EFFECTS OF CIRCULAR PERMUTATION ON THE CIS-CLEAVAGE REACTION OF A HEPATITIS-DELTA VIRUS RIBOZYME - APPLICATION TO TRANS-ACTING RIBOZYME DESIGN

In this study we investigated the effects of the relocation of the wil d type termini on the folding of a cis-cleaving RNA Rz 1 that was modi fied from the autolytic domains of hepatitis delta virus (HDV) RNA. Te n circularly permuted (CP) isomers of this ribozyme were synthesized. The structure homogeneity of RNA molecules, the molar ratio of the act ive species that undergoes cis-cleavage, and the rate of cis-cleavage were examined for each construct, CP isomers with new termini in H1 or at the junction of H2-H3, H1-J(1/4), J(1/4)-H4, or H4-J(4/2) of the p roposed pseudoknot-like structure were inactive. The single breaks of phosphodiester. bond in H2, J(1/2), Lp4, and at the 3'-end of Lp3 decr eased but did not abolish autolytic activity. The structural heterogen eity of RNA molecules may account for the limited cis-cleavage of the latter three isomers. The findings of circular permutation analysis we re used as the basis for designing an active trans-cleaving ribozyme b y dividing the cis-cleaving ribozyme into two subdomains at J(1/2) and Lp4. The ribozyme subdomain catalyzed the site-specific cleavage of t he circularly permuted composite substrate RNA in trans. Thus, the str ucture of HDV autolytic domain could be re-formed after two subdomains were associated through the base-pairing interactions of H1, H2, and H4.