Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1982 Mar 25;10(6):1877–1894. doi: 10.1093/nar/10.6.1877

The preparative synthesis of oligodeoxyribonucleotides using RNA ligase.

D M Hinton, C A Brennan, R I Gumport
PMCID: PMC320578  PMID: 7043396

Abstract

The synthesis of nmol quantities of defined sequences of oligodeoxyribonucleotides using T4 RNA ligase has been demonstrated. Reacting using from 18 to 200 nmol of substrates in which a single 2'-deoxyribonucleoside 3',5'-bisphosphate was added to an oligodeoxyribonucleotide resulted in yields from 13 to 95%. When two oligodeoxyribonucleotides were similarly joined using RNA ligase, the yields ranged from 10 to 50%. Although the reactions contained high concentrations of enzyme and were incubated from 5 to 21 days, there was little degradation of either substrates or products. We have also characterized an unusual product which arises when 3'-phosphate terminated oligodeoxyribonucleotides are incubated with RNA ligase and high concentrations of ATP. This product has an adenylyl group linked to the 3'-phosphate by an anhydride bond. The mechanistic and synthetic implications of forming this product are discussed.

Full text

PDF
1877

Images in this article

Selected References

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

  1. Barrio J. R., Barrio M. C., Leonard N. J., England T. E., Uhlenbeck O. C. Synthesis of modified nucleoside 3',5'-bisphosphates and their incorporation into oligoribonucleotides with T4 RNA ligase. Biochemistry. 1978 May 30;17(11):2077–2081. doi: 10.1021/bi00604a009. [DOI] [PubMed] [Google Scholar]
  2. Brownlee G. G., Sanger F. Chromatography of 32P-labelled oligonucleotides on thin layers of DEAE-cellulose. Eur J Biochem. 1969 Dec;11(2):395–399. doi: 10.1111/j.1432-1033.1969.tb00786.x. [DOI] [PubMed] [Google Scholar]
  3. Cameron V., Soltis D., Uhlenbeck O. C. Polynucleotide kinase from a T4 mutant which lacks the 3' phosphatase activity. Nucleic Acids Res. 1978 Mar;5(3):825–833. doi: 10.1093/nar/5.3.825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. England T. E., Uhlenbeck O. C. Enzymatic oligoribonucleotide synthesis with T4 RNA ligase. Biochemistry. 1978 May 30;17(11):2069–2076. doi: 10.1021/bi00604a008. [DOI] [PubMed] [Google Scholar]
  5. FELSENFELD G., HUANG S. L. Some effects of charge and structure upon ionic interactions of nucleic acids. Biochim Biophys Acta. 1961 Jul 22;51:19–32. doi: 10.1016/0006-3002(61)91012-5. [DOI] [PubMed] [Google Scholar]
  6. Gumport R. I., Hinton D. M., Pyle V. S., Richardson R. W. T4 RNA ligase as a nucleic acid synthesis and modification reagent. Nucleic Acids Symp Ser. 1980;(7):167–171. [PubMed] [Google Scholar]
  7. Gumport R. I., Uhlenbeck O. C. T4 RNA ligase as a nucleic acid synthesis and modification reagent. Gene Amplif Anal. 1981;2:313–345. [PubMed] [Google Scholar]
  8. Hinton D. M., Baez J. A., Gumport R. I. T4 RNA Ligase joins 2'-deoxyribonucleoside 3',5'-bisphosphates to oligodeoxyribonucleotides. Biochemistry. 1978 Nov 28;17(24):5091–5097. doi: 10.1021/bi00617a004. [DOI] [PubMed] [Google Scholar]
  9. Hinton D. M., Gumport R. I. The synthesis of oligodeoxyribonucleotides using RNA ligase. Nucleic Acids Res. 1979 Sep 25;7(2):453–464. doi: 10.1093/nar/7.2.453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kaufmann G., Kallenbach N. R. Determination of recognition sites of T4 RNA ligase on the 3'-OH and 5' -P termini of polyribonucleotide chains. Nature. 1975 Apr 3;254(5499):452–454. doi: 10.1038/254452a0. [DOI] [PubMed] [Google Scholar]
  11. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. McCoy M. I., Gumport R. I. T4 ribonucleic acid ligase joins single-strand oligo(deoxyribonucleotides). Biochemistry. 1980 Feb 19;19(4):635–642. doi: 10.1021/bi00545a005. [DOI] [PubMed] [Google Scholar]
  13. McCoy M. I., Lubben T. H., Gumport R. I. The purification of nuclease-free T4-RNA ligase. Biochim Biophys Acta. 1979 Mar 28;562(1):149–161. doi: 10.1016/0005-2787(79)90134-5. [DOI] [PubMed] [Google Scholar]
  14. Riley M., Paul A. Properties of synthetic polydeoxyribonucleotide complexes containing adenine and bromouracil. Biochemistry. 1971 Oct 12;10(21):3819–3825. doi: 10.1021/bi00797a003. [DOI] [PubMed] [Google Scholar]
  15. Silber R., Malathi V. G., Hurwitz J. Purification and properties of bacteriophage T4-induced RNA ligase. Proc Natl Acad Sci U S A. 1972 Oct;69(10):3009–3013. doi: 10.1073/pnas.69.10.3009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Wells R. D., Jacob T. M., Narang S. A., Khorana H. G. Studies on polynucleotides. LXIX. Synthetic deoxyribopolynucleotides as templates for the DNA polymerase of Escherichia coli: DNA-like polymers containing repeating trinucleotide sequences. J Mol Biol. 1967 Jul 28;27(2):237–263. doi: 10.1016/0022-2836(67)90018-6. [DOI] [PubMed] [Google Scholar]
  17. Winter G., Brownlee G. G. 3'End labelling of RNA with 32P suitable for rapid gel sequencing. Nucleic Acids Res. 1978 Sep;5(9):3129–3139. doi: 10.1093/nar/5.9.3129. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES