Related Experiment Video
Updated: Aug 19, 2026
Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection
Published on: July 21, 2022
[Preparation and identification of hammerhead ribozyme in vitro against caspase-12 mRNA fragments]
Shan Jiang1, Qing Xie, Wei Zhang
1Department of Infectious Diseases, Ruijin Hospital of Shanghai Second Medical University, Shanghai 200025, China.
Objective:
To design and synthesize ribozymes targeting 138 and 218 sites of the mRNA nucleotide of mouse caspase-12, a key intermedium of ER stress mediated apoptosis, and to identify their activities through in vitro transcription and cleavage.
Methods:
The mouse caspase-12 gene fragment was obtained by RT-PCR and cloned into the PGEM-T vector under the control of T7 RNA polymerase promoter. The transcription product of the target was labeled with a-32P UTP, while ribozymes were not labeled. Ribozyme and target RNA were incubated for 90 min at 37 degree C in a reaction buffer to perform the cleavage reaction.
Results:
It was found that under a condition of 37 degree C, pH 7.5 and with Mg2+ in a concentration of 10 mmol/L, Rz138 and Rz218 both cleaved targets at predicted sites, and the cleavage efficiency of Rz138 was 100%.
Conclusion:
Rz138 and Rz218 prepared in vitro possess the perfect specific catalytic cleavage activity. Rz138 has excellent cleavage efficiency. It may be a promising tool to prevent ER stress induced apoptosis through catalytic cleavage of caspase-12 mRNA in vivo. It also can be used to verify whether caspase-12 is necessary in ER stress induced apoptosis.
