A re-investigation of the thio effect at the hammerhead cleavage site

The effect of introducing a phosphorothioate at the hammerhead cleavage site was investigated using a kinetically well-characterized hammerhead, In buffers containing Mg ion, the R(P)-phosphorothioate isomer cleaved 2000- to 80 000-fold slower than the Sp isomer or the unmodified RNA substrate. Addition of low concentrations of several thiophilic metal ions, especially Cd(2+), to these reactions is sufficient to fully restore the cleavage rate of the R(P) substrate without affecting cleavage rate of the all-oxygen or S(P) substrate, Thus, a model proposing coordination of a divalent metal ion to the pro-R oxygen at the hammerhead cleavage site appears justified.