Zhang Ling, Zhang Kaixiang, Rauf Sana, Dong Duo, Liu Yang, Li Jinghong
Department of Chemistry, Beijing Key Laboratory for Analytical Methods and Instrumentation, Tsinghua University , Beijing 100084, China.
Anal Chem. 2016 Apr 19;88(8):4533-40. doi: 10.1021/acs.analchem.6b00555. Epub 2016 Apr 6.
Ligands that selectively promote the formation of G-quadruplexes in human telomeres have great potential for cancer treatment by inhibiting the telomere extension by telomerase. Thus, understanding the interactions of the G-quadruplex ligands with the telomere sequence at the single-molecule level is of significant importance. Here, human telomere sequence interactions with a small molecule ligand pyridostatin (PDS) were analyzed via α-hemolysin protein nanopore, and a nanopore thermodynamic analytical method was proposed. The prolonged unraveling time of the telomeric DNA G-quadruplex after PDS binding demonstrated the potent stabilization effect of ligand on G-quadruplex structure. The signature two-level electronic blocks generated by K(+)-PDS-G-quadruplex complexes suggested a two-state unraveling process, including the dissociation of the interquartet cation and the unraveling of the cation-free ligand-bound G-quadruplex. The translocation studies and the analysis of free-energy changes demonstrated a ligand-binding mode in which PDS molecule and K(+) were simultaneously bound to one G-quadruplex structure with the coordinated effect on G-quadruplex stabilization. The single-molecular nanopore platform permits the efficient and accurate determination of ligand affinity constants without the requirement for labeling, amplification, or ligand/receptor titration, which provides a general analytical tool for effectively monitoring and quantifying the G-quadruplex/ligand interactions, possessing important implications for the design and screen of anticancer drugs.
能够选择性促进人类端粒中G-四链体形成的配体，在通过抑制端粒酶的端粒延伸来治疗癌症方面具有巨大潜力。因此，在单分子水平上理解G-四链体配体与端粒序列之间的相互作用具有重要意义。在此，通过α-溶血素蛋白纳米孔分析了人类端粒序列与小分子配体吡啶硫酮（PDS）的相互作用，并提出了一种纳米孔热力学分析方法。PDS结合后端粒DNA G-四链体解旋时间的延长，证明了配体对G-四链体结构的有效稳定作用。由K(+)-PDS-G-四链体复合物产生的标志性双电平电子阻断表明了一个双态解旋过程，包括四重态间阳离子的解离和无阳离子配体结合的G-四链体的解旋。转位研究和自由能变化分析表明了一种配体结合模式，其中PDS分子和K(+)同时结合到一个G-四链体结构上，对G-四链体稳定具有协同作用。单分子纳米孔平台允许高效、准确地测定配体亲和常数，而无需标记、扩增或配体/受体滴定，这为有效监测和量化G-四链体/配体相互作用提供了一种通用分析工具，对抗癌药物的设计和筛选具有重要意义。
