Abstract
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.
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- Barabino S. M., Sproat B. S., Ryder U., Blencowe B. J., Lamond A. I. Mapping U2 snRNP--pre-mRNA interactions using biotinylated oligonucleotides made of 2'-OMe RNA. EMBO J. 1989 Dec 20;8(13):4171–4178. doi: 10.1002/j.1460-2075.1989.tb08602.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Blencowe B. J., Sproat B. S., Ryder U., Barabino S., Lamond A. I. Antisense probing of the human U4/U6 snRNP with biotinylated 2'-OMe RNA oligonucleotides. Cell. 1989 Nov 3;59(3):531–539. doi: 10.1016/0092-8674(89)90036-6. [DOI] [PubMed] [Google Scholar]
- Inoue H., Hayase Y., Asaka M., Imura A., Iwai S., Miura K., Ohtsuka E. Synthesis and properties of novel nucleic acid probes. Nucleic Acids Symp Ser. 1985;(16):165–168. [PubMed] [Google Scholar]
- Inoue H., Hayase Y., Imura A., Iwai S., Miura K., Ohtsuka E. Synthesis and hybridization studies on two complementary nona(2'-O-methyl)ribonucleotides. Nucleic Acids Res. 1987 Aug 11;15(15):6131–6148. doi: 10.1093/nar/15.15.6131. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Inoue H., Hayase Y., Iwai S., Ohtsuka E. Sequence-dependent hydrolysis of RNA using modified oligonucleotide splints and RNase H. FEBS Lett. 1987 May 11;215(2):327–330. doi: 10.1016/0014-5793(87)80171-0. [DOI] [PubMed] [Google Scholar]
- Inoue H., Hayase Y., Iwai S., Ohtsuka E. Sequence-specific cleavage of RNA using chimeric DNA splints and RNase H. Nucleic Acids Symp Ser. 1988;(19):135–138. [PubMed] [Google Scholar]
- Lamond A. I., Sproat B., Ryder U., Hamm J. Probing the structure and function of U2 snRNP with antisense oligonucleotides made of 2'-OMe RNA. Cell. 1989 Jul 28;58(2):383–390. doi: 10.1016/0092-8674(89)90852-0. [DOI] [PubMed] [Google Scholar]
- Mukai S., Shibahara S., Morisawa H. A new method for the unidirectional deletion of DNA with Bal 31 nuclease using 2'-O-MeRNA-DNA chimeric adaptors. Nucleic Acids Symp Ser. 1988;(19):117–120. [PubMed] [Google Scholar]
- Shibahara S., Mukai S., Nishihara T., Inoue H., Ohtsuka E., Morisawa H. Site-directed cleavage of RNA. Nucleic Acids Res. 1987 Jun 11;15(11):4403–4415. doi: 10.1093/nar/15.11.4403. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sproat B. S., Beijer B., Iribarren A. New synthetic routes to protected purine 2'-O-methylriboside-3'-O-phosphoramidites using a novel alkylation procedure. Nucleic Acids Res. 1990 Jan 11;18(1):41–49. doi: 10.1093/nar/18.1.41. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sproat B. S., Lamond A. I., Beijer B., Neuner P., Ryder U. Highly efficient chemical synthesis of 2'-O-methyloligoribonucleotides and tetrabiotinylated derivatives; novel probes that are resistant to degradation by RNA or DNA specific nucleases. Nucleic Acids Res. 1989 May 11;17(9):3373–3386. doi: 10.1093/nar/17.9.3373. [DOI] [PMC free article] [PubMed] [Google Scholar]