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首页> 外文期刊>The Analyst: The Analytical Journal of the Royal Society of Chemistry: A Monthly International Publication Dealing with All Branches of Analytical Chemistry >A unique dual recognition hairpin probe mediated fluorescence amplification method for sensitive detection of uracil-DNA glycosylase and endonuclease IV activities
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A unique dual recognition hairpin probe mediated fluorescence amplification method for sensitive detection of uracil-DNA glycosylase and endonuclease IV activities

机译：独特的双重识别发夹探针介导的荧光扩增方法，用于灵敏检测尿嘧啶-DNA糖基化酶和核酸内切酶IV活性

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摘要

Uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV) play cooperative roles in uracil base-excision repair (UBER) and inactivity of either will interrupt the UBER to cause disease. Detection of UDG and Endo IV activities is crucial to evaluate the UBER process in fundamental research and diagnostic application. Here, a unique dual recognition hairpin probe mediated fluorescence amplification method was developed for sensitively and selectively detecting UDG and Endo IV activities. For detecting UDG activity, the uracil base in the probe was excised by the target enzyme to generate an apurinic/apyrimidinic (AP) site, achieving the UDG recognition. Then, the AP site was cleaved by a tool enzyme Endo IV, releasing a primer to trigger rolling circle amplification (RCA) reaction. Finally, the RCA reaction produced numerous repeated G-quadruplex sequences, which interacted with N-methyl-mesoporphyrin IX to generate an enhanced fluorescence signal. Alternatively, for detecting Endo IV activity, the uracil base in the probe was first converted into an AP site by a tool enzyme UDG. Next, the AP site was cleaved by the target enzyme, achieving the Endo IV recognition. The signal was then generated and amplified in the same way as those in the UDG activity assay. The detection limits were as low as 0.00017 U mL(-1) for UDG and 0.11 U mL(-1) for Endo IV, respectively. Moreover, UDG and Endo IV can be well distinguished from their analogs. This method is beneficial for properly evaluating the UBER process in function studies and disease prognoses.
机译：尿嘧啶DNA糖基化酶（UDG）和核酸内切酶IV（Endo IV）在尿嘧啶碱基切除修复（UBER）中起着协同作用，而两者的失活都会打断UBER引起疾病。 UDG和Endo IV活动的检测对于评估基础研究和诊断应用中的UBER过程至关重要。在这里，开发了一种独特的双重识别发夹探针介导的荧光扩增方法，用于灵敏和选择性地检测UDG和Endo IV活性。为了检测UDG活性，探针中的尿嘧啶碱基被目标酶切除，生成了一个嘌呤/嘧啶（AP）位点，从而实现了UDG识别。然后，用工具酶Endo IV切割AP位点，释放引物以触发滚环扩增（RCA）反应。最后，RCA反应产生了许多重复的G-四链体序列，这些序列与N-甲基-间卟啉IX相互作用以产生增强的荧光信号。或者，为了检测Endo IV活性，首先通过工具酶UDG将探针中的尿嘧啶碱基转化为AP位点。接下来，AP位点被目标酶切割，实现Endo IV识别。然后以与UDG活性测定相同的方式生成并放大信号。 UDG的检出限低至0.00017 U mL（-1），Endo IV的检出限低至0.11 U mL（-1）。而且，UDG和Endo IV可以与它们的类似物很好地区分开。该方法有助于在功能研究和疾病预后中正确评估UBER过程。

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