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. • The reversible regulation of oligonucleotide secondary structure, particularly in the context of studying the role and function of G4 DNA is of significant importance. • A red-light-switchable amido-pyridinium dithienylethene (DTE) reversibly modulates G-quadruplex DNA and shows anticancer activity. • Incorporation of an amide linker into the DTE scaffold enhances photoisomer-dependent G4 binding, yielding up to 7-fold affinity differences. • Biophysical and docking experiments show the open isomer preferentially targets cancer-related G4s and is 5× more toxic to HeLa over healthy fibroblasts. • This study demonstrates proof of concept of bidirectional control of G4 binding and biological activity, advancing photo-pharmacology.
Allen et al. (Sun,) studied this question.