One-step highly sensitive florescence detection of T4 polynucleotide kinase activity and biological small molecules by ligation-nicking coupled reaction-mediated signal amplification
One-step highly sensitive florescence detection of T4 polynucleotide kinase activity and biological small molecules by ligation-nicking coupled reaction-mediated signal amplification
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通过连接-切口耦合反应介导的信号放大对T4多核苷酸激酶活性和生物小分子进行一步高灵敏荧光检测
DOI：
10.1016/j.bios.2013.03.034
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发表时间：
2013-09-15
影响因子：
12.6
通讯作者：
Fan, Chunhai
中科院分区：
文献类型：
--
作者：
Chen, Feng;Zhao, Yongxi;Fan, Chunhai
DNA磷酸化是由多核苷酸激酶（PNK）催化的，在许多生物事件中起重要的调控作用。本文以T4 PNK为模型靶点，描述了一种一步、高灵敏度、简单、快速的荧光检测其活性和抑制作用的方法。这种创新策略的灵感来自于结扎-缺口耦合反应介导信号放大的巨大放大能力。在T4 PNK存在下，与分子信标（MB）环序列互补的两个短寡核苷酸中的一个被磷酸化，然后通过DNA连接酶与另一个连接。在连接的DNA和MB之间形成稳定的双链后，荧光被恢复，并通过核酸内切酶辅助切割多个MB进一步显着扩增。同时，MBs的断裂也会产生新的切口，从而引发结扎反应。最终，当结扎和切口过程达到动态平衡时，荧光增强达到最大。除了基于单纳米颗粒计数的检测方法（均限于1:1的信号转导函数）外，与现有方法相比，该策略的灵敏度（0.00001 U/mL）提高了100-1700倍。该传感系统在复杂生物基质和T4 PNK抑制筛选中的应用取得了满意的结果。此外，该方法还成功用于三磷酸腺苷（ATP）等生物小分子的检测，并可进一步扩展用于烟酰胺腺嘌呤二核苷酸（NAD(+)）的检测。(C) 2013 Elsevier B.V.版权所有
DNA phosphorylation, catalyzed by polynucleotide kinase (PNK), plays significant regulatory roles in many biological events. Herein, using T4 PNK as a model target, we describe a one-step, highly sensitive, simple and rapid fluorescence approach for monitoring its activity and inhibition. This innovative strategy is inspired by the great amplification capability of ligation-nicking coupled reaction-mediated signal amplification. In the presence of T4 PNK, one of two short oligonucleotides complementary to the loop sequence of molecular beacon (MB) are phosphorylated, and then ligated with the other by DNA ligase. Upon formation of the stable duplex between the ligated DNA and MB, the fluorescence is restored and further significantly amplified through nicking endonuclease assisted cleavage of multiple MBs. Meanwhile, the cleavage of MBs will also generate new nicks to initiate the ligation reaction. Eventually, a maximum fluorescence enhancement is obtained when the ligation and nicking process reached a dynamic equilibrium. As compared to those of the existing approaches except for the assay based on single nanoparticle counting, all limited to 1:1 signal transduction function, the sensitivity (0.00001 U/mL) of the proposed strategy is 100-1700 times higher. The application of the sensing system in complex biological matrix and screening of T4 PNK inhibition are demonstrated with satisfactory results. Moreover, this approach is also successfully used to detect biological small molecules such as adenosine triphosphate (ATP), and can be further extended for nicotinamide adenine dinucleotide (NAD(+)) detection. (C) 2013 Elsevier B.V. All rights reserved.