Ratiometric NAD + Sensors Reveal Subcellular NAD + Modulators.

Mapping NAD + dynamics in live cells and human is essential for translating NAD + interventions into effective therapies. Yet, genetically encoded NAD + sensors with better specificity and pH resistance are still needed for the cost-effective monitoring of NAD + in both subcellular compartments and clinical samples. Here, we introduce multicolor, resonance energy transfer-based NAD + sensors covering nano- to millimolar concentration ranges for clinical NAD + measurement and subcellular NAD + visualization. The sensors captured the blood NAD + increase induced by NMN supplementation and revealed the distinct subcellular effects of NAD + precursors and modulators. The sensors then enabled high-throughput screenings for mitochondrial and nuclear NAD + modulators and identified α-GPC, a cognition-related metabolite that induces NAD + redistribution from mitochondria to the nucleus relative to the total adenine nucleotides, which was further confirmed by NAD + FRET microscopy.