RNA aptamer-induced fluorescence enhancement for NADH monitoring in cellular environment

Cellular redox homeostasis is tightly regulated by the oxidation-reduction reactions of nicotinamide metabolites, including NAD(H) and NADP(H), which serve as essential cofactors in enzymatic processes related to energy metabolism. Monitoring intracellular NADH levels is therefore of significant interest. Most chemosensor designs to date rely on fluorescence turn-on mechanisms triggered by NADH oxidation, but these reaction-based sensors are inherently limited by NADH concentration and reaction kinetics. While NADH exhibits intrinsic fluorescence, its low quantum yield has led to the development of redox-sensitive substrates that emit fluorescence upon NADH oxidation. Here, we report an alternative fluorescence enhancement strategy based on an NADH-binding RNA aptamer. The interaction between NADH and a 49-base-pair RNA aptamer induces a 1.4-fold increase in fluorescence emission in vitro and an 1.8-fold increase in live-cell imaging. This fluorescence enhancement arises from aptamer-induced structural rigidity, analogous to the mechanism by which 4-(p-hydroxybenzylidene)-5-imidazolidinone (HBI) enhances fluorescence in green fluorescent protein. Using our aptamer-based assay, we established a live-cell fluorescence emission assay for real-time monitoring of cellular NADH dynamics.