Hou Ting, Wang Xiuzhong, Lu Tingting, Liu Xiaojuan, Li Feng
College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao, 266109, China.
Anal Bioanal Chem. 2014 May;406(12):2943-8. doi: 10.1007/s00216-014-7719-6. Epub 2014 Mar 29.
As a prominent member of the 5'-kinase family, T4 polynucleotide kinase (PNK) plays an important role in gene function regulations, and the study of PNK activity and its potential inhibitors is significant for research related to the DNA phosphorylation process. Here, we proposed a novel strategy for the detection of PNK activity and its inhibition, which combines exonuclease enzyme reaction and nicking enzyme-assisted fluorescence signal amplification. Through recycling cleavage of DNA fluorescence probe for signal amplification, a highly sensitive PNK sensing platform is developed, and a very low detection limit of 0.05 mU/mL is achieved, which is better than or comparable to that of the previously reported PNK assays. The present approach adopts a simple separation-free procedure in which the enzyme assay is conducted in homogeneous solutions. Additionally, the inhibitory effects of several known kinase inhibitors on PNK have been successfully detected. Since the proposed assay exhibits the advantages of high sensitivity and simplicity, it holds great potential in providing a promising platform for convenient and highly sensitive detection of PNK activity and its inhibitors.
作为5'-激酶家族的重要成员，T4多核苷酸激酶（PNK）在基因功能调控中发挥着重要作用，对PNK活性及其潜在抑制剂的研究对于DNA磷酸化过程相关研究具有重要意义。在此，我们提出了一种检测PNK活性及其抑制作用的新策略，该策略结合了核酸外切酶反应和切口酶辅助荧光信号放大。通过对DNA荧光探针的循环切割进行信号放大，构建了一个高灵敏度的PNK传感平台，实现了0.05 mU/mL的极低检测限，优于或与先前报道的PNK检测方法相当。本方法采用简单的无需分离的程序，其中酶检测在均相溶液中进行。此外，已成功检测了几种已知激酶抑制剂对PNK的抑制作用。由于所提出的检测方法具有高灵敏度和简单性的优点，它在为方便、高灵敏地检测PNK活性及其抑制剂提供一个有前景的平台方面具有巨大潜力。
