Activity identification of anti-caspase-3 mRNA hammerhead ribozyme in both cell-free condition and BRL-3A cells.
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.
Xu
R
R
Department of Infectious Diseases, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China.
Liu
J
J
Zhou
X
X
Xie
Q
Q
Jin
Y
Y
Yu
H
H
Liao
D
D
D000818:Animals; D053148:Caspase 3; D020169:Caspases / Q000235:genetics*; D002460:Cell Line; D002474:Cell-Free System / Q000378:metabolism; D007700:Kinetics; D010957:Plasmids / Q000235:genetics; D012313:RNA / Q000378:metabolism; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D013696:Temperature; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection
D016337:RNA, Catalytic; D012333:RNA, Messenger; D012313:RNA; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases