Abstract
The attachment of reporter groups, drug derivatives, or chemically reactive species to DNA in a sequence-specific manner has the potential to provide new materials for detailed spectroscopic and structural analyses as well as new classes of DNA therapeutics and diagnostics. Sequence specific labeling of a nucleic acid sequence can be achieved by a number of procedures that employ either the nucleobase, the carbohydrate or the phosphate residue as a site for attachment (for a recent review see ref. 1). Specific functional groups (such as terminal phosphomonoesters, see Chapter 3) or the enhanced reactivity of selected sites on the purine or pyrimidine building blocks (such as the C5-position of pyrimidines, see Chapter 2) are often used in order to covalently attach an appropriate linker or the desired reporter group. The manner in which the nucleic acid is labeled may be dictated by the specific study involved, but in general the principles of simplicity and versatility are best employed to guide the choice of labeling procedure.
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References
Goodchild, J. (1990) Conjugates of oligonucleotides and modified oligonucleotides: a review of their synthesis and properties. Bioconjugate Chem. 1, 165–187.
Telser, J., Cruickshank, K. A., Morrison, L. E., and Netzel, T. L. (1989) Synthesis and characterization of DNA oligomers and duplexes containing covalently attached molecular labels: comparison of biotin, fluorescein, andpyrene labels by thermodynamic and optical spectroscopic measurements. J. Amer. Chem. Soc. 111, 6966–6976
Telser, J., Cruickshank, K A, Morrison, L. E., Netzel, T. L., and Chan, C.-K (1989) DNA duplexes covalently labeled at two sites: synthesis and characterization by steady-state and time-resolved optical spectroscopies. J. Amer. Chem. Soc. 111, 7226–7232.
MacMillian, A. M. and Verdine, G. L. (1990) Synthesis of functionally tethered oligodeoxynucleotides by the convertible nucleoside approach. J. Org. Chem. 55, 5931–5933.
Nemer, M. J and Olgilvie, K. K. (1980) Phosphoramidate analogues of diribonucleoside monophosphates. Tetrahedron Lett. 21, 4153–4154.
Froehler, B. C. (1986) Deoxynucleoside H-phosphonate diester intermediates in the synthesis of internucleotide phosphate analogues. Tetrahedron Lett. 27, 5575–5578.
Froehler, B. C, Ng, P. G., and Matteucci, M. D. (1986) Synthesis of DNA via deoxynucleoside H-phosphonate intermediates. Nucleic Acids Res. 14, 5399–5407.
Froehler, B.C., Ng, P G., and Matteucci, M D (1988) phosphoramidate analogues of DNA: synthesis and thermal stability of heteroduplexes. Nucleic Acids Res. 16, 4831–4839.
Letsinger, R. L. and Schott, M. E. (1981) Selectivity in binding a phenanthri-dinium-dinucleotide derivative to homopolynucleotides. J. Amer. Chem. Soc. 103, 7394–7398.
Yamana, K. and Letsinger, R. L. (1985) Synthesis and properties of oligonucleotides bearing a pendant pyrene group. Nucleic Acids Symp. Ser 16, 169–173.
Letsinger, R. L, Bach, S. A., and Eadie, J. S. (1986) Effects of pendant groups at phosphorus on binding properties of d-ApA analogues. Nucleic Acids. Res. 14, 3487–3497.
Jäger, A., Levy, M. J, and Hecht, S. M. (1988) Oligonucleotide N-alkylphosphoramidates: synthesis and binding to polynucleotides. Biochemistry 27, 7237–7246.
Letsinger, R. L., Zhang, G., Sun, D. K., Ikeuchi, T., and Sarin, P. S. (1989) Cholesteryl-conjugated oligonucleotides, synthesis, properties, and activity as inhibitors of replication of human immunodeficiency virus in cell culture. Proc. Natl. Acad. Sci.. USA 86, 6553–6556.
Agrawal, S. and Tang, J.-Y. (1990) Site-specific functionalization of oligonucleotides for non-radioactive labeling. Tetrahedron Lett. 31, 1543–1546.
Cosstick, R, McLaughlin, L. W., and Eckstein, F. (1984) Fluorescent labeling of tRNA and oligodeoxynucleotides using T4 RNA ligase Nucleic Acids Res 12, 1791–1810.
Fidanza, J. A and McLaughlin, L. W. (1989) Introduction of reporter groups at specific sites in DNA containing phosphorothioate diesters. J Amer. Chem. Soc. 111, 9117–9119.
Hodges, R., Conway, N. E., and McLaughlin L W (1989) “Post-assay” covalent labeling of phosphorothioate-containing nucleic acids with multiple fluorescent markers. Biochemistry 28, 261–267.
Conway, N. E., Fidanza, J. A.,and McLaughlin, L. W. (1989) The introduction of reporter group at multiple and/or specific sites in DNA containing phosphorothioate diesters. Nucleic Acids Res. Symp. Ser. 21, 43–44.
Conway, N. E., Fidanza, J. A., and McLaughlin, L. W (1990) Reaction of internucleotidic phosphorothioate diesters with reporter groups. Phosphorus and Sulfur 51, 27–30.
Agrawal, S. and Zamecmk, P C (1990) Site specific functionalization of oligonucleotides for attaching two different reporter groups. Nucleic Acids Res. 18, 5419–5423.
Connolly, B A, Potter, B. V. L., Eckstein, F, Pingoud, A., and Grotjahn, L. (1984) Synthesis and characterization of an octanucleotide containing the EcoRI recognition sequence with a phosphorothioate group at the cleavage site Biochemistry 23, 3443–3453
Stec, W. J., Zon, G., Egan, W, and Stec, B (1984) Automated solid-phase synthesis, separation, and stereochemistry of phosphorothioate analogues of oligodeoxyribonucleotides J. Amer. Chem Soc 106, 6077–6079.
Iyer, R. P., Egan, W., Regan, J, and Beaucage, S. L. (1990) 3H-l,2-benzodithiol-3-one 1,1-dioxide as an improved sulfunzing reagent in the solid-phase synthesis of oligodeoxyribonucleosidephosphorothioates. J. Amer. Chem. Soc. 112, 1253–1254.
Iyer, R. P., Phillips, L. R, Egan, W., Regan, J. B., and Beaucage, S. L. (1990) The automated synthesis of sulfur-containing ohgodeoxyribonucleotides using 3H-l,2-benzodithiol-3-one 1,1-dioxide as a sulfur-transfer reagent. J. Org. Chem. 55, 4693–4699.
Piel, N. and McLaughlin, L. W. (1984) Chromatographic purification of synthetic oligonucleotides, in Oligonucleotide Synthesis: A Practical Approach (Gait, M. J., ed.), IRL, New York, pp. 117–134
LaPlanche, L., James, T. L., Powell, C, Wilson, D. W, Uzhanski, B., Stec, W. J., Summers, M. F, and Zon, G. (1986) Phosphorothioate modified oligode-oxynucleotides III. NMR and UV spectroscopic studies of the Rp-Rp, Sp-Rp and Sp-Sp duplexes [d(GGsAATTCC)]2 derived from diastereoisomeric Oethyl phosphorothioate. Nucleic Acids Res. 14, 9081–9093.
Ott, J. and Eckstein, F. (1987) Protection of oligonucleotide primers against degradation by DNA polymerase I. Biochemistry 26, 8237–8241.
Stein, C. A., Subasinghe, C, Shinozuka, K., and Cohen, J. S. (1988) Physicochemical properties of phosphorothioate ohgodeoxy nucleotides Nucleic Acids Res. 16, 3209–3221.
Stein, C. A. and Cohen, J. S. (1988) Oligodeoxynucleotides as inhibitors of gene expression: a review. Cancer Res. 48, 2659–2668.
Stein, C. A. and Cohen, J. S. (1989) Phosphorothioate oligoxynucleotide analogues, in Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression (Cohen, J S., ed.), CRC, Boca Raton, FL, pp 97–118.
Eckstein, F. (1985)Nucleosidephosphorothioates. Ann. Rev. Biochem. 54, 367–402, and references therein.
Cosstick, R. and Eckstein, F. (1985) Synthesis of d(GC) and d(CG) octamers containing alternating phosphorothioate linkages: effect of the phosphorothioate group on the B-Z transition. Biochemistry 24, 3630–3638.
Potter, B. V. L, Romaniuk, P. J., and Eckstein, F. (1983) Stereochemical course of DNA hydrolysis by nuclease SI. J. Biol. Chem. 258, 1758–1773.
Stec, W. J., Zon, G., and Uznansky, B. (1985) Reversed-phase high-performance liquid chromatographic separation of diastereomeric phosphorothioate analogues of ohgodeoxyribonucleotides and other backbone-modified congeners of DNA. J Chromatog. 326, 263–280.
Atherton, F. R., Openshaw, H. T, and Todd, A. R. (1945) Studies on phosphorylation. Part U The reaction of dialkyl phosphites with polyhalogen compounds in presence of bases A new method for the phosphorylation of amines J. Chem. Soc 660–663.
ackburn, G. M., Cohen, J. S., and Todd, A. R (1966) Studies in phosphorylation. Part XXIX The synthesis of dialkyl phosphates from monoalkyl phosphonates: direct oxidative esterification. J. Chem. Soc. 239–245.
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Fidanza, J.A., Ozaki, H., McLaughlin, L.W. (1994). Functionalization of Oligonucleotides by the Incorporation of Thio-Specific Reporter Groups. In: Protocols for Oligonucleotide Conjugates. Methods in Molecular Biology, vol 26. Humana Press. https://doi.org/10.1007/978-1-59259-513-6_4
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DOI: https://doi.org/10.1007/978-1-59259-513-6_4
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