Intracellular Selection of Theophylline-Sensitive Hammerhead Aptazyme
Qinlin Pu1, Shan Zhou1, Xin Huang2
1Natural Products Research Center, Chengdu Institution of Biology, Chinese Academy of Science, Chengdu 610041, P.R. China; University of Chinese Academy of Sciences, Beijing 10049, P.R. China.
Abstract:
Hammerhead ribozyme-based aptazyme (HHAz), inheriting the advantages of small size and high efficiency from the RNA-cleaving ribozyme and the specific recognition ability of aptamers to specific targets, exhibits the huge potential to be a transgene expression regulator. Herein, we report a selection strategy for HHAz by using a toxin protein IbsC as the reporter to offer a positive phenotype, thus realizing an easy-operating, time- and labor-saving selection of HHAz variants with desired properties. Based on this strategy, we obtained a new HHAz (TAP-1), which could react sensitively toward the extracellular regulatory molecule, theophylline, both in prokaryotic and eukaryotic systems. With fluorescent protein reporter, the intracellular switching efficiencies of TAP-1 and other reported theophylline-dependent HHAzs has been quantitatively evaluated, showing that TAP-1 not only exhibits the best downregulating ability at high concentration of theophylline but also maintains high activity with 0.1 mM theophylline, which is a safe concentration in the human body.
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