Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.17.254771v1?rss=1 Authors: Chen, Z., Zhang, S., Li, X., Ai, H. Abstract: Peroxynitrite 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 {gamma} and lipopolysaccharide-induced immune responses in macrophages, and in amyloid {beta}-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. Copy rights belong to original authors. Visit the link for more info