COMPLETE IDENTIFICATION OF NONBRIDGING PHOSPHATE OXYGENS INVOLVED IN HAMMERHEAD CLEAVAGE

Phosphorothioate interference analysis is suited for the rapid identif ication of structurally and functionally important phosphate groups. P revious interference studies, however, have been limited to the analys is of pro-R(p) phosphate oxygens. We employed solid-phase oligonucleot ide synthesis and modification interference analysis to investigate ei ther of the nonbridging phosphate oxygens within the hammerhead ribozy me. Two novel sites of S-p phosphorothioate interference were identifi ed at positions A(6) and U-16.1 The results from interference experime nts were confirmed by single phosphorothioate substitutions at sites o f interest. Metal rescue experiments revealed that direct metal ion co ordination occurs in the functional ribozyme only at the site of cleav age and at the pro-R(p) oxygen of position A(9). The new approach may be generally useful in rapidly evaluating the functional importance of phosphate groups in nucleic acids.