Harnessing Light for G-Quadruplex Modulation: Dual Isomeric Effects of an <i>Ortho</i>-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 <i>ortho</i>-fluoroazobenzene derivative, <b>Py-Azo4F-3N</b>, 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 <i>trans</i> 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.