IMPORTANCE OF PURINE-PYRIMIDINE HYDROXYL AND PURINE AMINO-GROUPS FOR HAMMERHEAD RIBOZYME CLEAVAGE

The importance of the 2'-hydroxyl and 2-amino groups of guanosine resi dues for the catalytic efficiency of a hammerhead ribozyme has been in vestigated. The three guanosines in the central core of a hammerhead r ibozyme were replaced by deoxyinosine, inosine, and deoxyguanosine, an d ribozymes containing these analogues were chemically synthesized. Mo st of the modified ribozymes are drastically decreased in their cleava ge efficiency. However, deletion of the 2-amino group at Gs (replaceme nt with inosine, deoxyguanosine, deoxyinosine) caused little alteratio n in the catalytic activity relative to that obtained with the unmodif ied ribozyme. Whereas, deletion of the 2'-amino group at G(12) and Gs (replacement with inosine, deoxyinosine, and deoxyguanosine) resulted in ribozymes with drastic decrease in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, two uridin e residues, U7 and U4, in the ribozyme sequence were replaced by deoxy uridine (dU). The dU4 complex resulted in a decrease in the catalytic rate, with relative cleavage activity that was about half that observe d for the native complex. By comparison, the dU7 complex exhibited a r elative cleavage activity within 3.3-fold of that observed with native ribozyme/substrate complex. This result suggests that the 2'-hydroxyl group at U7 is not essential for activity