Loop Site Mutation-Enhanced Sensing Performance of G-Triplex Probe: Preliminary Exploration on Its Stability and “Structure-Efficiency” Relationship

G-triplex is a unique topological structure formed by G-rich nucleic acid sequences with three G-tracts. It can bind to small ligands and function as a label-free sensing probe. However, the number of discovered G-triplex/small ligands with stable structures and excellent performance is still limited, requiring deeper insight on their "structure-efficiency" relationships. In this paper, we systematically investigated the effects of loop bases on the structure stability and preliminarily explored the "structure-efficiency" relationship between the loop composition and sensing performance by using thioflavin T (ThT) and hemin as ligand models. We found that the number of "A" bases of loop1 was important for small ligands binding, and the short "C" bases of loop2 were critical for the stability of the G-triplex structure. Further molecular dynamics simulations between G-triplex and small ligands supported the argument. From the exploration, we sought a novel G-triplex sequence G31-2. Although the novel G31-2 sequence differed from the previously reported sequence G31 by only one base, significant performance enhancement, shorter self-assembling time, and higher binding affinity were unprecedentedly achieved in the binding with both the small ligands (ThT and hemin), and meanwhile, excellent structure stability and controllability were maintained. Finally, a straightforward, label-free fluorescent assay for alkaline phosphatase activity detection was designed and achieved by employing the G31-2/ThT probe. The discoveries observed in this work will serve as a new inspiring guideline for understanding G-rich sequences folding and developing new functional G-triplex/small ligand probes, which may facilitate and promote their advanced applications in diagnosis, therapy, and biosensing.