Decoding Mitochondrial CircRNA Dynamics with In Situ Super-Resolution Bioimaging and Quantification via a Ratiometric DNA Tetrahedral Amplifier

Functional circular RNAs (circRNAs) associated with disease often exhibit specific subcellular localization. Notably, mitochondrial circRNAs have emerged as a novel subgroup implicated in cellular physiology and pathology, but their precise functions remain largely unexplored. Hence, the precise study of mitochondrial circRNAs is highly desirable, yet it is a great challenge due to the low abundance of mitochondrial circRNAs and the absence of effective tools. Here, we developed a mitochondria-targeting ratiometric DNA tetrahedral amplifier (DNAT-TPP) for spatially resolved in situ super-resolution bioimaging and quantification of mitochondrial circRNAs. This DNAT-TPP combines a triphenylphosphonium (TPP)-modified DNA tetrahedron scaffold with catalytic hairpin assembly (CHA) amplification, enabling sensitive detection of the nonalcoholic steatohepatitis (NASH)-associated mitochondrial circRNA SCAR (circSCAR) in solution and in living cells via a fluorescence resonance energy transfer (FRET) signal readout. Importantly, circSCAR within the mitochondrial network was clearly visualized by super-resolution bioimaging using structured illumination microscopy (SIM), and the expression levels of mitochondrial circSCAR between normal and cancer cells were reliably distinguished by DNAT-TPP. In addition, in situ quantification of circSCAR in a cellular NASH model was established by the DNAT-TPP. Furthermore, we achieved in vivo visualization of endogenous mitochondrial circSCAR by DNAT-TPP and performed efficient fluorescence bioimaging of circSCAR in normal mice liver. This work establishes a robust amplifier for spatially resolved mitochondrial circRNA super-resolution bioimaging and quantification, paving a way for investigating functional and disease-associated mitochondrial circRNAs in pathogenesis.