Abstract In this study, a label-free ultrasensitive fluorescent biosensor for monitoring of EcoRI activity has been developed based on terminal deoxynucleotidyl transferase (TdT) directed G-quadruplexes formation. Double-stranded DNA is specifically cleaved by EcoRI into single-stranded fragments with the newly generated free 3′-OH terminus. In the condition of the polymerization pool containing 10% dTTP, 40% dATP and 50% dGTP, TdT can elongate the cleaved fragments at their 3′-OH ends to produce G-rich DNA sequences which could subsequently form into G-quadruplexes motif in the aid of K+. The obtained G-quadruplexes structure can be recognized by the typical G-quadruplex-selective probe N-methylporphyrin dipropionate IX (NMM). Thus, EcoRI activity could be easily and sensitively determined by using this detection platform. The detection limit for EcoRI is as low as 0.07 U/mL according to the linear concentration range of 0.1–30 U/mL. Moreover, the proposed assay showed high potential in real sample detection. This detection platform is label-free, highly sensitive, simple to operate and cost effective, possessing high application potential as a useful tool for endonuclease detection. Moreover, we also demonstrated the capability of this strategy to detect EcoRI in serum sample, showing high application potential as a useful tool for endonuclease detection.

中文翻译：

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基于末端脱氧核苷酸转移酶生成的 G-四链体对 EcoRI 活性的无标记超灵敏测定

摘要 在本研究中，基于末端脱氧核苷酸转移酶 (TdT) 定向 G-四链体形成，开发了一种用于监测 EcoRI 活性的无标记超灵敏荧光生物传感器。双链 DNA 被 EcoRI 特异性切割成具有新生成的游离 3'-OH 末端的单链片段。在含有 10% dTTP、40% dATP 和 50% dGTP 的聚合池条件下，TdT 可以延长其 3'-OH 末端的切割片段，产生富含 G 的 DNA 序列，随后形成 G-四链体基序。 K+的帮助。获得的G-四链体结构可以通过典型的G-四链体选择性探针N-甲基卟啉二丙酸酯IX（NMM）识别。因此，使用该检测平台可以轻松、灵敏地确定 EcoRI 活性。根据 0.1–30 U/mL 的线性浓度范围，EcoRI 的检测限低至 0.07 U/mL。此外，所提出的测定在实际样品检测中显示出很高的潜力。该检测平台无标记、灵敏度高、操作简单且成本效益高，作为核酸内切酶检测的有用工具具有很高的应用潜力。此外，我们还展示了该策略检测血清样品中 EcoRI 的能力，显示出作为核酸内切酶检测有用工具的高应用潜力。作为核酸内切酶检测的有用工具具有很高的应用潜力。此外，我们还证明了该策略检测血清样品中 EcoRI 的能力，显示出作为核酸内切酶检测有用工具的高应用潜力。作为核酸内切酶检测的有用工具具有很高的应用潜力。此外，我们还展示了该策略检测血清样品中 EcoRI 的能力，显示出作为核酸内切酶检测有用工具的高应用潜力。