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 designed 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 cleavage 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 ribozymes 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 multiribozyme expression system of the [Coat'A196Rz/Coat'B210Rz]₅ is more significantly superior to that of the [Coat'A196Rz/Coat'B210Rz]₁ in cleavage of RNA substrate. The fractions cleaved by [Coat'A196Rz/Coat'B210Rz]₅ on the MDR1/MRP1 substrate for 8 h at observed temperatures showed no marked difference. The studies of Mg²⁺ on cleavage efficiency indicate that cleavage reaction is dependent on Mg²⁺ ions concentration. The plot of lg(k_obs) vs. lgc(Mg²⁺) displays a linear relationship between 2.5 mmol/L and 20 mmol/L Mg²⁺. It suggests that Mg²⁺ ions play a crucial role in multi-ribozyme cleavage on the substrate.