Liu Lingling, Shao Yong, Peng Jian, Huang Chaobiao, Liu Hua, Zhang Lihua
Institute of Physical Chemistry, Zhejiang Normal University , Jinhua 321004, Zhejiang, People's Republic of China.
Anal Chem. 2014 Feb 4;86(3):1622-31. doi: 10.1021/ac403326m. Epub 2014 Jan 17.
This work demonstrates the significant fluorescence enhancement of thioflavin T (ThT) when binding to G-quadruplexes possessing hybrid structures by using UV-vis absorption spectra, fluorescence spectra, and Tm experiments to confirm the binding events. ThT binding does not disturb native G-quadruplex structures preformed in Na(+) and K(+) solutions. The fluorescence enhancement is caused by the rotation restriction of benzothiazole (BZT) and dimethylaminobenzene (DMAB) rings in the ThT excited state upon its G-quadruplex binding. This molecular rotor mechanism as a means of fluorescence enhancement is confirmed using a nonrotor analogue of ThT. Hydroxylation and electrolyte experiments demonstrate that ThT stacks on the tetrad of the hybrid G-quadruplexes, whereas electrostatic forces contribute more to ThT binding for other G-quadruplex structures. By stacking on the tetrad, the ThT binding favors selective identification of DNA hybrid G-quadruplex structures with enhanced fluorescence and can serve as a conformation probe to monitor G-quadruplex structure conversion between hybrid and other structures. Using these properties, we developed a selective and label-free fluorescent K(+) sensor with a detection limit of 1 mM for K(+) in the presence of 100 mM Na(+). The coexistence of other metal ions produces a fluorescence response comparable to K(+) alone. We believe that ThT can potentially provide structure identification of hybrid G-quadruplexes and aid in the construction of G-quadruplex-based sensors.
这项工作通过紫外可见吸收光谱、荧光光谱和熔解温度（Tm）实验证实结合事件，展示了硫黄素T（ThT）与具有杂化结构的G-四链体结合时显著的荧光增强。ThT结合不会干扰在Na⁺和K⁺溶液中预先形成的天然G-四链体结构。荧光增强是由于ThT在激发态下与G-四链体结合时，苯并噻唑（BZT）和二甲基氨基苯（DMAB）环的旋转受限所致。使用ThT的非旋转类似物证实了这种作为荧光增强手段的分子转子机制。羟基化和电解质实验表明，ThT堆积在杂化G-四链体的四分体上，而静电力对其他G-四链体结构的ThT结合贡献更大。通过堆积在四分体上，ThT结合有利于选择性识别具有增强荧光的DNA杂化G-四链体结构，并可作为构象探针监测杂化结构与其他结构之间的G-四链体结构转换。利用这些特性，我们开发了一种选择性且无标记的荧光钾离子传感器，在存在100 mM Na⁺的情况下，对K⁺的检测限为1 mM。其他金属离子的共存产生的荧光响应与单独的K⁺相当。我们相信，ThT有可能提供杂化G-四链体的结构识别，并有助于构建基于G-四链体的传感器。
