The effect of introducing a phosphorothioate at the hammerhead cleavage sit
e was investigated using a kinetically well-characterized hammerhead, In bu
ffers containing Mg ion, the R-P-phosphorothioate isomer cleaved 2000- to 8
0 000-fold slower than the Sp isomer or the unmodified RNA substrate. Addit
ion of low concentrations of several thiophilic metal ions, especially Cd2, to these reactions is sufficient to fully restore the cleavage rate of th
e R-P substrate without affecting cleavage rate of the all-oxygen or S-P su
bstrate, Thus, a model proposing coordination of a divalent metal ion to th
e pro-R oxygen at the hammerhead cleavage site appears justified.