TY - JOUR
T1 - Synthesis of RNA containing inosine
T2 - Analysis of the sequence requirements for the 5′ splice site of the Tetrahymena group I intron
AU - Green, Rachel
AU - Szostak, Jack W.
AU - Benner, Steven A.
AU - Rich, Alexander
AU - Usman, Nassim
N1 - Funding Information:
We thank Milligen/Biosearch for providing the Cyclone DNA synthesizer, ancillary reagents and RNA phosphoramidites, Mr Adam Foley for obtaining NMR spectra, Mr Steven Scaringe for technical assistance, and Drs Loren Williams, David Bartel and Jennifer Doudna for critical reading of this manuscript. This research was supported by funds from the National Institutes of Health, The National Science Foundation, the Office of Naval Research, the National Aeronautics & Space Administration, the Swiss National Science Foundation, and Hoechst A.G. NU was the recipient of an NIH Fogarty International Research Fellowship 1988-89.
PY - 1991/8/11
Y1 - 1991/8/11
N2 - Two protected derivatives of the ribonucleoside inosine have been prepared to serve as building blocks for phosphoramidite-based synthesis of RNA. Two different synthetic routes address the unusual solubility characteristics of inosine and its derivatives. The final products of the different synthetic pathways, 5′-O- (dimethoxytrityl)-2′-O-(t-butyldimethylsilyl) inosine 3′-O- (β-cyanoethyldiisopropylamino) phosphoramidite 5a, and O6-p-nitrophenylethyl-5′-O-(dimethoxytrityl)-2′-O-(t-butyldimethylsilyl) inosine 3′-O-(methyldiisopropyl- amino) phosphoramidite 5b, were chemically incorporated into short oligoribonucleotides which also contained the four standard ribonucleoside bases. The oligomers were chosen to study base-specific interactions between an RNA substrate and an RNA enzyme derived from the Group I Tetrahymena self- splicing intron. The oligomers were shown to be biochemically competent using a trans cleavage assay with the modified Tetrahymena intron. The results confirm the dependence of the catalytic activity on a wobble base pair, rather than a Watson-Crick base pair, in the helix at the 5′-splice site. Furthermore, comparison of guanosine and inosine in a wobble base pair allows one to assess the importance of the guanine 2-amino group for biological activity. The preparation of the inosine phosphoramidites adds to the repertoire of base analogues available for the study of RNA catalysis and RNA-protein interactions.
AB - Two protected derivatives of the ribonucleoside inosine have been prepared to serve as building blocks for phosphoramidite-based synthesis of RNA. Two different synthetic routes address the unusual solubility characteristics of inosine and its derivatives. The final products of the different synthetic pathways, 5′-O- (dimethoxytrityl)-2′-O-(t-butyldimethylsilyl) inosine 3′-O- (β-cyanoethyldiisopropylamino) phosphoramidite 5a, and O6-p-nitrophenylethyl-5′-O-(dimethoxytrityl)-2′-O-(t-butyldimethylsilyl) inosine 3′-O-(methyldiisopropyl- amino) phosphoramidite 5b, were chemically incorporated into short oligoribonucleotides which also contained the four standard ribonucleoside bases. The oligomers were chosen to study base-specific interactions between an RNA substrate and an RNA enzyme derived from the Group I Tetrahymena self- splicing intron. The oligomers were shown to be biochemically competent using a trans cleavage assay with the modified Tetrahymena intron. The results confirm the dependence of the catalytic activity on a wobble base pair, rather than a Watson-Crick base pair, in the helix at the 5′-splice site. Furthermore, comparison of guanosine and inosine in a wobble base pair allows one to assess the importance of the guanine 2-amino group for biological activity. The preparation of the inosine phosphoramidites adds to the repertoire of base analogues available for the study of RNA catalysis and RNA-protein interactions.
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U2 - 10.1093/nar/19.15.4161
DO - 10.1093/nar/19.15.4161
M3 - Article
C2 - 1714564
AN - SCOPUS:0025835642
SN - 0305-1048
VL - 19
SP - 4161
EP - 4166
JO - Nucleic acids research
JF - Nucleic acids research
IS - 15
ER -