Ratiometric NAD plus Sensors Reveal Subcellular NAD plus 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 costeffective 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 alpha-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.