NOckout: A DNA-Based Fluorescent Probe to Quantitatively Map NOS3 Activity with Subcellular Spatial Resolution

Compartmentalized nitric oxide (NO) production drives critical signaling pathways in cells, yet there are no methods to quantitatively image NO with sub-cellular spatial resolution in living systems. Here, we introduce a new DNA-based fluorescent reporter technology that maps NO with sub-cellular resolution in live cells. It combines small molecule NO detection chemistry with the sub-cellular targetability of DNA based scaffold to provide quantifiable NO maps using ratiometric imaging. We could thereby map the activity of Nitric Oxide Synthase 3 (NOS3) which resides at the plasma membrane and the trans-Golgi network. We find that despite its lower abundance, the pool of NOS3 at the plasma membrane is seven-fold more active than Golgi associated NOS3. The ability to quantitatively map NO dynamics with sub-cellular resolution provides the potential to discover selective regulators of distinct NOS3 populations.