可以通过成功插入体外选择的适体来构建对用户定义的分析物响应的人工核糖开关,与分析物结合,进入mRNA的非翻译区。其中,真核核糖开关作为生物传感器比细菌更有前途,因为它们在环境温度下功能良好。此外,无细胞表达系统允许以环境友好的方式更广泛地使用这些核糖开关作为无细胞生物传感器而没有细胞限制。当前最好的无细胞真核核糖开关通过植入的分析物响应性核酶介导的自我裂解来调节真核经典翻译起始(即,一种aptazyme,适体-核酶融合)。然而,它作为传感器有严重的缺陷:由于使用的核酶活性较低,自我切割和翻译反应必须分别和顺序进行,并且必须选择不同的aptazyme来改变分析物的特异性,即使下一个分析物的适体是可用的。我们在这里逐步设计新型的无细胞真核核糖开关,利用高活性的自我切割,因此不需要反应分配。尽管单步和一锅反应,与目前需要多步反应的最佳无细胞真核核糖开关相比,这些核糖开关显示出更高的分析物剂量依赖性和灵敏度。此外,分析物特异性可以以极其容易的方式改变,简单地通过适体替换(以及随后对巨型适体的简单微调)。鉴于无细胞系统可以冻干储存和运输,目前的一锅法和因此易于操作的利用真核核糖开关的无细胞生物传感器有望广泛用于环境温度下分析物的现场感测。
Artificial riboswitches responsive to user-defined analytes can be constructed by successfully inserting in vitro selected aptamers, which bind to the analytes, into untranslated regions of mRNA. Among them, eukaryotic riboswitches are more promising as biosensors than bacterial ones because they function well at ambient temperature. In addition, cell-free expression systems allow the broader use of these riboswitches as cell-free biosensors in an environmentally friendly manner without cellular limitations. The current best cell-free eukaryotic riboswitch regulates eukaryotic canonical translation initiation through self-cleavage mediated by an implanted analyte-responsive ribozyme (i.e., an aptazyme, an
aptamer-ribozyme fusion). However, it has critical flaws as a sensor: due to the less-active ribozyme used, self-cleavage and translation reactions must be conducted separately and sequentially, and a different aptazyme has to be selected to change the analyte specificity, even if an
aptamer for the next analyte is available. We here stepwise engineered novel types of cell-free eukaryotic riboswitches that harness highly active self-cleavage and thus require no reaction partitioning. Despite the single-step and one-pot reaction, these riboswitches showed higher analyte dose dependency and sensitivities than the current best cell-free eukaryotic riboswitch requiring multistep reactions. In addition, the analyte specificity can be changed in an extremely facile way, simply by
aptamer substitution (and the subsequent simple fine-tuning for giant aptamers). Given that cell-free systems can be lyophilized for storage and transport, the present one-pot and thus easy-to-handle cell-free biosensors utilizing eukaryotic riboswitches are expected to be widely used for on-the-spot sensing of analytes at ambient temperature.