RESUMO
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 Rp-A(2'ps5'G)pC, Sp-A(2'ps5'G)pC and Sp-ApA(2'ps5'G)pC, were prepared via solution-phase methods. Unlike the all-phosphodiester control, A(2'p5'G)pC, the Rp-thioated trimer was not cleaved by yDBR, demonstrating that changing the pro-Rp oxygen at the 2',5' phosphodiester bond averts hydrolysis by the enzyme. In contrast, the Sp 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.
