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Loop Site Mutation-Enhanced Sensing Performance of G-Triplex Probe: Preliminary Exploration on Its Stability and

Hui Zhou1,2, Feng Huang1, Yingyan Xie1

  • 1School of Pharmacy, Guangdong Medical University, Dongguan 523808, China.

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|August 11, 2025
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Summary

Researchers optimized G-rich nucleic acid structures (G-triplexes) for biosensing by analyzing loop base effects. A new G-triplex sequence (G31-2) showed enhanced ligand binding and stability, enabling a novel fluorescent assay for alkaline phosphatase activity.

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Area of Science:

  • Biochemistry and Molecular Biology
  • Nucleic Acid Chemistry
  • Biosensing Technologies

Background:

  • G-triplexes are unique G-rich nucleic acid structures with potential as label-free sensing probes.
  • Limited understanding of G-triplex/small ligand structure-efficiency relationships hinders probe development.
  • Optimization is needed for stable G-triplex structures with enhanced sensing capabilities.

Purpose of the Study:

  • To systematically investigate the impact of loop bases on G-triplex structure stability.
  • To explore the relationship between loop composition and sensing performance using thioflavin T (ThT) and hemin.
  • To develop a novel G-triplex sequence with improved binding affinity, stability, and sensing performance.

Main Methods:

  • Systematic analysis of loop base effects on G-triplex structure stability.
  • Preliminary exploration of structure-efficiency relationships using ThT and hemin as ligand models.
  • Molecular dynamics simulations to support findings on G-triplex and small ligand interactions.
  • Design and validation of a label-free fluorescent assay for alkaline phosphatase activity.

Main Results:

  • Loop1 'A' bases are important for small ligand binding; short loop2 'C' bases are critical for G-triplex stability.
  • A novel G-triplex sequence (G31-2) demonstrated significantly enhanced performance, faster self-assembly, and higher binding affinity compared to G31.
  • The G31-2 sequence maintained excellent structure stability and controllability.
  • A straightforward, label-free fluorescent assay for alkaline phosphatase detection was successfully developed using the G31-2/ThT probe.

Conclusions:

  • Loop base composition critically influences G-triplex stability and ligand binding efficiency.
  • The novel G31-2 G-triplex sequence offers superior performance for biosensing applications.
  • This work provides guidelines for developing advanced G-triplex/small ligand probes for diagnosis, therapy, and biosensing.