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. 1989 May 11;17(9):3373–3386. doi: 10.1093/nar/17.9.3373

Highly efficient chemical synthesis of 2'-O-methyloligoribonucleotides and tetrabiotinylated derivatives; novel probes that are resistant to degradation by RNA or DNA specific nucleases.

B S Sproat 1, A I Lamond 1, B Beijer 1, P Neuner 1, U Ryder 1
PMCID: PMC317781  PMID: 2726482

Abstract

2'-O-Methyloligoribonucleotides have been synthesised on solid phase from base protected 5'-O-dimethoxytrityl-2'-O-methylribonucleoside-3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidites) using 5-(4-nitrophenyl)-1H-tetrazole as activator. Coupling yields greater than 99% were achieved, as judged by trityl cation release. The preparation of a modified 2'-deoxycytidine building block bearing an N4-(5-trifluoroacetylaminopentyl) spacer is also described. The latter compound enabled the chemical synthesis of 2'-O-methyloligoribonucleotide probes carrying several 5'- terminal biotinylation sites (in general four modified residues were used), which can be conveniently 32P end-labelled enzymatically using polynucleotide kinase. Used in conjunction with streptavidin-containing derivatives, such biotinylated probes have important applications in biochemical purification and electron microscopy of RNA-protein complexes. The 2'-O-methyloligoribonucleotides are completely resistant to degradation by either RNA or DNA specific nucleases. In contrast, nucleases with dual RNA/DNA specificity show a complete spectrum of cleavage rates.

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Selected References

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

  1. Berkower I., Leis J., Hurwitz J. Isolation and characterization of an endonuclease from Escherichia coli specific for ribonucleic acid in ribonucleic acid-deoxyribonucleic acid hybrid structures. J Biol Chem. 1973 Sep 10;248(17):5914–5921. [PubMed] [Google Scholar]
  2. 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]
  3. Inoue H., Hayase Y., Asaka M., Imura A., Iwai S., Miura K., Ohtsuka E. Synthesis and properties of novel nucleic acid probes. Nucleic Acids Symp Ser. 1985;(16):165–168. [PubMed] [Google Scholar]
  4. Inoue H., Hayase Y., Imura A., Iwai S., Miura K., Ohtsuka E. Synthesis and hybridization studies on two complementary nona(2'-O-methyl)ribonucleotides. Nucleic Acids Res. 1987 Aug 11;15(15):6131–6148. doi: 10.1093/nar/15.15.6131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Inoue H., Hayase Y., Iwai S., Ohtsuka E. Sequence-dependent hydrolysis of RNA using modified oligonucleotide splints and RNase H. FEBS Lett. 1987 May 11;215(2):327–330. doi: 10.1016/0014-5793(87)80171-0. [DOI] [PubMed] [Google Scholar]
  6. Inoue H., Hayase Y., Iwai S., Ohtsuka E. Sequence-specific cleavage of RNA using chimeric DNA splints and RNase H. Nucleic Acids Symp Ser. 1988;(19):135–138. [PubMed] [Google Scholar]
  7. Martin F. H., Castro M. M., Aboul-ela F., Tinoco I., Jr Base pairing involving deoxyinosine: implications for probe design. Nucleic Acids Res. 1985 Dec 20;13(24):8927–8938. doi: 10.1093/nar/13.24.8927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Mukai S., Shibahara S., Morisawa H. A new method for the unidirectional deletion of DNA with Bal 31 nuclease using 2'-O-MeRNA-DNA chimeric adaptors. Nucleic Acids Symp Ser. 1988;(19):117–120. [PubMed] [Google Scholar]
  9. Nyilas A., Chattopadhyaya J. Synthesis of O2'-methyluridine, O2'-methylcytidine, N4,O2'-dimethylcytidine and N4,N4,O2'-trimethylcytidine from a common intermediate. Acta Chem Scand B. 1986 Nov;40(10):826–830. doi: 10.3891/acta.chem.scand.40b-0826. [DOI] [PubMed] [Google Scholar]
  10. Ohtsuka E., Tozuka Z., Iwai S., Ikehara M. A new condensing reagent, 1-(2,4,6-triisopropylbenzenesulfonyl)-5-(pyridin-2-yl)tetrazolide and its use in the synthesis of lambda cro binding heptadecanucleotide on a polymer support. Nucleic Acids Res. 1982 Oct 25;10(20):6235–6241. doi: 10.1093/nar/10.20.6235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Shibahara S., Mukai S., Nishihara T., Inoue H., Ohtsuka E., Morisawa H. Site-directed cleavage of RNA. Nucleic Acids Res. 1987 Jun 11;15(11):4403–4415. doi: 10.1093/nar/15.11.4403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Sproat B. S., Brown D. M. A new linkage for solid phase synthesis of oligodeoxyribonucleotides. Nucleic Acids Res. 1985 Apr 25;13(8):2979–2987. doi: 10.1093/nar/13.8.2979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Takahashi Y., Kato K., Hayashizaki Y., Wakabayashi T., Ohtsuka E., Matsuki S., Ikehara M., Matsubara K. Molecular cloning of the human cholecystokinin gene by use of a synthetic probe containing deoxyinosine. Proc Natl Acad Sci U S A. 1985 Apr;82(7):1931–1935. doi: 10.1073/pnas.82.7.1931. [DOI] [PMC free article] [PubMed] [Google Scholar]

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