Ultrasensitive Genetically Encoded Indicator for Hydrogen Peroxide Identifies Roles for the Oxidant in Cell Migration and Mitochondrial Function

Abstract

Hydrogen peroxide (H₂O₂) is a key redox intermediate generated within cells. Existing probes for H₂O₂ have not solved the problem of detection of the ultra-low concentrations of the oxidant: these reporters are not sensitive enough, or pH-dependent, or insufficiently bright, or not functional in mammalian cells, or have poor dynamic range. Here we present HyPer7, the first bright, pH-stable, ultrafast, and ultrasensitive ratiometric H₂O₂ probe. HyPer7 is fully functional in mammalian cells and in other higher eukaryotes. The probe consists of a circularly permuted GFP integrated into the ultrasensitive OxyR domain from Neisseria meningitidis. Using HyPer7, we were able to uncover the details of H₂O₂ diffusion from the mitochondrial matrix, to find a functional output of H₂O₂ gradients in polarized cells, and to prove the existence of H₂O₂ gradients in wounded tissue in vivo. Overall, HyPer7 is a probe of choice for real-time H₂O₂ imaging in various biological contexts.

Keywords: D-amino acid oxidase; H(2)O(2); H(2)O(2) gradients; HyPer7; cell migration; chemogenetics; genetically encoded probes; hydrogen peroxide; mitochondria; redox signaling.