Lin Ju-Sheng, Song Yu-Hu, Kong Xin-Juan, Li Bin, Liu Nan-Zhi, Wu Xiao-Li, Jin You-Xin
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031,China.
World J Gastroenterol. 2003 Mar;9(3):572-7. doi: 10.3748/wjg.v9.i3.572.
To study the preparation and cleavage activity of anti-transforming growth factor (TGF)beta1 U1 small nuclear (sn) RNA chimeric hammerhead ribozymes in vitro.
TGFbeta1 partial gene fragment was cloned into T-vector at the downstream of T7 promoter. (32)p-labeled TGFbeta1 partial transcripts as target RNA were transcribed in vitro and purified by denaturing polyacrylamide gel electrophoresis (PAGE). Anti-TGFbeta1 ribozymes were designed by computer, then synthetic ribozyme fragments were cloned into the U1 ribozyme vector pZeoU1EcoSpe containing U1 snRNA promoter/enhancer and terminator. (32)p-labeled U1 snRNA chimeric ribozyme transcripts were gel-purified, incubated with target-RNAs at different conditions and autoradiographed after running denaturing PAGE.
Active U1snRNA chimeric ribozyme (U1Rz803) had the best cleavage activity at 50 degrees; at 37 degrees, it was active, K(m)=34.48 nmol/L, K(cat)=0.14 min(-1); while the point mutant ribozyme U1Rz803(m) had no cleavage activity, so these indicated the design of U1Rz803 was correct.
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 TGFbeta1 in vivo, therefore it may provide a new avenue for the treatment of liver fibrosis in the future.
研究抗转化生长因子（TGF）β1 U1小核（sn）RNA嵌合锤头状核酶的体外制备及切割活性。
将TGFβ1部分基因片段克隆到T7启动子下游的T载体中。以（32）P标记的TGFβ1部分转录本作为靶RNA进行体外转录，并通过变性聚丙烯酰胺凝胶电泳（PAGE）纯化。利用计算机设计抗TGFβ1核酶，然后将合成的核酶片段克隆到含有U1 snRNA启动子/增强子和终止子的U1核酶载体pZeoU1EcoSpe中。对（32）P标记的U1 snRNA嵌合核酶转录本进行凝胶纯化，在不同条件下与靶RNA孵育，经变性PAGE电泳后进行放射自显影。
活性U1snRNA嵌合核酶（U1Rz803）在50℃时切割活性最佳；在37℃时具有活性，K（m）=34.48 nmol/L，K（cat）=0.14 min（-1）；而点突变核酶U1Rz803（m）无切割活性，这表明U1Rz803的设计是正确的。
本研究制备的U1Rz803具有良好的特异性催化切割活性。这些结果表明U1 snRNA嵌合核酶U1Rz803可能在体内抑制TGFβ1的表达，因此可能为未来肝纤维化的治疗提供新途径。
