Synthesis of RNA containing inosine: Analysis of the sequence requirements for the 5′ splice site of the Tetrahymena group I intron

Rachel Green, Jack W. Szostak, Steven A. Benner, Alexander Rich, Nassim Usman

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)4161-4166
Number of pages6
JournalNucleic acids research
Volume19
Issue number15
DOIs
StatePublished - Aug 11 1991
Externally publishedYes

ASJC Scopus subject areas

  • Genetics

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