Harnessing Light for G‑Quadruplex Modulation: Dual Isomeric Effects of an Ortho-Fluoroazobenzene Derivative

G-quadruplexes (G4s) are important therapeutic and photopharmacological targets in cancer research. Small-molecule ligands targeting G4s offer a promising strategy to block DNA transactions and induce genetic instability in cancer cells. While numerous G4-ligands have been reported, relatively few examples exist of compounds whose G4-interactive binding properties can be modulated using light. Herein, we report the photophysical characterization of a novel ortho-fluoroazobenzene derivative, Py-Azo4F-3N, that undergoes reversible two-way isomerization upon visible light exposure. Using a combination of biophysical techniques, including affinity and selectivity assays, structural and computational analysis, and cytotoxicity experiments in cancer cell lines, we carefully characterized the G4-interactive binding properties of both isomers. We identify the trans isomer as the most promising form of interacting and stabilizing G4s, enhancing their ablation capability in cancer cells. Our research highlights the importance of light-responsive molecules in achieving precise control over G4 structures, demonstrating their potential in innovative anticancer strategies.