides. Antisense Nucleic Acid Drug Development, 9, 171–181.
Kennedy, A. K., Haniford, D. B., & Mizuuchi, K. (2000). Single active site catalysis of the successive phosphoryl transfer steps by DNA transposases: insights from phosphorothioate stereoselectivity. Cell, 101, 295–305.
Potter, B. V., Connolly, B. A., & Eckstein, F. (1983). Synthesis and configurational analysis of a dinucleoside phosphate isotopically chiral at phosphorus. Stereochemical course of Penicillium citrum nuclease P1 reaction. Biochemistry, 22, 1369–1377.
Koziołkiewicz, M., Wójcik, M., Kobylańska, A., Karwowski, B., Rębowska, B., Guga, P., et al. (1997). Stability of stereoregular oligo(nucleoside phosphorothioate)s in human plasma: Diastereoselectivity of plasma 3′-exonuclease. Antisense Nucleic Acid Drug Development, 7, 43–48.
Gupta, A., DeBrosse, C., & Benkovic, S. J. (1982). Template-prime-dependent turnover of (Sp)-dATP alpha S by T4 DNA polymerase. The stereochemistry of the associated 3′ goes to 5′-exonuclease. Journal of Biological Chemistry, 257, 7689–7692.
Skerra, A. (1992). Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity. Nucleic Acids Research, 20, 3551–3554.
Zhang, J., Chen, L. L., Guo, Z. F., Peng, C. Y., Liao, D. F., & Li, K. (2003). On/off switch mediated by exo+ polymerases: Experimental analysis for its physiological and technological implications. Journal of Biochemistry and Molecular Biology, 36, 529–532.
Zhang, J., & Li, K. (2003). Single-base discrimination mediated by proofreading 3′ phosphorothioate-modified primers. Molecular Biotechnology, 25, 223–228.
Nawrot, B., Rebowska, B., Cieślińska, K., & Stec, W. J. (2005). New approach to the synthesis of oligodeoxyribonucleotides modified with phosphorothioates of predetermined sense of P-chirality. Tetrahedron Letters, 46, 6641–6644.
Brown, T., Pritchard, C. E., Turner, G., & Salisbury, S. A. (1989). A new base-stable linker for solid-phase oligonucleotide synthesis. Journal of the Chemical Society-Chemical Communications, 14, 891–893.
Stec, W. J., Grajkowski, A., Koziolkiewicz, M., & Uznański, B. (1991). Novel route to oligo(deoxyribonucleoside phosphorothioates). Stereocontrolled synthesis of P-chiral oligo(deoxyribonucleoside phosphorothioates). Nucleic Acids Research, 19, 5883–5888.
Uznański, B., Grajkowski, A., & Wilk, A. (1989). The isopropoxyacetic group for convenient base protection during solid-support synthesis of oligodeoxyribonucleotides and their triester analogs. Nucleic Acids Research, 17, 4863–4871.
Caruthers, M. H. (1985). Gene synthesis machines: DNA chemistry and its uses. Science, 230, 281–285.
Azhayev, A. V. (1999). A new universal solid support for oligonucleotide synthesis. Tetrahedron, 55, 787–800.
Iyer, R. P., Phillips, L. R., Egan, W., Regan, J. B., & Beaucage, S. L. (1990). The automated synthesis of sulfur-containing oligodeoxyribonucleotides using 3H–1, 2-benzodithiol-3-one 1, 1-dioxide as a sulfur-transfer reagent. Journal of Organic Chemistry, 55, 4693–4699.
Di Giusto, D., & King, G. C. (2003). Single base extension (SBE) with proofreading polymerases and phosphorothioate primers: Improved fidelity in single-substrate assays. Nucleic Acids Research, 31, e7.
Braithwaite, D. K., & Ito, J. (1993). Compilation, alignment, and phylogenetic relationships of DNA polymerases. Nucleic Acids Research, 21, 787–802.
Stivers, J. T., Nawrot, B., Jagadeesh, G. J., Stec, W. J., & Shuman, S. (2000). Stereochemical outcome and kinetic effects of Rp- and Sp-phosphorothioate substitutions at the cleavage site of vaccinia type I DNA topoisomerase. Biochemistry, 39, 5561–5572.
Eckstein, F. (2000). Phosphorothioate oligodeoxynucleotides: What is their origin and what is unique about them? Antisense Nucleic Acid Drug Development, 10, 117–121.
Guga, P. (2007). P-chiral oligonucleotides in biological recognition processes. Current Topics in Medicinal Chemistry, 7, 695–713.
Tsuchihashi, Z., & Dracopoli, N. C. (2002). Progress in high throughput SNP genotyping methods. The Pharmacogenomics Journal, 2, 103–110.
Syvänen, A. (2005). Toward genome-wide SNP genotyping. Nature Genetics, 37, S5–S10.
Acknowledgments
This work was supported by the Ministry of Science and Higher Education through the Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, under Decision 70/E-63/SN-014/2006 and PBZ-MNiSW-07/I/2007 for years 2008-2010. The authors are grateful to Richard Hogrefe, Gerald Zon, and Alexandre Lebedev for advice and helpful discussions.
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Nawrot, B., Paul, N., Rebowska, B. et al. Significance of Stereochemistry of 3′-Terminal Phosphorothioate-modified Primer in DNA Polymerase-mediated Chain Extension. Mol Biotechnol 40, 119–126 (2008). https://doi.org/10.1007/s12033-008-9096-x
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DOI: https://doi.org/10.1007/s12033-008-9096-x