SPECIFICITY OF HAMMERHEAD RIBOZYME CLEAVAGE

To be effective in gene inactivation, the hammerhead ribozyme must cle ave a complementary RNA target without deleterious effects from cleavi ng non-target RNAs that contain mismatches and shorter stretches of co mplementarity. The specificity of hammerhead cleavage was evaluated us ing HH16, a well-characterized ribozyme designed to cleave a target of 17 residues, Under standard reaction conditions, HH16 is unable to di scriminate between its full-length substrate and 3'-truncated substrat es, even when six fewer base pairs are formed between HH16 and the sub strate, This striking lack of specificity arises because all the subst rates bind to the ribozyme with sufficient affinity so that cleavage o ccurs before their affinity differences are manifested. In contrast, H H16 does exhibit high specificity towards certain 3'-truncated version s of altered substrates that either also contain a single base mismatc h or are shortened at the 5' end. In addition, the specificity of HH16 is improved in the presence of p7 nucleocapsid protein from human imm unodeficiency virus (HIV)-1, which accelerates the association and dis sociation of RNA helices, These results support the view that the hamm erhead has an intrinsic ability to discriminate against incorrect base s, but emphasizes that the high specificity is only observed in a cert ain range of helix lengths.