Abstract：
Cell-free protein synthesis (CFPS) reactions can be used to detect nucleic acids. However, most CFPS systems rely on a toehold switch and exhibit the following critical limitations: (i) off-target signals due to leaky translation in the absence of target nucleic acids, (ii) a suboptimal detection limit of approximately 30 nM without pre-amplification, and (iii) labor-intensive screening processes due to sequence constraints for the target nucleic acids. To overcome these shortcomings, we developed a new split T7 switch-mediated CFPS system in which the split T7 promoter was applied to a three-way junction structure to selectively initiate transcription-translation only in the presence of target nucleic acids. Both fluorescence and colorimetric detection systems were constructed by employing different reporter proteins. Notably, we introduced the self-complementation of split fluorescent proteins to streamline preparation of the proposed system, enabling versatile applications. Operation of this one-pot approach under isothermal conditions enabled the detection of target nucleic acids at concentrations as low as 10 pM, representing more than a thousand times improvement over previous toehold switch-based approaches. Furthermore, the proposed system demonstrated high specificity in detecting target nucleic acids and compatibility with various reporter proteins encoded in the expression region. By eliminating issues associated with the previous toehold switch system, our split T7 switch-mediated CFPS system could become a core platform for detecting various target nucleic acids.
摘要：
无细胞蛋白质合成(CFPS)反应可用于检测核酸。然而,大多数CFPS系统依赖于支点开关，并表现出以下关键限制：（i）在不存在靶核酸的情况下，由于泄漏翻译而导致的脱靶信号，（ii）在没有预扩增的情况下，约30nM的次优检测限，和(iii)由于靶核酸的序列约束而导致的劳动密集型筛选过程。为了克服这些缺点，我们开发了一种新的分裂T7开关介导的CFPS系统,其中将分裂T7启动子应用于三通连接结构,仅在靶核酸存在的情况下选择性启动转录-翻译.荧光和比色检测系统都是通过使用不同的报告蛋白来构建的。值得注意的是,我们引入了分裂荧光蛋白的自我互补，以简化拟议系统的制备，启用多功能应用程序。这种一锅法在等温条件下的操作使得能够以低至10μM的浓度检测靶核酸。与以前的基于脚趾切换的方法相比，改进了一千倍以上。此外，所提出的系统在检测靶核酸方面具有很高的特异性，并且与表达区编码的各种报告蛋白具有兼容性。通过消除与以前的足托开关系统相关的问题，我们的分裂T7开关介导CFPS系统可能成为检测各种靶核酸的核心平台.
