Construction of Multi-ribozyme Expression System and Its Characterization of Cleavage on the MDR1/MRP1 Double Target Substrate in vitro

To improve catalytic activity of ribozyme on its substrate, the multi-ribozyme expression system was de- signed and constructed from 20 cis-acting hammerhead ribozymes undergoing self-cleavage with 10 trans-acting hammerhead ribozymes inserted alternatively regularly and the plasmid of pGEM-MDR1/MRP1 used to transcribe the MDR1/MRP1(196/210) substrate containing double target sites was also constructed by DNA recombination. Endonuclease digestion analysis and DNA sequencing indicate all the recombinant plasmids were correct. The clea- vage activities were evaluated for the multi-ribozyme expression system on the MDR1/MRP1 substrate in the cell free system. The results demonstrate that the cis-acting hammerhead ribozymes in the multi-ribozyme expression system were able to cleave themselves and the 72 nt of 196Rz and the 71 nt of 210Rz trans-acting hammerhead ri- bozymes were liberated effectively, and the trans-acting hammerhead ribozymes released were able to act on the MDR1/MRP1 double target RNA substrate and cleave the target RNA at specific sites effectively. The multi- ribozyme expression system of the (CoatA196Rz/CoatB210Rz)5 is more significantly superior to that of the (CoatA196Rz/CoatB210Rz)1 in cleavage of RNA substrate. The fractions cleaved by (CoatA196Rz/CoatB210Rz)5 on the MDR1/MRP1 substrate for 8 h at observed temperatures showed no marked difference. The studies of Mg 2+ on cleavage efficiency indicate that cleavage reaction is dependent on Mg 2+ ions concentration. The plot of lg(kobs) vs. lgc(Mg 2+ ) displays a linear relationship between 2.5 mmol/L and 20 mmol/L Mg 2+ . It suggests that Mg 2+ ions play a