Activity identification of chimeric anti-caspase-3 mRNA hammerhead ribozymein vitro andin vivo

Abstract

To study the expression activity of various vectors containing anti-caspase-3 ribozyme cassettesin vivo, and to further study the role of caspas-3 in the apoptotic pathway, we constructed anti-caspase-3 hammerhead ribozyme embedded into the human snRNA U6, and detected the activity of the ribozymein vitro andin vivo. Meanwhile we compared it with the self-cleaving hammerhead ribozymes that we previously studied, and with the general ribozyme, cloned into RNA polymerase II expression systems. The results showed that the three ribozymes, p1.5RZ107, pRZ107 and pU6RZ107 had the correct structure, and that they could cleave caspase-3 mRNA exactly to produce two fragments: 143nt/553nt. p1.5RZ107 has the highest cleavage efficiencyin vitro, almost 80%. However, the U6 chimeric ribozyme, pU6RZ107, has the highest cleavage activityin vivo, almost to 65%, though it has lower cleavage activityin vitro. The cleavage results demonstrated that the pU6RZ107, the U6 chimeric ribozyme, could more efficiently express and downregulate the level of caspase-3in vivo, and the ribozyme could provide an alternative approach to the research into the mechanism of apoptosis and human gene therapy also.