CLEAVAGE PROPERTIES OF AN OLIGONUCLEOTIDE CONTAINING A BRIDGED INTERNUCLEOTIDE 5'-PHOSPHOROTHIOATE RNA LINKAGE

An oligonucleotide has been synthesized that contains a single bridgin g 5'-phosphorothioate at an RNA linkage (5'-ApCpGpGpTpCpTprCpsApCpGpAp GpC-3'). This new phosphodiester linkage is found to be particularly s usceptible to cleavage when compared with the corresponding oxo, deoxy and thiodeoxy derivatives. Divalent metal cations were observed to dr amatically increase the cleavage rate. The products of the cleavage un der a variety of conditions are a 5'-thiol-containing fragment (6mer) and a 2',3'-cyclic phosphate-containing fragment (8mer). The pseudofir st order rate constant, k(obs), for cleavage at pH 7.5 (50 mM Tris-HCl ) in the presence of 5 mM EDTA is 1.5 x 10(-4)/min. In the presence of 5 mM metal dichloride and 50 mM Tris-HCl, pH 7.5, the relative cleava ge rate enhancements are 10, 24, 71, 98, 370 and 3400 for Mg2+, Ca2+, Mn2+, Co2+, Zn2+ and Cd2+ respectively. The rate enhancements correlat e well with Pearson's HSAB principle, suggesting that cleavage is medi ated in part by coordination of the metal to the 5'-mercapto leaving g roup. RNA linkages containing bridging 5'-phosphorothioates should pro ve valuable for studying the mechanistic details of a variety of RNA c leaving agents, such as ribozymes.