SummaryPeroxynitrite is a highly 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 non-invasive detection of peroxynitrite is 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 123-fold fluorescence turn-on response towards peroxynitrite while displaying virtually no cross-reaction with other reactive oxygen/nitrogen species, including hydrogen peroxide. To facilitate the expression of pnGFP-Ultra in mammalian cells, we engineered a highly efficient noncanonical amino acid (ncAA) expression system that is broadly applicable to the mammalian expression of proteins containing various ncAAs. pnGFP-Ultra robustly detected peroxynitrite production during interferon γ and lipopolysaccharide-induced immune responses in macrophages, and in amyloid β-activated primary glial cells. Thus, pnGFP-Ultra fills an important technical gap and represents an important new addition to the molecular toolbox in probing RNS biology.In BriefChen et al. report pnGFP-Ultra, a high-performance fluorescent biosensor for minimally invasive and selective imaging of peroxynitrite production in live cells.HighlightspnGFP-Ultra is a genetically encoded peroxynitrite biosensor with a 123-fold fluorescence turn-on responsepnGFP-Ultra exhibits high selectivity toward peroxynitrite, with virtually no crossreaction with hydrogen peroxideAn optimized plasmid-based system increases noncanonical amino acid incorporation in mammalian cells by >10 foldpnGFP-Ultra robustly detects peroxynitrite production in macrophages and primary glial cells
Sensitive and fast fluorescence-based indirect sensing of TATP