Red-light-activated and thermally-stable amido-dithienylethenes for the reversible control of G-quadruplex binding and anticancer activity.

Photo-responsive G-quadruplex (G4) ligands offer a powerful means to achieve spatiotemporal control over nucleic acid targeting, yet many existing scaffolds suffer from limited thermal stability or modest differences in affinity between photo-isomers. Here we report a thermally stable amido-pyridinium dithienylethene ligand (2) designed to enhance photo-isomer-dependent G4 recognition and increased structural modulation upon switching. Ligand 2 undergoes efficient and reversible photo-isomerization between its open and closed forms under near-UV and red-light irradiation, with long-lived photo-stationary states. Biophysical assays demonstrate that the open-isomer binds cancer-relevant G4 oligonucleotide sequences with 2-7-fold higher affinity than the closed form, while retaining high selectivity over duplex DNA. NMR studies reveal widespread perturbations across multiple G4 topologies, consistent with groove-associated interactions, and confirm that structural changes can be reversibly modulated by alternating irradiation. Importantly, the open-isomer exhibits a five-fold increase in cytotoxicity toward HeLa cells compared to the closed form, while showing negligible toxicity in healthy fibroblasts. Overall, ligand 2 represents a red-light-activated and thermally robust photo-switchable scaffold capable of reversible control over G4 binding and anticancer activity. These findings highlight the potential of amide-modified DTE frameworks as next-generation photo-responsive agents for precise regulation of G4-binding activity and anticancer activity.