[Preparation and identification of hammerhead ribozyme in vitro against rat caspase-3 mRNA fragment]
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Published on: July 21, 2022
1Department of Infectious Diseases, Ruijin Hospital of Shanghai Second Medical University, Shanghai 200025, China.
Objective:
To study the transcript effect and cleavage activity in vitro of rat Caspase-3 specific ribozyme (Rz107 and Rz544).
Methods:
Rat caspase-3 gene fragment was cloned into T-vector under the control of T7 promoter. (32)P-labeled caspase-3 transcript was target-RNA. Rz107 and Rz544 genes against caspase-3 mRNA were cloned and transcribed in vitro. Cleavage reaction was detected.
Results:
It was found that Rz107 was active at 37 degrees C and more so at higher temperature within allowing temperature range. The optimal temperature was 50 degrees C. For Rz107, Km and kcat was 14.13 nmol/L and 2.31 min(-1), respectively. However, the Rz544 had no cleavage activity at all.
Conclusion:
Rz107 prepared in vitro possesses the perfect specific catalytic cleavage activity. It is hopeful that Rz107 would be developed to be a new nucleic acid drug that could effectively inhibit the inflammation of hepatitis through cleaving the key gene, caspase-3, in apoptosis in vivo.
