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

TY - JOUR

T1 - Highly efficient chemical synthesis of 2′-O-methyloligoribonucleotides and tetrabiotinylated derivatives; novel probes that are resistant to degradation by RNA or DNA specific nucleases

AU - Sproat, Brian S.

AU - Lamond, Angus I.

AU - Beijer, Barbro

AU - Neuner, Philippe

AU - Ryder, Ursula

PY - 1989/5/11

Y1 - 1989/5/11

N2 - 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.

AB - 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.

UR - https://www.scopus.com/pages/publications/0024519705

U2 - 10.1093/nar/17.9.3373

DO - 10.1093/nar/17.9.3373

M3 - Article

C2 - 2726482

AN - SCOPUS:0024519705

SN - 0305-1048

VL - 17

SP - 3373

EP - 3386

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 9

ER -