MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Collaborative Innovation Centre of Chemistry for Energy Materials, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Chronic Liver Disease and Hepatocellular Carcinoma of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University , Xiamen 361005, China.
The Key Lab of Analysis and Detection Technology for Food Safety of MOE, State Key Laboratory of Photocatalysis on Energy and Environment, College of Biological Science and Engineering, Fuzhou University , Fuzhou 350116, China.
ACS Appl Mater Interfaces. 2017 Nov 8;9(44):38356-38363. doi: 10.1021/acsami.7b14185. Epub 2017 Oct 24.
As a vital enzyme in DNA phosphorylation and restoration, T4 polynucleotide kinase (T4 PNK) has aroused great interest in recent years. Therefore, numerous strategies have been established for highly sensitive detection of T4 PNK based on diverse signal amplification techniques. However, they often need sophisticated design, a variety of auxiliary reagents and enzymes, or cumbersome manipulations. We have designed a new kind of allosteric aptamer probe (AAP) consisting of streptavidin (SA) aptamer and the complementary DNA (cDNA) for simple detection of T4 PNK without signal amplification and with minimized interference in complex biological samples. When the 5'-terminus of the cDNA is phosphorylated by T4 PNK, the cDNA is degraded by lambda exonuclease to release the fluorescein amidite (FAM)-labeled SA aptamer, which subsequently binds to streptavidin beads. The enhancement of the fluorescence signal on SA beads can be detected precisely and easily by a microscope or flow cytometer. Our method performs well in complex biological samples as a result of the enrichment of the signaling molecules on beads, as well as simple manipulations to discard the background interference and nonbinding molecules. Without signal amplification techniques, our AAP method not only avoids complicated manipulations but also decreases the time required. With the advantages of ease of operation, reliability, and robustness for T4 PNK detection in buffer as well as real biological samples, the AAP has great potential for clinical diagnostics, inhibitor screening, and drug discovery.
作为 DNA 磷酸化和修复过程中的一种重要酶，T4 多核苷酸激酶（T4 PNK）近年来引起了极大的兴趣。因此，已经建立了许多基于各种信号放大技术的高灵敏度 T4 PNK 检测策略。然而，它们通常需要复杂的设计、各种辅助试剂和酶，或者繁琐的操作。我们设计了一种新型的别构适体探针（AAP），由链霉亲和素（SA）适体和互补 DNA（cDNA）组成，用于简单检测 T4 PNK，无需信号放大，并且可以最小化复杂生物样品中的干扰。当 cDNA 的 5'-末端被 T4 PNK 磷酸化时，cDNA 被 λ 核酸外切酶降解，释放出荧光素胺（FAM）标记的 SA 适体，随后该适体与链霉亲和素珠结合。通过显微镜或流式细胞仪可以精确、轻松地检测到 SA 珠上荧光信号的增强。由于珠上信号分子的富集以及简单的操作来去除背景干扰和非结合分子，我们的方法在复杂的生物样品中表现良好。无需信号放大技术，我们的 AAP 方法不仅避免了复杂的操作，还减少了所需的时间。AAP 在缓冲液和真实生物样品中具有操作简单、可靠性高、T4 PNK 检测稳健等优点，在临床诊断、抑制剂筛选和药物发现方面具有巨大的潜力。
