Department of Chemical and Biomolecular Engineering (BK21+ Program), KAIST, Daehak-ro 291, Yuseong-gu, Daejeon, 34141, Republic of Korea.
Department of Biological Engineering, College of Engineering, Konkuk University, Seoul, 05029, Republic of Korea.
Anal Chim Acta. 2019 Jun 20;1059:80-85. doi: 10.1016/j.aca.2019.01.025. Epub 2019 Jan 25.
We herein describe a novel method for the detection of target protein based on the target-responsive DNA polymerase activity. In the sensor, two different types of DNA aptamers with the respective functions: one binds to the target protein and the other binds to DNA polymerase, are rationally engineered and combined to form the detection probe that regulates DNA polymerase activity in response to the target protein. In the presence of target protein, the detection probe becomes destabilized and stops the inhibition of DNA polymerase activity. Consequently, the active DNA polymerase initiates the primer extension reaction on the target-specific DNA aptamer, which recycles the target protein to promote another activation cycle of DNA polymerase. In addition, DNA polymerase also catalyzes the primer extension reaction on the primer/template complex in conjugation with TaqMan probe, leading to the significantly enhanced fluorescence intensities. With this novel strategy, we detected a model target protein, lysozyme with a limit of detection as low as 0.80 nM. In addition, the practical applicability of this system was successfully demonstrated by determining lysozyme in human serum.
我们在此描述了一种基于目标响应 DNA 聚合酶活性的检测目标蛋白的新方法。在传感器中，两种不同类型的 DNA 适体经过合理设计并结合在一起，分别具有以下功能：一种与目标蛋白结合，另一种与 DNA 聚合酶结合，形成检测探针，以响应目标蛋白调节 DNA 聚合酶活性。在存在目标蛋白的情况下，检测探针变得不稳定，并停止抑制 DNA 聚合酶活性。因此，活性 DNA 聚合酶在目标特异性 DNA 适体上启动引物延伸反应，将目标蛋白循环，以促进 DNA 聚合酶的另一个激活循环。此外，DNA 聚合酶还在与 TaqMan 探针结合的引物/模板复合物上催化引物延伸反应，导致荧光强度显著增强。利用这种新策略，我们检测到了模型靶蛋白溶菌酶，其检测限低至 0.80 nM。此外，通过测定人血清中的溶菌酶，成功地证明了该系统的实际应用。
