Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1988 May 11;16(9):3671–3691. doi: 10.1093/nar/16.9.3671

Ligation of oligonucleotides to nucleic acids or proteins via disulfide bonds.

B C Chu 1, L E Orgel 1
PMCID: PMC336549  PMID: 3375070

Abstract

We have developed general methods for joining together, via cleavable disulfide bonds, either two unprotected polynucleotides or a polynucleotide and a peptide or protein. To join two oligonucleotides, each is first converted to an adduct in which cystamine is joined to the 5'-terminal phosphate of the oligonucleotide by a phosphoramidate bond. The adducts are mixed and reduced with dithiothreitol. The dithiothreitol is then removed by dialysis. Oxidation by atmospheric oxygen occurs to yield the required dimer. To join an oligonucleotide to a cysteine-containing peptide or protein, the 5'-cystamine oligomer is first converted to a 2'-pyridyldisulfide adduct and then reacted with an excess of the peptide or protein. If the peptide does not contain a free cysteine residue, it is first treated with iminothiolane to introduce one or more sulfhydryl groups. We have used these procedures to join a 16 mer deoxynucleotide probe and MDV-1 RNA, a substrate of Q beta RNA polymerase. This adduct hybridizes with a complementary target DNA. We have also joined a 16mer probe to peroxidase and MDV-1 RNA to human IgG. The probe-peroxidase adduct maintains enzymatic activity and the MDV-1 RNA-IgG adduct binds to a complementary anti-IgG.

Full text

PDF
3671

Images in this article

3674Image
on p.3674
3675Image
on p.3675
3676Image
on p.3676
3677Image
on p.3677
3678Image
on p.3678
3679Image
on p.3679
3680Image
on p.3680
3681Image
on p.3681
3686Image
on p.3686
3686Image
on p.3686

Selected References

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

  1. Bridson P. K., Orgel L. E. Catalysis of accurate poly(C)-directed synthesis of 3'-5'-linked oligoguanylates by Zn2+. J Mol Biol. 1980 Dec 25;144(4):567–577. doi: 10.1016/0022-2836(80)90337-x. [DOI] [PubMed] [Google Scholar]
  2. Brocklehurst K., Little G. Reactions of papain and of low-molecular-weight thiols with some aromatic disulphides. 2,2'-Dipyridyl disulphide as a convenient active-site titrant for papain even in the presence of other thiols. Biochem J. 1973 May;133(1):67–80. doi: 10.1042/bj1330067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cheng S., Merlino G. T., Pastan I. H. A versatile method for the coupling of protein to DNA: synthesis of alpha 2-macroglobulin-DNA conjugates. Nucleic Acids Res. 1983 Feb 11;11(3):659–669. doi: 10.1093/nar/11.3.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chu B. C., Kramer F. R., Orgel L. E. Synthesis of an amplifiable reporter RNA for bioassays. Nucleic Acids Res. 1986 Jul 25;14(14):5591–5603. doi: 10.1093/nar/14.14.5591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Chu B. C., Orgel L. E. Nonenzymatic sequence-specific cleavage of single-stranded DNA. Proc Natl Acad Sci U S A. 1985 Feb;82(4):963–967. doi: 10.1073/pnas.82.4.963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Corey D. R., Schultz P. G. Generation of a hybrid sequence-specific single-stranded deoxyribonuclease. Science. 1987 Dec 4;238(4832):1401–1403. doi: 10.1126/science.3685986. [DOI] [PubMed] [Google Scholar]
  10. ELLMAN G. L. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959 May;82(1):70–77. doi: 10.1016/0003-9861(59)90090-6. [DOI] [PubMed] [Google Scholar]
  11. Jablonski E., Moomaw E. W., Tullis R. H., Ruth J. L. Preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes. Nucleic Acids Res. 1986 Aug 11;14(15):6115–6128. doi: 10.1093/nar/14.15.6115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jay E., Bambara R., Padmanabhan R., Wu R. DNA sequence analysis: a general, simple and rapid method for sequencing large oligodeoxyribonucleotide fragments by mapping. Nucleic Acids Res. 1974 Mar;1(3):331–353. doi: 10.1093/nar/1.3.331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. King T. P., Li Y., Kochoumian L. Preparation of protein conjugates via intermolecular disulfide bond formation. Biochemistry. 1978 Apr 18;17(8):1499–1506. doi: 10.1021/bi00601a022. [DOI] [PubMed] [Google Scholar]
  14. Kremsky J. N., Wooters J. L., Dougherty J. P., Meyers R. E., Collins M., Brown E. L. Immobilization of DNA via oligonucleotides containing an aldehyde or carboxylic acid group at the 5' terminus. Nucleic Acids Res. 1987 Apr 10;15(7):2891–2909. doi: 10.1093/nar/15.7.2891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Meinkoth J., Wahl G. Hybridization of nucleic acids immobilized on solid supports. Anal Biochem. 1984 May 1;138(2):267–284. doi: 10.1016/0003-2697(84)90808-x. [DOI] [PubMed] [Google Scholar]
  16. Nitta N., Kuge O., Yui S., Tsugawa A., Negishi K., Hayatsu H. A new reaction useful for chemical cross-linking between nucleic acids and proteins. FEBS Lett. 1984 Jan 23;166(1):194–198. doi: 10.1016/0014-5793(84)80071-x. [DOI] [PubMed] [Google Scholar]
  17. Tsang V. C., Peralta J. M., Simons A. R. Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis. Methods Enzymol. 1983;92:377–391. doi: 10.1016/0076-6879(83)92032-3. [DOI] [PubMed] [Google Scholar]
  18. 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