L-DNA-Based Framework Nucleic Acid Nanodevice for Lysosomal ATP Imaging

Real-time monitoring of biomolecular dynamics at the subcellular level is essential for understanding organellar functions. DNA nanoprobes represent a powerful tool, yet their rapid degradation upon lysosomal entry results in short imaging windows that preclude long-term dynamic monitoring. To address this challenge, we developed an L-DNA-based framework nucleic acid (L-FNA) nanodevice that leverages mirror-image chirality for intrinsic nuclease resistance. By intergrating an ATP-responsive aptamer module into this L-FNA platform, we constructed the ATP-sensing probe L-FNA-apt, enabling prolonged and stable imaging of intraluminal lysosomal ATP. Compared to conventional right-handed DNA probes, L-FNA exhibited an approximately 7-fold longer intracellular retention half-life (17.6 h vs 2.5 h) and L-FNA-apt maintained a functional integrity window exceeding 24 h. In contrast, D-FNA-apt lost most of its responsiveness by 6 h, revealing a 6 h window in which fluorescence persists without functional activity. Using this platform, we demonstrated that L-FNA-apt remains responsive to energy stress for at least 24 h in living cells. By enabling long-term monitoring of intraluminal metabolites through chiral nuclease resistance, this work establishes a framework to distinguish structural presence from functional integrity, providing a reliable tool for studying organelle energy metabolism and related diseases.