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Updated: Oct 3, 2026
Evaluation of Caspase Activation to Assess Innate Immune Cell Death
Published on: January 20, 2023
Activity identification of anti-caspase-3 mRNA hammerhead ribozyme in both cell-free condition and BRL-3A cells
1Department of Infectious Diseases, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China.
Objective:
To study the transcription effects and cleavage activities of rat caspase-3 specific hammerhead ribozyme (Rz107) in both cell-free conditions and BRL-3A cells.
Methods:
Rat caspase-3 gene fragment was cloned into the pGEM-T EASY vector under the T7 promoter control. The 32P-labeled caspase-3 transcript was the target-RNA. Rz107 genes designed against caspase-3 mRNA were cloned into vector p1.5 between 5'-cis-Rz and 3'-cis-Rz. 32P-labeled ribozyme transcripts were incubated with target-RNAs at different conditions and autoradiographed after denaturing gel-electrophoresis. Rz107 was electroporated into BRL-3A cells and the Rz107 expression was analyzed by RT-PCR.
Results:
In cell-free conditions, Rz107 was active at 37 degrees C. The optimal temperature was 50 degrees C. The Km and Kcat were 14.13 nmol/L and 2.31.min-1 respectively. Intracellular cleavage efficiency of Rz107 was 37%, as analyzed by RT-PCR. This indicated that the design of Rz107 was correct, and Rz107 had the activity of common enzymes.
Conclusions:
Rz107 in cell-free conditions possessed perfect specific catalytic cleavage activity, and it can also cleave the target RNA successfully in cells. The results illustrate the feasibility of ribozyme therapy as a potential alternative approach for treating liver disease caused by apoptosis.
