Acta Phys. -Chim. Sin. ›› 2022, Vol. 38 ›› Issue (4): 2004017.doi: 10.3866/PKU.WHXB202004017

• ARTICLE • Previous Articles     Next Articles

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

Wei Zhou, Yunchao Li, Louzhen Fan, Xiaohong Li()   

  • Received:2020-04-04 Accepted:2020-05-21 Published:2020-05-27
  • Contact: Xiaohong Li
  • About author:Xiaohong Li, Email:; Tel.: +86-10-58802741
  • Supported by:
    the National Natural Science Foundation of China(21673022)


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: Thioflavin T, G-quadruplex, Potassium ion, Sodium ion, Double-stranded DNA