Preparation and identification of anti-transforming growth factor beta1 U1 small nuclear RNA chimeric ribozyme in vitro

Abstract

Aim: To study the preparation and cleavage activity of anti-transforming growth factor (TGF)beta1 U1 small nuclear (sn) RNA chimeric hammerhead ribozymes in vitro.

Methods: 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.

Results: 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.

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 TGFbeta1 in vivo, therefore it may provide a new avenue for the treatment of liver fibrosis in the future.

Figure 1

Sequence and predicted structure of U1 snRNA chimeric ribozymes. arrows represent cleavage site and inactivating mutation.

Figure 2

In vitro transcripts of target RNA and U1snRNA chi-meric ribozymes. 1: transcript of target RNA (220 nt), 2: tran-scripts of U1Rz803(205 nt), 3: transcripts of U1Rz803_m (205 nt).

Figure 3

Cleavage of U1Rz803 and U1Rz803_m in vitro. 1: Tar-get RNA, 2: target RNA incubated with U1Rz803, 3: target RNA incubated with U1Rz803_m. The ribozymes (205 nt) were shown in this figure because the transcripts were incorporated into alpha ³²p-UTP in the preparation of ribozymes.

Figure 4

Temperature curve of the cleavage reactions of U1Rz803 prepared in vitro.

Figure 5

Time course. (A) Specific cleavage of target RNA by U1Rz803 in vitro at 37 °C for different times. Lane 1: substrate control; lane 2 : incubated for 10 min; lane 3: 20 min; lane 4: 40 min; lane 5: 60 min; lane 6: 90 min; lane 7: 120 min. (B) Time curse of cleavage reactions of U1Rz803 prepared in vitro.

Figure 6

The kinetic of cleavage reaction for U1Rz803. (A) Specific cleavage of target RNA by U1Rz803 for the kinetic of U1Rz803 in vitro. U1Rz803 concentration was 5 nmol·L^-1, sub-strate concentration was 10, 20, 40, 80, 160 nmol·L^-1 from left to right. (B) Lineweaver-Burk kinetic plots of cleavage reaction for U1Rz803 in vitro. U1Rz803 concentration was 5 nmol·L^-1, substrate concentration was 160, 80, 40, 20, 10 nmo·L^-1 for each dot from left to right. Reactions were performed at 37 °C for 20 minutes.