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Nucleic Acids Research, 1989, Vol. 17, No. 9 3373-3386
© 1989

CHEMISTRY

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*, Angus I. Lamond, Barbro Beijer, Philippe Neuner and Ursula Ryder

European Molecular Biology Laboratory Postfach 10 2209, D-6900 Heidelberg, FRG

*To whom correspondence should be addressed

Received February 21, 1989. Accepted April 3, 1989.

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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