Highly efficient chemical synthesis of 2′-O-methyloligoribonucleotides and tetrabiotinylated derivatives; novel probes that are resistant to degradation by RNA or DNA specific nucleases
Open Access
- 1 January 1989
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 17 (9) , 3373-3386
- https://doi.org/10.1093/nar/17.9.3373
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.Keywords
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