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...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1990-10, Vol.87 (19), p.7747-7751
Main Authors: Iribarren, Adolfo M., Sproat, Brian S., Neuner, Philippe, Sulston, Ingrid, Ryder, Ursula, Lamond, Angus I.
Format: Article
Language:English
Subjects:
Alkylation
Antisense elements
Base Sequence
Biological and medical sciences
Chromatography, Affinity
Deoxyribonucleases
Fundamental and applied biological sciences. Psychology
Gels
HeLa cells
HeLa Cells - metabolism
Humans
Indicators and Reagents
Methylation
Molecular and cellular biology
Molecular genetics
Molecular Sequence Data
Oligonucleotide probes
Oligonucleotide Probes - chemical synthesis
Oligonucleotides
Oligonucleotides, Antisense - chemical synthesis
Oligoribonucleotides
Ribonucleases
Ribonucleoproteins - analysis
Ribonucleoproteins - genetics
Ribonucleoproteins - isolation & purification
Ribonucleoproteins, Small Nuclear
RNA
RNA probes
Small nuclear ribonucleoproteins
Small nuclear RNA
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Summary: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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.87.19.7747