[Preparation and identification of hammerhead ribozyme in vitro against caspase-12 mRNA fragments].
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%.


CONCLUSIONS
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.
Jiang
Shan
S
Department of Infectious Diseases, Ruijin Hospital of Shanghai Second Medical University, Shanghai 200025, China.
Xie
Qing
Q
Zhang
Wei
W
Zhou
Xia-Qiu
XQ
Yu
Hong
H
Jin
You-Xin
YX
D000818:Animals; D001483:Base Sequence; D053454:Caspase 12 / Q000235:genetics*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D018384:Oxidative Stress / Q000235:genetics*; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics
D016337:RNA, Catalytic; D012333:RNA, Messenger; C427204:hammerhead ribozyme; C506440:Casp12 protein, mouse; D053454:Caspase 12