Topology-Resolved Imaging of Mitochondrial Nucleoid Condensates Uncovers Dynamic G-Quadruplex Remodeling

G-quadruplex structures in mitochondrial DNA (mtG4DNA) have been implicated in mitochondrial genome regulation and cellular metabolism, yet their spatial organization within mitochondrial nucleoids remains poorly understood. A central challenge is that mtG4DNA cannot be readily distinguished from mitochondrial double-stranded DNA (mt-dsDNA) in living cells, which has limited direct analysis of topological remodeling under stress and disease conditions. Here, we report SDMNA, a mitochondria-targeted fluorescent probe that enables topology-resolved imaging of mtDNA in situ. Built on a Y-shaped triphenylamine scaffold, SDMNA generates distinct optical responses to different mtDNA conformations. Binding to G4DNA imposes stronger conformational restriction on the probe, resulting in increased fluorescence intensity and a longer fluorescence lifetime relative to duplex binding. Together with its large Stokes shift and high photostability, these properties support fluorescence lifetime imaging microscopy and STED nanoscopy for quantitative discrimination of mtG4DNA and mt-dsDNA in living cells. Using this approach, we identify condition-dependent remodeling of mitochondrial DNA topology in oxidative stress, replicative senescence, and FUS-mutant amyotrophic lateral sclerosis patient-derived fibroblasts. These findings establish SDMNA as a platform for probing mitochondrial nucleoid organization and mtDNA structural remodeling in aging- and disease-associated mitochondrial dysfunction.