AIM: To study the preparation and cleavage activity of antitransforming growth factor (TGF)β1 U1 small nuclear (sn)RNA chimeric hammerhead ribozymesin vitro.METHODS: TGFβ1 partial gene fragment was cloned into T-vec...AIM: To study the preparation and cleavage activity of antitransforming growth factor (TGF)β1 U1 small nuclear (sn)RNA chimeric hammerhead ribozymesin vitro.METHODS: TGFβ1 partial gene fragment was cloned into T-vector at the downstream of T7 promoter. 32p-labeled TGFβ1 partial transcripts as target RNA were transcribed in vitro and purified by denaturing polyacrylamide gel electrophoresis (PAGE). Anti-TGFβ1 ribozymes were designed by computer, then synthetic ribozyme fragments were cloned into the U1 ribozyme vector pZeoU1EcoSpe containing U1 snRNA promoter/enhancer and terminator.32p-labeled U1 snRNA chimeric ribozyme transcripts were gel-purified, incubated with target-RNAs at different conditions and autoradiographed after running denaturing PAGE.RESULTS: Active UlsnRNA chimeric ribozyme (U1Rz803)had the best cleavage activity at 50 °C; at 37 °C, it was active, Km=34.48 nmol/L, Kcat=0.14 min-1; while the point mutant ribozyme U1Rz803m had no cleavage activity, so these indicated the design of U1Rz803 was correct.CONCLUSION: U1Rz803 prepared in this study possessed the perfect specific catalytic cleavage activity. These results indicate U1 snRNA chimeric ribozyme U1Rz803 may suppress the expression of TGFβ1in vivo, therefore it may provide a new avenue for the treatment of liver fibrosis in the future.展开更多
AIM: To investigate the designation, synthesis and biological activity of against vascular endothelial growth factor165 (VEGF165) ribozyme.METHODS: The ribozyme against VEGF165 was designed with computer. The transcri...AIM: To investigate the designation, synthesis and biological activity of against vascular endothelial growth factor165 (VEGF165) ribozyme.METHODS: The ribozyme against VEGF165 was designed with computer. The transcriptional vector was constructed which included the anti-VEGF165 ribozyme and 5', 3' selfsplicing ribozymes. The hammerhead ribozyme and substrate VEGF165 mRNA were synthesized through transcription in vitro.The cleavage activity of the ribozyme on target RNA was observed in a cell-free system.PESULTS: The anti-VEGF165 ribozyme was released properly from the transcription of pGEMRz212 cleaved by 5' and 3' self-splicing ribozymes which retained its catalytic activity,and the cleavage efficiency of ribozyme reached 90.7%.CONCLUSION: The anti-VEGF165 ribozyme designed with computer can cleave VEGF165 mRNA effectively.展开更多
基金the grant from Chinese Academy of Sciences,No.KSCX2-2-204
文摘AIM: To study the preparation and cleavage activity of antitransforming growth factor (TGF)β1 U1 small nuclear (sn)RNA chimeric hammerhead ribozymesin vitro.METHODS: TGFβ1 partial gene fragment was cloned into T-vector at the downstream of T7 promoter. 32p-labeled TGFβ1 partial transcripts as target RNA were transcribed in vitro and purified by denaturing polyacrylamide gel electrophoresis (PAGE). Anti-TGFβ1 ribozymes were designed by computer, then synthetic ribozyme fragments were cloned into the U1 ribozyme vector pZeoU1EcoSpe containing U1 snRNA promoter/enhancer and terminator.32p-labeled U1 snRNA chimeric ribozyme transcripts were gel-purified, incubated with target-RNAs at different conditions and autoradiographed after running denaturing PAGE.RESULTS: Active UlsnRNA chimeric ribozyme (U1Rz803)had the best cleavage activity at 50 °C; at 37 °C, it was active, Km=34.48 nmol/L, Kcat=0.14 min-1; while the point mutant ribozyme U1Rz803m had no cleavage activity, so these indicated the design of U1Rz803 was correct.CONCLUSION: U1Rz803 prepared in this study possessed the perfect specific catalytic cleavage activity. These results indicate U1 snRNA chimeric ribozyme U1Rz803 may suppress the expression of TGFβ1in vivo, therefore it may provide a new avenue for the treatment of liver fibrosis in the future.
文摘AIM: To investigate the designation, synthesis and biological activity of against vascular endothelial growth factor165 (VEGF165) ribozyme.METHODS: The ribozyme against VEGF165 was designed with computer. The transcriptional vector was constructed which included the anti-VEGF165 ribozyme and 5', 3' selfsplicing ribozymes. The hammerhead ribozyme and substrate VEGF165 mRNA were synthesized through transcription in vitro.The cleavage activity of the ribozyme on target RNA was observed in a cell-free system.PESULTS: The anti-VEGF165 ribozyme was released properly from the transcription of pGEMRz212 cleaved by 5' and 3' self-splicing ribozymes which retained its catalytic activity,and the cleavage efficiency of ribozyme reached 90.7%.CONCLUSION: The anti-VEGF165 ribozyme designed with computer can cleave VEGF165 mRNA effectively.
