Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1978 Jul;5(7):2297–2311. doi: 10.1093/nar/5.7.2297

Simplified methods for large scale enzymatic synthesis of oligoribonucleotides.

B W Shum, D M Crothers
PMCID: PMC342164  PMID: 673855

Abstract

We report simplified methods for large scale enzymatic synthesis of oligoribonucleotides using polynucleotide phosphorylase. The main features of the method are use of RPC-5 chromatography, including chromatography at two pH values to deal with the problem of primer phosphorolysis, rapid dialysis for large scale desalting, simplified methods for enzyme removal, and high resolution 1H and 31P NMR for product identification and demonstration of purity. The capacity of the method is adequate to allow beginning with grams of material in the first polymerization step, so that product yields of several milligrams, sufficient for many physical studies, are possible after as many as three separate polymerization reactions.

Full text

PDF
2297

Selected References

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

  1. Augustyniak J., Janowicz Z., Labuda D., Wower J. Desalting of RPC-5 oligonucleotides on Dowex 50. Anal Biochem. 1975 Nov;69(1):310–311. doi: 10.1016/0003-2697(75)90594-1. [DOI] [PubMed] [Google Scholar]
  2. Bahl C. P., Wu R., Itakura K., Katagiri N., Narang S. A. Chemical and enzymatic synthesis of lactose operator of Escherichia coli and its binding to lactose repressor. Proc Natl Acad Sci U S A. 1976 Jan;73(1):91–94. doi: 10.1073/pnas.73.1.91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bennett G. N., Mackey J. K., Wiebers J. L., Gilham P. T. 2'-O-(alpha-methoxyethyl)nucleoside 5'-diphosphates as "single-addition" substrates in the synthesis of specific oligoribonucleotides with polynucleotide phosphorylase. Biochemistry. 1973 Sep 25;12(20):3956–3962. doi: 10.1021/bi00744a027. [DOI] [PubMed] [Google Scholar]
  4. Brahms J., Aubertin A. M., Dirheimer G., Grunberg-Manago M. Studies of trinucleotide conformations. Role of guanine residues in an oligonucleotide chain. Biochemistry. 1969 Aug;8(8):3269–3278. doi: 10.1021/bi00836a021. [DOI] [PubMed] [Google Scholar]
  5. Cozzone P. J., Jardetzky O. Phosphorus-31 Fourier transform nuclear magnetic resonance study of mononucleotides and dinucleotides. 1. Chemical shifts. Biochemistry. 1976 Nov 2;15(22):4853–4859. doi: 10.1021/bi00667a016. [DOI] [PubMed] [Google Scholar]
  6. Craine J. E., Klee C. B. A deoxyadenylate kinase activity associated with polynucleotide phosphorylase from Micrococcus luteus. Nucleic Acids Res. 1976 Nov;3(11):2923–2928. doi: 10.1093/nar/3.11.2923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Egan B. Z. Separation of oligonucleotides by reversed-phase chromatography. Biochim Biophys Acta. 1973 Mar 19;299(2):245–252. doi: 10.1016/0005-2787(73)90347-x. [DOI] [PubMed] [Google Scholar]
  8. Gillam S., Waterman K., Smith M. Enzymatic synthesis of oligonucleotides of defined sequence. Addition of short blocks of nucleotide residues to oligonucleotide primers. Nucleic Acids Res. 1975 May;2(5):613–624. doi: 10.1093/nar/2.5.613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Khym J. X. Separation of nucleic acid components on polyacrylamide gel columns. Anal Biochem. 1976 Mar;71(1):231–242. doi: 10.1016/0003-2697(76)90032-4. [DOI] [PubMed] [Google Scholar]
  10. Klee C. B., Singer M. F. A convenient method for the preparation of primer-dependent polynucleotide phosphorylase from Micrococcus lysodeikticus. Biochem Biophys Res Commun. 1967 Nov 17;29(3):356–361. doi: 10.1016/0006-291x(67)90462-7. [DOI] [PubMed] [Google Scholar]
  11. Mackey J. K., Gilham P. T. New approach to the synthesis of polyribonucleotides of defined sequence. Nature. 1971 Oct 22;233(5321):551–553. doi: 10.1038/233551a0. [DOI] [PubMed] [Google Scholar]
  12. Pearson R. L., Weiss J. F., Kelmers A. D. Improved separation of transfer RNA's on polychlorotrifuoroethylene-supported reversed-phase chromatography columns. Biochim Biophys Acta. 1971 Feb 11;228(3):770–774. doi: 10.1016/0005-2787(71)90748-9. [DOI] [PubMed] [Google Scholar]
  13. Roe B., Marcu K., Dudock B. The isolation and sequence analysis of transfer RNA: the use of plaskon chromatography (RPC-5). Biochim Biophys Acta. 1973 Aug 10;319(1):25–36. doi: 10.1016/0005-2787(73)90037-3. [DOI] [PubMed] [Google Scholar]
  14. Singhal R. P. Anion-exchange chromatography on reversed-phase columns: isolation and assay of nucleosides, nucleotides, and oligonucleotides from nucleic acids and from cytoplasm. Biochim Biophys Acta. 1973 Aug 10;319(1):11–24. doi: 10.1016/0005-2787(73)90036-1. [DOI] [PubMed] [Google Scholar]
  15. Uhlenbeck O. C., Cameron V. Equimolar addition of oligoribonucleotides with T4 RNA ligase. Nucleic Acids Res. 1977 Jan;4(1):85–98. doi: 10.1093/nar/4.1.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Walker G. C., Uhlenbeck O. C., Bedows E., Gumport R. I. T4-induced RNA ligase joins single-stranded oligoribonucleotides. Proc Natl Acad Sci U S A. 1975 Jan;72(1):122–126. doi: 10.1073/pnas.72.1.122. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES