Trans-acting aptazyme for conditional gene knockdown in eukaryotic cells
Shan Zhou, Meiyi Chen, Yi Yuan, Yan Xu, Qinlin Pu, Xilei Ai, Shuai Liu, Feng Du, Xin Huang, Juan Dong, Xin Cui, Zhuo Tang
Abstract
Trans -acting hammerhead ribozyme inherits the advantages of being the smallest and best-characterized RNA-cleaving ribozyme, offering high modularity and the ability to cleave any desired sequence without the aid of any protein, as long as the target sequence contains a cleavage site. However, achieving precise control over the trans- acting hammerhead ribozyme would enable safer and more accurate regulation of gene expression. Herein, we described an intracellular selection of hammerhead aptazyme that contains a theophylline aptamer on stem II based on toxin protein IbsC. Based on the intracellular selection, we obtained three new cis- acting hammerhead aptazymes. Moreover, the corresponding trans- acting aptazymes could be efficiently induced by theophylline to knock down different targeted genes in eukaryotic cells. Notably, the best one, T195, exhibited a ligand-dependent and dose-dependent response to theophylline, and the cleavage efficiency could be enhanced by incorporating multiplex aptazymes.