A RAPID AND EFFICIENT SYSTEM FOR SCREENING HIV-1 POL MESSENGER-RNA-SPECIFIC RIBOZYMES

Hammerhead ribozymes are potentially important tools for suppressing i ntracellular expression of unwanted RNAs. However, the reports that ex ist on their activity against different targets have described mixed s uccess. As an initial step towards developing a rapid and effective sy stem for in vivo testing of ribozymes, two human immunodeficiency viru s type-1 (HIV-1) polymerase (Pol) mRNA-specific ribozymes, Rz(Pro) dir ected against the protease (Pro) coding region and Rz(RT) directed aga inst the reverse transcriptase (RT) coding region, were designed and t ested in Escherichia coil. Both ribozymes displayed similar efficienci es in cleaving their target RNAs in vitro. RNA polymerase chain reacti on was adapted to demonstrate the in vivo cleavage of Rz(Pro) and Rz(R T) target sites. The resultant drop in HIV-1 RT activity was measured as well. The degree of suppression of RT activity was more apparent in vivo in cells expressing Rz(RT). The RT activity in cells expressing Rz(RT) was shown to decrease by up to 96%. This system will be useful for rapid screening of (i) other ribozyme target sites within the Pol mRNA so that multitargeted ribozymes could be designed for use in anti -HIV-l gene therapy, (ii) ribozymes with improved stability and cataly tic activity, and (iii) cofactors, if any, that could enhance ribozyme activity in vivo.