Design mitochondria-specific fluorescent turn-on probes targeting G-quadruplexes for live cell imaging and mitophagy monitoring study

A series of new mitochondrial-selective fluorescent probe was designed and synthesized based on the integration of two planar molecular scaffolds of benzo-indole/indole with a p-substituted styrene moiety. The small-sized probes are flexible and rotatable via an ethylene bridge. The restriction of free rotation upon interaction with targeting biomolecules of G-quadruplexes in mitochondria generates strong emission in visible range (575–615 nm). Cell imaging study showed that the ligands are targeting mitochondria but not nucleus. Competition experiments showed that the ligand BYM is highly selective towards mitochondrial G4-DNA structures against other non-G4 nucleic acid structures including single-/double-stranded DNA and hairpin. The equilibrium binding constant (K_eq) of BYM interacting with mitochondrial G4-DNA (mt6363, K_eq = 10.8 × 10⁶ M⁻¹) is almost 1000-fold higher than that of mitochondrial double-stranded DNA (K_eq = 0.01 × 10⁶ M⁻¹). The probe also showed high sensitivity (LOD = 1.52 nM) and good linear relationship (R² = 0.9983) with the mitochondrial G4-DNA. The delivery of BYM to mitochondria is not mitochondrial membrane potential dependent but mainly through the permeability transition pore on the mitochondrial inner membrane. In addition, BYM exhibits low cytotoxicity against a number of human cancer and noncancerous cells, indicating that BYM could be a good ligand for bioorthogonal study in live cells by targeting the mitochondrial G4-DNA structures. In the present study, BYM was demonstrated in monitoring the dynamic process of mitochondrial autophagy. The small-sized fluorescent probe possessing high photostability, selectivity and sensitivity targeting mitochondrial G-quadruplexes may able to provide a useful chemical tool for real-time study of mitochondrial functions in live cells.