Aptamer-mediated universal enzyme assay based on target-triggered DNA polymerase activity
Aptamer-mediated universal enzyme assay based on target-triggered DNA polymerase activity
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DOI：
10.1016/j.bios.2016.07.038
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发表时间：
2017-02-15
影响因子：
12.6
通讯作者：
Park, Hyun Gyu
中科院分区：
文献类型：
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作者：
Park, Ki Soo;Lee, Chang Yeol;Park, Hyun Gyu
我们在此描述了一种用于通用荧光开启酶测定的创新方法，该方法依赖于靶酶触发的DNA聚合酶活性。在第一靶识别步骤中，靶酶被设计成使源自对含有突出端序列和互补阻断剂DNA的DNA聚合酶具有特异性的适体的检测探针不稳定，从而导致被检测探针抑制的DNA聚合酶活性的恢复。在第二信号转导步骤中，基于与TaqMan探针偶联的引物延伸反应监测该靶触发的聚合酶活性。利用这一设计原理，我们成功地检测了两个模型酶，核酸外切酶I和尿嘧啶DNA糖基化酶的活性，具有高灵敏度和高选择性。由于该策略由分离的靶点识别和信号转导模块组成，因此可以通过简单地重新设计检测探针的突出端序列而普遍用于许多不同靶酶的灵敏测定，同时保持基于TaqMan探针的信号转导模块作为通用信号工具。(C)2016爱思唯尔B.V.保留所有权利。
We herein describe an innovative method for a universal fluorescence turn-on enzyme assay, which relies on the target enzyme-triggered DNA polymerase activity. In the first target recognition step, the target enzyme is designed to destabilize detection probe derived from an aptamer specific to DNA polymerase containing the overhang sequence and the complementary blocker DNA, which consequently leads to the recovery of DNA polymerase activity inhibited by the detection probe. This target triggered polymerase activity is monitored in the second signal transduction step based on primer extension reaction coupled with TaqMan probe. Utilizing this design principle, we have successfully detected the activities of two model enzymes, exonuclease I and uracil DNA glycosylase with high sensitivity and selectivity. Since this strategy is composed of separated target recognition and signal transduction modules, it could be universally employed for the sensitive determination of numerous different target enzymes by simply redesigning the overhang sequence of detection probe, while keeping TaqMan probe-based signal transduction module as a universal signaling tool. (C) 2016 Elsevier B.V. All rights reserved.