RNA
We synthesize our RNA on solid-phase using suitably protected 2´-O-t.-butyldimethylsilyl
ribonucleoside-3´-O-(b-cyanoethyl-N,N-diisopropylamino)
phosphoramidites largely according to the standard procedures (Usman,
N., Ogilvie, K. K., Jiang, M.-Y. and Cedergren, R. J., J. Am.
Chem. Soc., 1987, 109, 7845-7854 and Scaringe, S. A.,
Francklyn, C. and Usman, N., Nucleic Acids Research, 1990,
18, 5433-5441). In addition, the protection of the exocyclic
amino groups of the nucleobases by labile protecting groups such
as phenoxyacetyl or tert.-butylphenoxyacetyl (Sinha, N. D., Davis,
P., Usman, N., Pérez, J., Hodge, R., Kremsky, J. and Casale, R.,
Biochimie, 1993, 75, 13-23) enables rapid deprotection
of synthetic RNA under very mild conditions using aqueous ammonia/ethanolic
methylamine (Wincott, F., DiRenzo, A., Shaffer, C., Grimm, S., Tracz,
D., Workman, C., Sweedler, D., Gonzalez, C., Scaringe, S. and Usman,
N., Nucleic Acids Research, 1995, 23, 2677-2684) which
also prevents any premature loss of the silyl protecting groups.
2´-O-Methyl RNA
2´-O-Methyloligoribonucleotides are extremely useful reagents
for a variety of biological experiments. Their utility stems largely
from the following two properties:
- A 2´-O-methylRNA-RNA duplex is more stable thermally
than the corresponding DNA-RNA one (Inoue, H. et al., Nucleic
Acids Research, 1987, 15, 6131-6148) ·
- The former duplex is not a substrate for RNase H (Inoue, H.
et al., FEBS Lett., 1987, 215, 327-330)
In addition 2´-O-methyloligoribonucleotides are chemically
more stable than either DNA or RNA and are resistant to degradation
by RNA- or DNA-specific nucleases (Sproat, B. S. et al., Nucleic
Acids Research, 1989, 17, 3373-3386). Applications range
from simple antisense type experiments to the correction of aberrant
splicing. Many groups have made use of biotinylated 2´-O-methyloligoribonucleotides
for the affinity selection or affinity depletion of ribonucleoprotein
complexes, in particular in the field of RNA processing. The reader
is urged to read the articles of Lamond, A. I. et al., Cell,
1989, 58, 383-390, Barabino, S. et al., The EMBO J.,
1989, 8, 4171-4178 and Blencowe, B. J. et al., Cell,
1989, 59, 531-539 for details of affinity depletion of RNA-protein
complexes from crude nuclear extracts in the field of RNA splicing. Selected bibliography of articles describing the chemistry and
applications of 2´-O-methylRNA
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