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

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_min 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.