In vitro and in vivo comparison of hammerhead, hairpin, and hepatitis delta virus self-processing ribozyme cassettes

Ribozyme expression cassettes were constructed which generate trimmed, trans-acting ribozymes from longer transcripts through the action of a downstream cis-acting ribozyme. This self-processing system produces small, well-defined trans-acting ribozymes with minimal, nonproductive, intramolecular structure. These cassettes also permit direct comparison of different ribozyme expression vectors without the need to compensate for different transcription initiation and termination sequences. Expression cassettes were created that contain a T7 promoter and that encode a single trans-acting ribozyme followed by either a hammerhead, hairpin, or hepatitis delta virus cis-acting ribozyme. All three ribozyme motifs function efficiently when transcribed in vitro, although slight differences are observed in the efficiency of self-processing for the different motifs. When transiently expressed in cultured mouse cells, the same specific cleavage products are observed. In addition, the relative efficiencies of in vitro self-processing between the three ribozyme constructs was maintained in vivo. Thus, the cellular milieu does not differentially alter the activity of the three ribozyme motifs. Detection of ribozyme-catalyzed RNA cleavage products from cultured cells is direct proof of ribozyme action in vivo.