2'-O-alkyl oligoribonucleotides as antisense probes.
2'-O-Methyl oligoribonucleotides have recently been introduced as antisense probes for studying RNA processing and for affinity purification of RNA-protein complexes. To identify RNA analogues with improved properties for antisense analysis, 2'-O-alkyl oligoribonucleotides were synthesized in which the alkyl moiety was either the three-carbon linear allyl group or the five-carbon branched 3,3-dimethylallyl group. Both these analogues were found to be completely resistant to degradation by either DNA- or RNA-specific nucleases. Use of biotinylated derivatives of the probes to affinity-select ribonucleoprotein particles from crude HeLa cell nuclear extracts showed that the presence of the bulky 3,3-dimethylallyl group significantly reduces affinity selection, whereas the allyl derivative binds rapidly and stably to targeted sequences and affinity-selects efficiently. The allyl derivatives also showed an increase in the level of specific binding to targeted sequences compared with 2'-O-methyl probes of identical sequence. These properties indicate that the 2'-O-allyl oligoribonucleotides are particularly well suited for use as antisense probes.
Iribarren
A M
AM
European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
Sproat
B S
BS
Neuner
P
P
Sulston
I
I
Ryder
U
U
Lamond
A I
AI
D000478:Alkylation; D001483:Base Sequence; D002846:Chromatography, Affinity; D003851:Deoxyribonucleases; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents; D008745:Methylation; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000138:chemical synthesis*; D016376:Oligonucleotides, Antisense / Q000138:chemical synthesis*; D012260:Ribonucleases; D012261:Ribonucleoproteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D017411:Ribonucleoproteins, Small Nuclear
D007202:Indicators and Reagents; D015345:Oligonucleotide Probes; D016376:Oligonucleotides, Antisense; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D003851:Deoxyribonucleases; D012260:Ribonucleases