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.

基于锤头状核酶的适体酶（HHAz）继承了RNA切割核酶的体积小，效率高的优势，以及适体对特定靶标的特异性识别能力，具有成为转基因表达调节剂的巨大潜力。本文中，我们通过使用毒素蛋白IbsC作为报告基因提供阳性表型来报告HHAz的选择策略，从而实现了具有所需特性的HHAz变体的易于操作，省时省力的选择。基于此策略，我们获得了一种新的HHAz（TAP-1），可以在原核和真核系统中对细胞外调节分子茶碱敏感反应。使用荧光蛋白报道分子