Nucleoside triphosphates (NTPs) play important roles in living organisms. However, no fluorescent assays are currently available to simply and rapidly detect multiple NTPs with satisfactory selectivity, sensitivity and low cost. Here we demonstrate for the first time a target-triggered in-vitro transcription machinery for ultra-selective, sensitive and instant fluorescence detection of multiple NTPs. The machinery assembles RNA polymerase, DNA template and non-target NTPs to convert the target NTP into equivalent RNA signal sequences which are monitored by the fluorescence enhancement of molecular beacon. The machinery offers excellent selectivity for the target NTP against NDP, NMP and dNTP. Notably, to accelerate the kinetics of the machinery while maintain its high specificity, we investigated the sequence of DNA templates systematically and established a set of guidelines for the design of the optimum DNA templates, which allowed for instant detection of the target NTP at fmol level in less than 1 min. Furthermore, the machinery could be transformed into logic gates to study the coeffects of two NTPs in biosynthesis and real-time monitoring systems to reflect the distribution of NTP in nucleotide pools. These results provide very useful and low-cost tools for both biochemical tests and point-of-care analysis.

核苷三磷酸酯（NTPs）在活生物体中起重要作用。然而，目前尚无荧光测定法可用于以令人满意的选择性，灵敏度和低成本简单快速地检测多种NTP。在这里，我们首次展示了目标触发的体外转录机器，用于对多个NTP进行超选择性，灵敏和即时的荧光检测。该设备组装RNA聚合酶，DNA模板和非目标NTP，以将目标NTP转换为等效的RNA信号序列，并通过分子信标的荧光增强对其进行监测。该机器对目标NTP具有针对NDP，NMP和dNTP的出色选择性。值得注意的是，为了在维持其高特异性的同时加快机器的动力学，我们系统地研究了DNA模板的序列，并建立了一套设计最佳DNA模板的准则，从而可以在fmol水平上立即检测目标NTP。在不到1分钟的时间内 此外，该机器可以转换成逻辑门，以研究两种NTP在生物合成和实时监控系统中的协同作用，以反映NTP在核苷酸库中的分布。这些结果为生化测试和即时分析提供了非常有用且低成本的工具。