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.