Monitoring oxidative inflammatory processes in live cells and tissue with Hypocrates, a genetically encoded biosensor for hypochlorite

Abstract

Hypochlorous acid, an aggressive oxidant, is important in immune defense against pathogens. The current lack of tools to monitor the dynamics of hypochlorous acid in live cells and tissue hinders a better understanding of inflammatory processes. We engineered a genetically encoded biosensor, Hypocrates, for the visualization of hypochlorous acid. Hypocrates consists of a circularly permuted yellow fluorescent protein integrated into the structure of the transcription repressor NemR from E. coli. We determined sensitivity, selectivity, reaction rates, and the X-ray structure of this ratiometric redox biosensor, and tested the response of Hypocrates in HeLa Kyoto cells at varying hypochlorite concentrations. By combining Hypocrates with the biosensor HyperRed, we visualized the dynamics of hypochlorous acid and hydrogen peroxide in a zebrafish tail fin injury model.

Single‐beam dual‐color alternate‐pathway two‐photon spectroscopy: Toward an optical toolbox for redox biology

“…The spectra of 2PA cross sections for rhodamine B (a), coumarin 540A (b), coumarin 334 (c), and fluorescein (d) as measured in this study (data points with error bars connected by solid lines) and as presented in Pochechuev et al [59] and Kostyuk et al [60] (asterisks). One-photon extinction spectra ε(λ 0 ), in accordance with Pochechuev et al, [59] are presented by the dashed lines with counts of ε shown along the right axis and with the wavelength λ 0 shown along the top abscissa axis and rescaled, for the convenience of comparison, such that λ 0 = λ/2 reference, Figure 3a,b presents the spectra of one-photon excitation, ε(λ 0 ), for the same set of dyes, with counts of ε shown along the right axis and with the wavelength λ 0 shown along the top abscissa axis and rescaled, for the convenience of comparison, such that λ 0 = λ/2.…”

“…The spectra of 2PA cross sections for rhodamine B (a), coumarin 540A (b), coumarin 334 (c), and fluorescein (d) as measured in this study (data points with error bars connected by solid lines) and as presented in Pochechuev et al [ 59 ] and Kostyuk et al [ 60 ] (asterisks). One‐photon extinction spectra ε ( λ ′), in accordance with Pochechuev et al, [ 59 ] are presented by the dashed lines with counts of ε shown along the right axis and with the wavelength λ ′ shown along the top abscissa axis and rescaled, for the convenience of comparison, such that λ ′ = λ /2…”

“…[35,59] HyPer7, on its part, provides a highly sensitive, pH-stable probe for hydrogen peroxide, [56,57] offering means to understand the role of H 2 O 2 as an oxidant in cell migration and mitochondrial function. To complete the picture, Hypocrates holds much promise as a next-generation genetically encoded fluorescent sensor of sodium hypochlorite, [60] offering a meaningful complement to SypHer3s and HyPer7 as a highly sensitive fluorescent probe for oxidant detection as a part of oxidative-stress diagnosis and redox-signaling studies.…”