Design, characterization and testing of tRNA3(Lys)-based hammerhead ribozymes

Abstract

A hammerhead ribozyme targeted against the HIV-1 env coding region was expressed as part of the anticodon loop of human tRNA3(LYS) without sacrificing tRNA stability or ribozyme catalytic activity. These tRNA-ribozymes were isolated from a library which was designed to contain linkers (sequences connecting the ribozyme to the anticodon loop) of random sequence and variable length. The ribozyme target site was provided in cis during selection and in trans during subsequent characterization. tRNA-ribozymes that possessed ideal combinations of linkers were expected to recognize the cis target site more freely and undergo cleavage. The cleaved molecules were isolated, cloned and characterized. Active tRNA-ribozymes were identified and the structural features conducive to cleavage were defined. The selected tRNA-ribozymes were stable, possessed cleavage rates lower or similar to the linear hammerhead ribozyme, and could be transcribed by an extract containing RNA polymerase III. Retroviral vectors expressing tRNA-ribozymes were tested in a human CD4+ T cell line and were shown to inhibit HIV-1 replication. These tRNA3(Lys)-based hammerhead ribozymes should therefore prove to be valuable for both basic and applied research. Special application is sought in HIV-1 or HIV-2 gene therapy.