硫黄素T与侧翼连接双链DNA的G-四链体高特异性作用

引用本文: 周蔚, 李运超, 范楼珍, 李晓宏. 硫黄素T与侧翼连接双链DNA的G-四链体高特异性作用[J]. 物理化学学报, 2022, 38(4): 200401. doi: 10.3866/PKU.WHXB202004017 shu

Citation: Wei Zhou, Yunchao Li, Louzhen Fan, Xiaohong Li. Thioflavin T Specifically Binding with G-Quadruplex Flanked by DoubleStranded DNA[J]. Acta Physico-Chimica Sinica, 2022, 38(4): 200401. doi: 10.3866/PKU.WHXB202004017 shu

硫黄素T与侧翼连接双链DNA的G-四链体高特异性作用

北京师范大学化学学院，北京 100875

通讯作者: 李晓宏, lxhxiao@bnu.edu.cn

基金项目:
国家自然科学基金 21673022

摘要: 富G碱基的DNA序列在离子诱导下可形成G-四链体(G4)，基于这一构型转化设计了大量的传感检测平台。其中的荧光检测平台是基于G4与荧光小分子的相互作用。但是，G4与荧光小分子的有效结合依赖于G4构型和体系中存在的离子种类和离子浓度，尤其是高Na⁺浓度(140 mmol·L^-1)。那么如何实现G4与荧光小分子普适性地有效结合，并不依赖于体系中的Na⁺和Na⁺浓度，是一个难题。在本研究中，以最简单的富G DNA序列凝血酶适体链TBA (thrombin binding aptamer)为例，在3’端和5’端分别增加10个碱基(TBA-10 bp)，K⁺诱导TBA-10 bp形成K⁺稳定TBA (K⁺-TBA，G4)并衔接含有10个互补碱基对的双链DNA (K⁺-TBA-10 bp)。相较于K⁺-TBA，硫磺素T与K⁺-TBA-10 bp结合后的荧光强度增加了100倍，相互作用强度增加了1000倍，而且与体系中的Na⁺ (5-140 mmol·L^-1)无关。结合荧光光谱，紫外吸收光谱和圆二色光谱发现硫磺素T特异性的嵌合于K⁺-TBA和双链DNA衔接处的空腔内。有趣的是，这一结合模式不受G4构型的影响。该研究结果为研究G4与荧光小分子的有效结合提供了新视角，也为拓展G4在生物功能和生化检测领域的应用提供了实验依据。

关键词:

English

Thioflavin T Specifically Binding with G-Quadruplex Flanked by DoubleStranded DNA

College of Chemistry, Beijing Normal University, Beijing 100875, China

Corresponding author: Xiaohong Li, Email: lxhxiao@bnu.edu.cn; Tel.: +86-10-58802741

Received Date: 04 April 2020
Accepted Date: 21 May 2020
Revised Date: 20 May 2020
Available Online: 15 April 2022
Fund Project:
the National Natural Science Foundation of China

Abstract: G-rich DNA sequences can transform into G-quadruplexes (G4s) in the presence of metal ions. Based on the structural switches, G4 has been recognized as an attractive signal-transducing element for constructing colorimetric, electrochemical, and fluorescent sensing platforms capable of recognizing ions, small biological molecules, proteins, and even cells. For fluorescent sensing platforms, fluorescent small molecules (FSMs) specifically binding with G4s, such as crystal violet (CV), protoporphyrin IX (PPIX), zinc protoporphyrin IX (ZnPPIX), and Thioflavin T (ThT), are usually applied as fluorescent signal readout probes. It was noticed that the binding affinity of FSM with G4 is highly dependent on G4 morphologies because G-rich DNA sequences can fold into multiple G4 conformations, such as parallel, antiparallel, or hybrid. For example, CV only binds with antiparallel G4, PPIX or ZnPPIX preferentially interacts with parallel G4, and ThT displays high affinity for hybrid G4. Furthermore, the binding affinity of FSMs with G4 is also dependent on co-existing ions and ion concentrations, especially elevated Na⁺ level (140 mmol·L^-1). It is the reason why the performance of G4-based sensors in biological and environmental samples is decreased with different extents. Therefore, how to design G-rich DNA sequences to generally achieve FSMs specifically binding with G4, which is independent of G4 morphologies and co-existing Na⁺ and Na⁺ concentrations remains a challenge. In this study, a simple G-rich DNA sequence (thrombin binding aptamer, TBA) flanked by 10-mer single-stranded DNA at the 3' and 5' termini (TBA-10 bp) is designed. In the presence of K⁺, TBA transforms into antiparallel G4 (K⁺-TBA) and TBA-10 bp transforms into antiparallel K⁺-TBA flanked by fully hybridized double-stranded DNA (ds-DNA) (K⁺-TBA-10 bp). Actually, ThT cannot effectively bind with antiparallel K⁺-TBA. Compared with K⁺-TBA, upon K⁺-TBA-10 bp binding with ThT, ThT emission fluorescence increased by 100-fold. Importantly, the binding affinity improved by 1000-fold, which is independent of co-existing Na⁺ and Na⁺ concentrations (5-140 mmol·L^-1). Integrated with UV-Vis spectroscopy, fluorescent spectroscopy, and circular dichroism spectroscopy, it is believed that ThT can specifically and efficiently imbed in the junction between K⁺-TBA and ds-DNA. To corroborate the binding mode, TBA in TBA-10 bp is substituted by other G-rich DNA sequences transforming into parallel and antiparallel G4 in the presence of K⁺, respectively. The resulting improved ThT emission fluorescence indicated that such a specific binding mode generally improved the binding affinity of FSMs with G4. Our findings provide new insights into the improvement of the binding affinity of FSMs and G4, and reveal potential biochemical and bioanalytical applications of G4.

Key words:

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通讯作者: 陈斌, bchen63@163.com

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沈阳化工大学材料科学与工程学院沈阳 110142

硫黄素T与侧翼连接双链DNA的G-四链体高特异性作用

通讯作者: 李晓宏, lxhxiao@bnu.edu.cn

English

Thioflavin T Specifically Binding with G-Quadruplex Flanked by DoubleStranded DNA

Corresponding author: Xiaohong Li, Email: lxhxiao@bnu.edu.cn; Tel.: +86-10-58802741

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通讯作者: 陈斌, bchen63@163.com

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硫黄素T与侧翼连接双链DNA的G-四链体高特异性作用

通讯作者: 李晓宏, lxhxiao@bnu.edu.cn

English

Thioflavin T Specifically Binding with G-Quadruplex Flanked by DoubleStranded DNA

Corresponding author: Xiaohong Li, Email: lxhxiao@bnu.edu.cn; Tel.: +86-10-58802741

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南： 你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

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