Analytical and Bioanalytical Chemistry ( IF 3.8 ) Pub Date : 2022-09-22 , DOI: 10.1007/s00216-022-04327-6 Liuya Wei 1 , Xianglong Kong 1 , Mengran Wang 1 , Yixin Zhang 2 , Ruiyan Pan 1 , Yuanzheng Cheng 1 , Zhihua Lv 3 , Jin Zhou 1 , Jingjing Ming 1
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The phosphorylation process of DNA by T4 polynucleotide kinase (T4 PNK) plays a crucial role in DNA recombination, DNA replication, and DNA repair. Traditional monomeric G-quadruplex (G4) systems are always activated by single cation such as K+ or Na+. The conformation transformation caused by the coexistence of multiple cations may interfere with the signal readout and limit their applications in physiological system. In view of the stability of dimeric G4 in multiple cation solution, we reported a label-free T4 PNK fluorescence sensor based on split dimeric G4 and ligation-induced dimeric G4/thioflavin T (ThT) conformation. The dimeric G4 was divided into two independent pieces of one normal monomeric G4 and the other monomeric G4 fragment phosphorylated by T4 PNK in order to decrease the background signal. With the introduction of template DNA, DNA ligase, and invasive DNA, the dimeric G4 could be generated and liberated to combine with ThT to show obvious fluorescence signal. Using our strategy, the linear range from 0.005 to 0.5 U mL−1, and the detection limit of 0.0021 U mL−1 could be achieved without the consideration of interference caused by the coexistence of multiple cations. Additionally, research in real sample determination and inhibition effect investigations indicated its further potential application value in biochemical process research and clinic diagnostics.
Graphical abstract
中文翻译：
基于分裂二聚体G-四链体和连接诱导二聚体G-四链体/硫黄素T构象的无标记T4多核苷酸激酶荧光传感器
T4 多核苷酸激酶 (T4 PNK) 对 DNA 的磷酸化过程在 DNA 重组、DNA 复制和 DNA 修复中起着至关重要的作用。传统的单体 G-四链体 (G4) 系统总是由单一阳离子如 K +或 Na +激活. 多种阳离子共存引起的构象转变可能会干扰信号读出，限制其在生理系统中的应用。鉴于二聚体 G4 在多阳离子溶液中的稳定性，我们报道了一种基于分裂二聚体 G4 和连接诱导二聚体 G4/硫黄素 T (ThT) 构象的无标记 T4 PNK 荧光传感器。二聚体 G4 被分成两个独立的片段，一个是正常单体 G4，另一个是被 T4 PNK 磷酸化的单体 G4 片段，以降低背景信号。随着模板DNA、DNA连接酶和侵入性DNA的引入，可以产生并释放出二聚体G4，与ThT结合，显示出明显的荧光信号。使用我们的策略，线性范围从 0.005 到 0.5 U mL -1，在不考虑多种阳离子共存干扰的情况下，可以达到0.0021 U mL -1的检测限。此外，在实际样品测定和抑制作用研究方面的研究表明其在生化过程研究和临床诊断方面的进一步潜在应用价值。
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