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.