Hammerhead cleavage of the phosphorodithioate linkage

Under standard reaction conditions, a hammerhead ribozyme with a phosphorod ithioate Linkage at the cleavage site cleaved to the expected products with a rate about 500-fold slower than the corresponding phosphodiester linkage . When the greater stability of the dithioate linkage to nonenzymatic nucle ophilic attack is taken into account, the hammerhead is remarkably effectiv e at cleaving the dithioate linkage considering that the R-P-phosphoromonot hioate linkage is virtually inactive. On the basis of experiments determini ng the Mg2+ concentration dependence of the cleavage rate and the stimulati on of cleavage by thiophilic Cd2+ ion, the lesser catalytic rate enhancemen t of the dithioate linkage is primarily due to the loss of a single Mg2+ io n bound near the cleavage site. These results are qualitatively similar to, but quantitatively different from, similar experiments examining the hamme rhead cleavage properties of the R-P-phosphoromonothioate linkage. The dith ioate linkage thus promises to be a valuable alternative phosphate analogue to the monothioate linkage in studying the mechanisms of RNA catalysis.