Development of a high quantum yield probe for detection of mitochondrial G-quadruplexes in live cells based on fluorescence lifetime imaging microscopy

Mitochondrial G-quadruplexes are components that are potentially involved in regulating mitochondrial function and play crucial roles in the replication and transcription of mitochondrial genes. Consequently, it is imperative to develop probes that can detect mitochondrial G-quadruplexes to understand their functions and mechanisms. In this study, a triphenylamine fluorescent probe, TPPE, which has excellent cytocompatibility and does not affect the natural state of G-quadruplexes, was designed and demonstrated to localize primarily to the mitochondria. Owing to the unique binding mode between TPPE and G-quadruplexes, TPPE was able to distinguish G-quadruplexes from other substances due to the higher fluorescence lifetime and quantum yield. On the basis of the photon counts determined via fluorescence lifetime imaging microscopy, we analyzed the differences in the numbers of mitochondrial G-quadruplexes in various cell lines. We observed reductions in the number of mitochondrial G-quadruplexes during apoptosis, ferroptosis and glycolysis inhibition. This study shows the great potential of using TPPE to track and analyze mitochondrial G-quadruplexes and presents a novel perspective in the development of probes to detect mitochondrial G-quadruplexes in live cells.