IngentaConnect Synthesis, characterization, and biological properties of small b...

Authors: Mourani, Rawan¹; Damha, Masad¹

Source: Nucleosides, Nucleotides & Nucleic Acids, Volume 25, Number 2, Number 2/January 2006 , pp. 203-229(27)

Abstract:

Synthetic branched RNA fragments were prepared to examine the stereochemical requirements for hydrolysis of RNA lariats by the yeast debranching enzyme (yDBR). Specifically, two branched trinucleoside diphosphates and a tetranucleoside triphosphate containing a 2',5'-linked phosphorothioate linkage of defined stereochemistry, namely R p -A(2'ps5'G)pC, S p -A(2'ps5'G)pC and S p -ApA(2'ps5'G)pC, were prepared via solution-phase methods. Unlike the all-phosphodiester control, A(2'p5'G)pC, the R p -thioated trimer was not cleaved by yDBR, demonstrating that changing the pro-R p oxygen at the 2',5'-phosphodiester bond averts hydrolysis by the enzyme. In contrast, the S p branched compounds (trimer and tetramer) were cleaved yDBR, albeit with reduced efficiency relative to the corresponding all-phosphodiester branched compounds. Furthermore, the small branched RNAs (5 nt) were not cleaved as efficiently as a 18-nt bRNA, suggesting that the enzyme appears to have a stronger preference for larger bRNA substrates. The non-hydrolyzable branched RNA fragments prepared during these studies may be promising candidates for the future co-crystallization and X-ray analyses of DBR:bRNA complexes.

Keywords: Branched RNA; Debranching enzyme

Document Type: Research article

DOI: 10.1080/15257770500447004

Affiliations: 1: Department of Chemistry, McGill University, Montreal, Quebec, Canada

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Synthesis, characterization, and biological properties of small branched RNA fragments containing chiral (R p and S p ) 2′,5′-phosphorothioate linkages