Abstract
<jats:p>G-quadruplex (G4) DNA structures play crucial roles in gene regulation and are implicated in diseases such as cancer, making them compelling targets for chemical biology. Selective detection of specific G4 topologies, especially the parallel conformation, in live cells remains a significant challenge. We report two cyclometalated platinum(II) complexes, OPtC1 and OPtC2, as conformation-specific fluorescent probes for parallel G4 DNA. They exhibit high selectivity (~27-fold) for parallel G4s over duplex DNA via a synergistic end-stacking and groove-binding mode. In live cells, OPtC1 shows excellent photostability, low cytotoxicity, and precise nuclear localization. Crucially, by employing fluorescence lifetime imaging microscopy (FLIM), we demonstrate that OPtC1 selectively visualizes parallel G4 structures. The binding is confirmed by a distinct lifetime attenuation mediated via Förster resonance energy transfer (FRET), providing a robust, intensity-independent signature. This work establishes OPtC1 as a powerful probe for parallel G4s and showcases fluorescence lifetime as a conformation-specific reporter for validating target engagement in living systems, advancing beyond conventional intensity-based microscopy.</jats:p>