Preparation and identification of anti-transforming growth factor β1 U1 small nuclear RNA chimeric ribozyme in vitro

AIM To study the preparation and cleavage activity of anti-transforming growth factor(TGF)β1 U1 small nuclear(sn)RNA chimeric hammerhead ribozyrnes in vitro.METHODS:TGFβ1 partial gene fragment was cloned intoT-vector at the downstream of T7 promoter.32p-labeledTGFβ1 partial transcripts as target RNA were transcribed invitro and purified by denaturing polyacrylamide gelelectrophoresis(PAGE).Anti-TGFβl ribozymes weredesigned by computer,then synthetic ribozyme fragmentswere cloned into the U1 ribozyme vector pZeoUiEcoSpecontaining U1 snRNA promoter/enhancer and terminator.32p-labeled U1 snRNA chimeric ribozyme transcripts weregel-purified,incubated with target-RNAs at different conditionsand autoradiographed after running denaturing PAGE.RESULTS:Active UlsnRNA chimeric ribozyme(UIRz803)had the best cleavage activity at 50℃;at 37℃,it wasactive,Km=34.48 nmol/L,Kcat=0.14 min-1;while the pointmutant ribozyme U1Rz803mhad no cleavage activity,so theseindicated the design of U1P,z803 was correct.CONCLUSION:U1Rz803 prepared in this study possessedthe perfect specific catalytic cleavage activity.These resultsindicate U1 snRNA chimeric ribozyme UIRz803 may suppressthe expression of TGFβ1 in vivo,therefore it may provide anew avenue for the treatment of liver fibrosis in the future.