Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1991 Jun 11;19(11):3019–3025. doi: 10.1093/nar/19.11.3019

A general method for the synthesis of 3'-sulfhydryl and phosphate group containing oligonucleotides.

K C Gupta 1, P Sharma 1, P Kumar 1, S Sathyanarayana 1
PMCID: PMC328265  PMID: 2057359

Abstract

The syntheses are described of polymer supports useful for the synthesis of 3'-partially protected sulfhydryl, free sulfhydryl or phosphate group containing oligonucleotides. The supports are compatible with established phosphoramidite chemistry of oligonucleotide synthesis giving rise to oligonucleotides with terminal 3'-partially protected sulfhydryl, free sulfhydryl or phosphate function during final deprotection. Crosslinking of the thiol group containing oligonucleotide to sulfhydryl group specific fluorescent probes was carried out with high selectivity, in high yields under mild conditions. 3-Aminopropylated Controlled Pore Glass (CPG) was succinylated with succinic anhydride followed by the reaction with S-(2-thio-5-nitropyridyl)-2-mercaptoethanol in the presence of dicyclohexylcarbodiimide (DCC). The resultant polymer support was reacted with 4,4'-dimethoxytrityloxyalkanthiol 5(a - c) to yield the derivatized polymer supports 5(a - c). The support 5a directly leads to oligonucleotide-3'-phosphate on deprotection with ammonical DTT at 55 degrees C while the supports 5b and 5c lead to oligonucleotide-3'-thiols or partially protected 3'-sulfhydryl group containing oligonucleotides during final deprotection.

Full text

PDF
3019

Selected References

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

  1. Agrawal S., Christodoulou C., Gait M. J. Efficient methods for attaching non-radioactive labels to the 5' ends of synthetic oligodeoxyribonucleotides. Nucleic Acids Res. 1986 Aug 11;14(15):6227–6245. doi: 10.1093/nar/14.15.6227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bischoff R., Coull J. M., Regnier F. E. Introduction of 5'-terminal functional groups into synthetic oligonucleotides for selective immobilization. Anal Biochem. 1987 Aug 1;164(2):336–344. doi: 10.1016/0003-2697(87)90502-1. [DOI] [PubMed] [Google Scholar]
  3. Chollet A., Kawashima E. H. Biotin-labeled synthetic oligodeoxyribonucleotides: chemical synthesis and uses as hybridization probes. Nucleic Acids Res. 1985 Mar 11;13(5):1529–1541. doi: 10.1093/nar/13.5.1529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chu B. C., Orgel L. E. Detection of specific DNA sequences with short biotin-labeled probes. DNA. 1985 Aug;4(4):327–331. doi: 10.1089/dna.1985.4.327. [DOI] [PubMed] [Google Scholar]
  5. Chu B. C., Wahl G. M., Orgel L. E. Derivatization of unprotected polynucleotides. Nucleic Acids Res. 1983 Sep 24;11(18):6513–6529. doi: 10.1093/nar/11.18.6513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Connolly B. A., Rider P. Chemical synthesis of oligonucleotides containing a free sulphydryl group and subsequent attachment of thiol specific probes. Nucleic Acids Res. 1985 Jun 25;13(12):4485–4502. doi: 10.1093/nar/13.12.4485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Connolly B. A. The synthesis of oligonucleotides containing a primary amino group at the 5'-terminus. Nucleic Acids Res. 1987 Apr 10;15(7):3131–3139. doi: 10.1093/nar/15.7.3131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cosstick R., McLaughlin L. W., Eckstein F. Fluorescent labelling of tRNA and oligodeoxynucleotides using T4 RNA ligase. Nucleic Acids Res. 1984 Feb 24;12(4):1791–1810. doi: 10.1093/nar/12.4.1791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gaur R. K., Sharma P., Gupta K. C. A simple method for the introduction of thiol group at 5'-termini of oligodeoxynucleotides. Nucleic Acids Res. 1989 Jun 12;17(11):4404–4404. doi: 10.1093/nar/17.11.4404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hudson E. N., Weber G. Synthesis and characterization of two fluorescent sulfhydryl reagents. Biochemistry. 1973 Oct 9;12(21):4154–4161. doi: 10.1021/bi00745a019. [DOI] [PubMed] [Google Scholar]
  11. Kempe T., Sundquist W. I., Chow F., Hu S. L. Chemical and enzymatic biotin-labeling of oligodeoxyribonucleotides. Nucleic Acids Res. 1985 Jan 11;13(1):45–57. doi: 10.1093/nar/13.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Leary J. J., Brigati D. J., Ward D. C. Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots. Proc Natl Acad Sci U S A. 1983 Jul;80(13):4045–4049. doi: 10.1073/pnas.80.13.4045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Marugg J. E., Piel N., McLaughlin L. W., Tromp M., Veeneman G. H., van der Marel G. A., van Boom J. H. Polymer supported DNA synthesis using hydroxybenzotriazole activated phosphotriester intermediates. Nucleic Acids Res. 1984 Nov 26;12(22):8639–8651. doi: 10.1093/nar/12.22.8639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Murasugi A., Wallace R. B. Biotin-labeled oligonucleotides: enzymatic synthesis and use as hybridization probes. DNA. 1984 Jun;3(3):269–277. doi: 10.1089/dna.1.1984.3.269. [DOI] [PubMed] [Google Scholar]
  15. Richardson R. W., Gumport R. I. Biotin and fluorescent labeling of RNA using T4 RNA ligase. Nucleic Acids Res. 1983 Sep 24;11(18):6167–6184. doi: 10.1093/nar/11.18.6167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Russo A., Bump E. A. Detection and quantitation of biological sulfhydryls. Methods Biochem Anal. 1988;33:165–241. doi: 10.1002/9780470110546.ch5. [DOI] [PubMed] [Google Scholar]
  17. Sinha N. D., Biernat J., McManus J., Köster H. Polymer support oligonucleotide synthesis XVIII: use of beta-cyanoethyl-N,N-dialkylamino-/N-morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product. Nucleic Acids Res. 1984 Jun 11;12(11):4539–4557. doi: 10.1093/nar/12.11.4539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Smith L. M., Fung S., Hunkapiller M. W., Hunkapiller T. J., Hood L. E. The synthesis of oligonucleotides containing an aliphatic amino group at the 5' terminus: synthesis of fluorescent DNA primers for use in DNA sequence analysis. Nucleic Acids Res. 1985 Apr 11;13(7):2399–2412. doi: 10.1093/nar/13.7.2399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sproat B. S., Beijer B., Rider P., Neuner P. The synthesis of protected 5'-mercapto-2',5'-dideoxyribonucleoside-3'-O-phosphoramidites; uses of 5'-mercapto-oligodeoxyribonucleotides. Nucleic Acids Res. 1987 Jun 25;15(12):4837–4848. doi: 10.1093/nar/15.12.4837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Swatditat A., Tsen C. C. Determining simple sulfhydryl compounds (low molecular weight) and their contents in biological samples by using 2,2'-dithiobis-(5-nitropyridine). Anal Biochem. 1972 Feb;45(2):349–356. doi: 10.1016/0003-2697(72)90198-4. [DOI] [PubMed] [Google Scholar]
  21. Tanaka T., Sakata T., Fujimoto K., Ikehara M. Synthesis of oligodeoxyribonucleotide with aliphatic amino or phosphate group at the 5' end by the phosphotriester method on a polystyrene support. Nucleic Acids Res. 1987 Aug 11;15(15):6209–6224. doi: 10.1093/nar/15.15.6209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Zuckermann R., Corey D., Schultz P. Efficient methods for attachment of thiol specific probes to the 3'-ends of synthetic oligodeoxyribonucleotides. Nucleic Acids Res. 1987 Jul 10;15(13):5305–5321. doi: 10.1093/nar/15.13.5305. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES