Highly efficient chemical synthesis of 2'-O-methyloligoribonucleotides and tetrabiotinylated derivatives; novel probes that are resistant to degradation by RNA or DNA specific nucleases.
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.
Sproat
B S
BS
European Molecular Biology Laboratory, Heidelberg, FRG.
Lamond
A I
AI
Beijer
B
B
Neuner
P
P
Ryder
U
U
D001710:Biotin; D003851:Deoxyribonucleases / Q000378:metabolism*; D007202:Indicators and Reagents; D008745:Methylation; D009843:Oligoribonucleotides / Q000138:chemical synthesis*; D012260:Ribonucleases / Q000378:metabolism*; D013329:Structure-Activity Relationship
D007202:Indicators and Reagents; D009843:Oligoribonucleotides; D001710:Biotin; D003851:Deoxyribonucleases; D012260:Ribonucleases