English

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

• Wei Zhou ,
• Yunchao Li ,
• Louzhen Fan ,
• Xiaohong Li ^,

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

• 1. [1]

     Davis, J. T. Angew. Chem. Int. Ed. 2004, 43, 668. doi: 10.1002/anie.200300589

  2. [2]

     Maizels, N. Nat. Struct. Mol. Biol. 2006, 13, 1055. doi: 10.1038/nsmb1171

  3. [3]

     Sun, H.; Li, X.; Li, Y.; Fan, L.; Kraatz, H. B. Analyst 2013, 138, 856. doi: 10.1039/c2an36564b

  4. [4]

     Xu, L.; Shen, X.; Hong, S.; Wang, J.; Zhang, Y.; Wang, H.; Zhang, J.; Pei, R. Chem. Commun. 2015, 51, 8165. doi: 10.1039/c5cc01590a

  5. [5]

     Liu, J.; Lu, Y. J. Am. Chem. Soc. 2003, 125, 6642. doi: 10.1021/ja034775u

  6. [6]

     Li, C. L.; Liu, K. T.; Lin, Y. W.; Chang, H. T. Anal. Chem. 2011, 83, 225. doi: 10.1021/ac1028787

  7. [7]

     Kim, H. N.; Ren, W. X.; Kim, J. S.; Yoon, J. Chem. Soc. Rev. 2012, 41, 3210. doi: 10.1039/c1cs15245a

  8. [8]

     Hwang, K.; Wu, P.; Kim, T.; Lei, L.; Tian, S.; Wang, Y.; Lu, Y. Angew. Chem. Int. Ed. 2014, 53, 13798. doi: 10.1002/anie.201408333

  9. [9]

     Yang, L.; Qing, Z.; Liu, C.; Tang, Q.; Li, J.; Yang, S.; Zheng, J.; Yang, R.; Tan, W. Anal. Chem. 2016, 88, 9285. doi: 10.1021/acs.analchem.6b02667

  10. [10]

      Yang, J.; Dou, B.; Yuan, R.; Xiang, Y. Anal. Chem. 2016, 88, 8218. doi: 10.1021/acs.analchem.6b02035

  11. [11]

      Liu, Z.; Luo, X.; Li, Z.; Huang, Y.; Nie, Z.; Wang, H. H.; Yao, S. Anal. Chem. 2017, 89, 1892. doi: 10.1021/acs.analchem.6b04360

  12. [12]

      Li, X. M.; Zheng, K. W.; Hao, Y. H.; Tan, Z. Angew. Chem. Int. Ed. 2016, 55, 13759. doi: 10.1002/anie.201607195

  13. [13]

      Hansel-Hertsch, R.; Antonio, M. D.; Balasubramanian, S. Nat. Rev. Mol. Cell Biol. 2017, 18, 279. doi: 10.1038/nrm.2017.3

  14. [14]

      Ge, L.; Wang, W.; Sun, X.; Hou, T.; Li, F. Anal. Chem. 2016, 88, 9691. doi: 10.1021/acs.analchem.6b02584

  15. [15]

      李晓宏, 于泽, 李运超, 叶明富. 物理化学学报2018, 34, 1293. doi: 10.3866/PKU.WHXB201804111Li, X. H.; Yu, Z.; Li, Y. C.; Ye, M. F. Acta Phys. -Chim. Sin. 2018, 34, 1293. doi: 10.3866/PKU.WHXB201804111

  16. [16]

      欧植泽, 高云燕, 蔡温姣, 马拖拖, 倚娜, 李志远. 物理化学学报2019, 35, 230. doi: 10.3866/PKU.WHXB201711281Ou, Z. Z.; Gao, Y. Y.; Cai, W. J.; Ma, T. T.; Yi, N.; Li, Z. Y. Acta Phys. -Chim. Sin. 2019, 35, 230. doi: 10.3866/PKU.WHXB201711281

  17. [17]

      Wang, M.; Wang, W.; Kang, T. S.; Leung, C. H.; Ma, D. L. Anal. Chem. 2016, 88, 981. doi: 10.1021/acs.analchem.5b04064

  18. [18]

      Liu, Z.; Luo, X.; Li, Z.; Huang, Y.; Nie, Z.; Wang, H. H.; Yao, S. Anal. Chem. 2017, 89, 1892. doi: 10.1021/acs.analchem.6b04360

  19. [19]

      Ge, B.; Huang, Y. C.; Sen, D.; Yu, H. Z., Angew. Chem. Int. Ed. 2010, 49, 9965. doi: 10.1002/anie.201004946

  20. [20]

      Leung, K. H.; He, B.; Yang, C.; Leung, C. H.; Wang, H. M.; Ma, D. L. ACS Appl. Mater. Interfaces 2015, 7, 24046. doi: 10.1021/acsami.5b08314

  21. [21]

      Zhang, L.; Zhu, J.; Li, T.; Wang, E. Anal. Chem. 2011, 83, 8871. doi: 10.1021/ac2006763

  22. [22]

      Hud, N. V. Nucleic Acid-Metal Ion Interactions; Royal Society of Chemistry: Cambridge, UK, 2009.

  23. [23]

      Neidle, S.; Balasubramanian, S. Quadruplex Nucleic Acids; Royal Society of Chemistry: Cambridge, UK, 2006; Vol. 7.

  24. [24]

      Kong, D. M.; Ma, Y. E.; Guo, J. H.; Yang, W.; Shen, H. X. Anal. Chem. 2009, 81, 2678. doi: 10.1021/ac802558f

  25. [25]

      Li, T.; Wang, E.; Dong, S. Anal. Chem. 2010, 82, 7576. doi: 10.1021/ac1019446

  26. [26]

      Liu, L.; Shao, Y.; Peng, J.; Huang, C.; Liu, H.; Zhang, L. Anal. Chem. 2014, 86, 1622. doi: 10.1021/ac403326m

  27. [27]

      Hu, M. H.; Zhou, J.; Luo, W. H.; Chen, S. B.; Huang, Z. S.; Wu, R.; Tan, J. H. Anal. Chem. 2019, 91, 2480. doi: 10.1021/acs.analchem.8b05298

  28. [28]

      Wang, M.; Wang, W.; Kang, T. S.; Leung, C. H.; Ma, D. L. Anal. Chem. 2016, 88, 981. doi: 10.1021/acs.analchem.5b04064

  29. [29]

      Wu, S.; Wang, L.; Zhang, N.; Liu, Y.; Zheng, W.; Chang, A.; Wang, F.; Li, S.; Shangguan, D. Chem. -Eur. J. 2016, 22, 6037. doi: 10.1002/chem.201505170

  30. [30]

      Zhao, D.; Dong, X.; Jiang, N.; Zhang, D.; Liu, C. Nucleic Acids Res. 2014, 42, 11612. doi: 10.1093/nar/gku833

  31. [31]

      Renaud de la Faverie, A.; Guedin, A.; Bedrat, A.; Yatsunyk, L. A.; Mergny, J. L. Nucleic Acids Res. 2014, 42, e65. doi: 10.1093/nar/gku111

  32. [32]

      Mohanty, J.; Barooah, N.; Dhamodharan, V.; Harikrishna, S.; Pradeepkumar, P. I.; Bhasikuttan, A. C. J. Am. Chem. Soc. 2013, 135, 367. doi: 10.1021/ja309588h

  33. [33]

      Zhou, W.; Yu, Z.; Ma, G.; Jin, T.; Li, Y.; Fan, L.; Li, X. Analyst 2019, 144, 2284. doi: 10.1039/C8AN02430H

  34. [34]

      De Rache, A.; Kejnovska, I.; Vorlickova, M.; Buess-Herman, C. Chem. -Eur. J. 2012, 18, 4392. doi: 10.1002/chem.201103381

  35. [35]

      Vorlíčková, M.; Kejnovská, I.; Bednářová, K.; Renčiuk, D.; Kypr, J. Chirality 2012, 24, 691. doi: 10.1002/chir.22064

  36. [36]

      Dutta, K.; Fujimoto, T.; Inoue, M.; Miyoshi, D.; Sugimoto, N. Chem. Commun. 2010, 46, 7772. doi: 10.1039/c0cc00710b

  37. [37]

      Zhang, D.; Han, J.; Li, Y.; Fan, L.; Li, X. J. Phys. Chem. B 2016, 120, 6606. doi: 10.1021/acs.jpcb.6b05002

  38. [38]

      Kong, D. M.; Ma, Y. E.; Guo, J. H.; Yang, W.; Shen, H. X. Anal. Chem. 2009, 81, 2678. doi: 10.1021/ac802558f

  39. [39]

      Waller, Z. A. E.; Sewitz, S. A.; Hsu, S. T. D.; Balasubramanian, S. J. Am. Chem. Soc. 2009, 131, 12628. doi: 10.1021/ja901892u

  40. [40]

      Zhang, R.; Cheng, M.; Zhang, L. M.; Zhu, L. N.; Kong, D. M. ACS Appl. Mater. Interfaces 2018, 10, 13350. doi: 10.1021/acsami.8b01901

  41. [41]

      Liu, L.; Shao, Y.; Peng, J.; Huang, C.; Liu, H.; Zhang, L. Anal. Chem. 2014, 86, 1622. doi: 10.1021/ac403326m

•