Exogenously and Endogenously Sequential Regulation of DNA Nanodevices Enables Organelle‐Specific Signal Amplification in Subcellular ATP Profiling

Adenosine triphosphate (ATP), the primary energy currency in cells, is dynamically regulated across different subcellular compartments. The ATP interplay between mitochondria and endoplasmic reticulum (ER) underscores their coordinated roles in various biochemical processes, highlighting the necessity for precise profiling of subcellular ATP dynamics. Here we present an exogenously and endogenously dual-regulated DNA nanodevice for spatiotemporally selective, subcellular-compartment specific signal amplification in ATP sensing. The system allows for exogenous NIR light-controlled spatiotemporal localization and activation of the aptamer sensor in mitochondria or ER, while a specific endogenous enzyme in the organelles further drives signal amplification via the consumption of molecular beacon fuels, resulting in significantly enhanced sensitivity and spatial precision for the subcellular ATP profiling in the organelle of interest. Furthermore, we demonstrate the application of this system for robust monitoring of ATP fluctuations in mitochondria and ER following drug interventions. This advancement provides a powerful tool for improving our understanding of cellular energetics at the subcellular level and holds potential for the development of targeted therapeutics.