A high-performance genetically encoded fluorescent biosensor for imaging physiological peroxynitrite. Issue 11 (18th November 2021)

• Record Type: Journal Article
• Authors: Chen, Zhijie; Zhang, Shen; Li, Xinyu; Ai, Hui-wang
• Is Part Of: Cell chemical biology. Volume 28:Issue 11(2021)
• Page Start: 1542
• Page End: 1553.e5
• Publication Date: 2021-11-18
• Subjects: peroxynitrite -- reactive nitrogen species -- biosensor -- fluorescent probes -- genetic code expansion -- noncanonical amino acids -- live-cell imaging -- protein engineering -- peroxynitrite production -- fluorescence microscopy; Biochemistry -- Periodicals; 572.05
• DOI: 10.1016/j.chembiol.2021.01.013
• Languages: English
• ISSNs: 2451-9456

Abstract

Summary: 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. Graphical abstract: Highlights: pnGFP-Ultra is a genetically encoded biosensor with an ~110-fold turn-on response pnGFP-Ultra exhibits high selectivity and sensitivity toward peroxynitrite An optimized plasmid-based system increases noncanonical amino acid incorporation pnGFP-Ultra robustly detects peroxynitrite in macrophages and primary glial cells Abstract : Advanced molecular tools that permit the selective, sensitive, and noninvasive detection of peroxynitrite are essential for understanding its pathophysiological functions. Chen et al. report on pnGFP-Ultra, a high-performance fluorescent turn-on biosensor for minimally invasive and selective imaging of peroxynitrite production in live cells.