Shi Zhilu, Zhang Xiafei, Cheng Rui, Li Baoxin, Jin Yan
Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Materials Science, Shaanxi Normal University, Xi'an 710062, China.
Analyst. 2015 Sep 7;140(17):6124-30. doi: 10.1039/c5an00968e.
Several fluorescence methods have been developed for sensitive detection of PNK activity based on signal amplification techniques, but they need fluorescently labeled DNA probes and superabundant assistant enzymes. We have addressed these limitations and report here a label-free and enzyme-free amplification strategy for sensitively and specifically studying PNK activity and inhibition via hybridization chain reaction (HCR). First, the phosphorylation of hairpin DNA H1 by T4 PNK makes it be specifically digested by lambda exonuclease (λ exo) from 5' to 3' direction to generate a single-stranded initiator which can successively open hairpins H2 and H3 to trigger an autonomous assembly of long DNA nanowires. Meanwhile, an intermolecular G-quadruplex is formed between H2 and H3, thereby providing fluorescence enhancement of N-methyl mesoporphyrin IX (NMM) which is a highly quadruplex-selective fluorophore. So, the PNK activity can be facilely and sensitively detected by using NMM as a signal probe which provides a low background signal to improve the overall sensitivity, resulting in the detection limit of 3.37 × 10(-4) U mL(-1). More importantly, its successful application for detecting PNK activity in a complex biological matrix and studying the inhibition effects of PNK inhibitors demonstrated that it provides a promising platform for screening PNK inhibitors as well as detecting PNK activity. Therefore, it is a highly sensitive, specific, reliable and cost-effective strategy which shows great potential for biological process research, drug discovery, and clinical diagnostics.
基于信号放大技术，已经开发了几种荧光方法用于灵敏检测PNK活性，但它们需要荧光标记的DNA探针和大量辅助酶。我们解决了这些局限性，并在此报告一种无标记且无酶的扩增策略，用于通过杂交链式反应（HCR）灵敏且特异性地研究PNK活性和抑制作用。首先，T4 PNK对发夹DNA H1进行磷酸化，使其被λ外切核酸酶（λ exo）从5'到3'方向特异性消化，产生单链引发剂，该引发剂可依次打开发夹H2和H3，触发长DNA纳米线的自主组装。同时，在H2和H3之间形成分子间G-四链体，从而增强N-甲基中卟啉IX（NMM）的荧光，NMM是一种高度四链体选择性荧光团。因此，使用NMM作为信号探针可以轻松灵敏地检测PNK活性，NMM提供低背景信号以提高整体灵敏度，检测限为3.37×10（-4）U mL（-1）。更重要的是，它在复杂生物基质中检测PNK活性以及研究PNK抑制剂抑制作用方面的成功应用表明，它为筛选PNK抑制剂以及检测PNK活性提供了一个有前景的平台。因此，这是一种高度灵敏、特异、可靠且经济高效的策略，在生物过程研究、药物发现和临床诊断方面显示出巨大潜力。
