Expression and in vitro cleavage activity of anti-caspase-7 hammerhead ribozymes

Abstract

Aim: To prepare hammerhead ribozymes against mouse caspase-7 and identify their cleavage activity in vitro, in order to select a ribozyme with specific cleavage activity against mouse caspase-7 as a potential gene therapy for apoptosis-related diseases. Methods: Anti-caspase-7 ribozymes targeting sites 333 and 394 (named Rz333 and Rz394) were designed by computer software, and their DNA sequences encoding ribozymes were synthesized. Caspase-7 DNA sequence was acquired by RT-PCR. Ribozymes and caspase-7 DNA obtained by in vitro transcription were cloned into pBSKneo U6′ and pGEM-T vectors, respectively. The cleavage activity of ribozymes against mouse caspase-7 was identified by cleavage experiments in vitro. Results: Rz333 and Rz394 were designed and their DNA sequences were synthesized respectively. The expression vector of caspase-7 and plasmids containing Rz333 and Rz394 were reconstructed successfully. Ribozymes and caspase-7 mRNA were expressed by in vitro transcription. In vitro cleavage experiment showed that 243-nt and 744-nt segments were produced after caspase-7 mRNA was mixed with Rz333 in equivalent, and the cleavage efficiency was 67.98%. No cleaved segment was observed when caspase-7 mRNA was mixed with Rz394. Conclusion: Rz333 can site-specific cleave mouse caspase-7 mRNA, and it shows a potential for gene therapy of a poptosis-related diseases by down-regulating gene expression of caspase-7. Copyright © 2004 by The WJG Press.