A high-performance genetically encoded fluorescent biosensor for imaging physiological peroxynitrite.

Peroxynitrite is a reactive nitrogen species (RNS) that plays critical roles in signal transduction, stress response, and numerous human diseases. Advanced molecular tools that permit the selective, sensitive, and noninvasive detection of peroxynitrite are essential for understanding its pathophysiological functions. Here, we present pnGFP-Ultra, a high-performance, reaction-based, genetically encodable biosensor for imaging peroxynitrite in live cells. pnGFP-Ultra features a p-boronophenylalanine-modified chromophore as the sensing moiety and exhibits a remarkable ~110-fold fluorescence turn-on response toward peroxynitrite while displaying virtually no cross-reaction with other reactive oxygen/nitrogen species. To facilitate the expression of pnGFP-Ultra in mammalian cells, we engineered an efficient noncanonical amino acid (ncAA) expression system that is broadly applicable to the mammalian expression of ncAA-containing proteins. pnGFP-Ultra robustly detected peroxynitrite production in activated macrophages and primary glial cells. pnGFP-Ultra fills an important technical gap and represents a valuable addition to the molecular toolbox for probing RNS biology.