Document Type
Article
Publication Date
3-1-2003
Abstract
Aim: To study the preparation and cleavage activity of anti-transforming growth factor (TGF)β1 U1 small nuclear (sn) RNA chimeric hammerhead ribozymes in 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 U1snRNA 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 TGFβ1 in vivo, therefore it may provide a new avenue for the treatment of liver fibrosis in the future.
Publication Source (Journal or Book title)
World Journal of Gastroenterology
First Page
572
Last Page
577
Recommended Citation
Lin, J., Song, Y., Kong, X., Li, B., Liu, N., Wu, X., & Jin, Y. (2003). Preparation and identification of anti-transforming growth factor β1 U1 small nuclear RNA chimeric ribozyme in vitro. World Journal of Gastroenterology, 9 (3), 572-577. https://doi.org/10.3748/wjg.v9.i3.572