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 hamme...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/MRPl（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 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 multi- ribozyme expression system of the [Coat＇A196Rz/Coat＇B210Rz]5 is more significantly superior to that of the [Coat＇A 196Rz/Coat＇B210Rz] 1 in cleavage of RNA substrate. The fractions cleaved by [Coat＇A 196Rz/Coat＇B210Rz]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 crucial role in multi-ribozyme cleavage on the substrate.展开更多
基金Supported by Fund of Shenzhen Bureau of Science and Technology, China(No.20008).
文摘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/MRPl（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 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 multi- ribozyme expression system of the [Coat＇A196Rz/Coat＇B210Rz]5 is more significantly superior to that of the [Coat＇A 196Rz/Coat＇B210Rz] 1 in cleavage of RNA substrate. The fractions cleaved by [Coat＇A 196Rz/Coat＇B210Rz]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 crucial role in multi-ribozyme cleavage on the substrate.
