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

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.