Development of highly potent fluorogenic triplex-specific ligands

Triplex-forming oligonucleotides (TFOs) have long attracted interest due to their remarkable sequence specificity in targeting duplex DNA, allowing recognition of extended DNA sequences. However, the formation of triplex structures is both thermodynamically and kinetically unfavorable under physiological conditions. To address these limitations while preserving the specificity of TFOs, researchers have developed small-molecule ligands that selectively stabilize triplex DNA. In this study, we present a novel class of highly potent fluorogenic ligands specific to triplex DNA, derived from natural flavone scaffolds. We evaluated their binding affinity and specificity for triplex DNA using a range of biophysical techniques, including UV thermal denaturation, fluorescence spectroscopy, circular dichroism, and gel mobility shift assays. These ligands effectively inhibit enzymatic activity through ligand-mediated triplex formation. Notably, they exhibit excellent selectivity for triplex DNA, inducing intense blue fluorescence, whereas they show negligible fluorescence enhancement with duplex DNA. Overall, our studies show that these ligands have strong potential as fluorogenic probes for detecting triplex DNA and as therapeutic agents for gene-regulation applications.