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

Brian S. Sproat (Lead / Corresponding author), Angus I. Lamond, Barbro Beijer, Philippe Neuner, Ursula Ryder

Research output: Contribution to journalArticle

202 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)3373-3386
Number of pages14
JournalNucleic Acids Research
Volume17
Issue number9
DOIs
Publication statusPublished - 11 May 1989

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