Harnessing fluorescent chemogenetic reporter technology for the detection of hydrogen peroxide

Daria Ezeriņa, Daria Smolyarova, Dina Bass, Ekaterina Potekhina, Vsevolod Belousov, Joris Messens

Research output: Unpublished contribution to conferencePoster


Hydrogen peroxide (H2O2) is a secondary messenger molecule that signals through the oxidation of specific cysteine residues on proteins. Its dysregulation has been associated with various diseases
including cancer and neurological diseases, making its detection in cells and tissues germane to the development of novel therapeutic options to combat those diseases. A great leap in its intracellular detection was made approximately 15 years ago with the advent of genetically encoded fluorescent protein-based biosensors, including the HyPer family. HyPer biosensors consist of OxyR, a prokaryotic redox-sensitive transcription factor, whose oxidation by H2O2 leads to a structural change that allosterically alters the excitation spectrum of the coupled circularly permuted YFP (cpYFP). The state-of- the-art HyPer7 is an ultrasensitive and selective H2O2 biosensor that is also ratiometrically pH independent1. However, being based on YFP, its usage in hypoxic conditions, as well as its application in multiparametric imaging remains limited. To circumvent these issues, here we replaced cpYFP in HyPer7 by cpFAST (Fluorogen-activating and Absorption-Shifting Tag) in the same OxyR position to create HyPerFAST. cpFAST is a 14 kDa protein that exhibits immediate fluorescence upon non-covalent association with synthetic fluorogens, which mimic fluorophores and are non-fluorescent until they bind to FAST. As a result, HyPerFAST gains the added benefits of almost no photobleaching and customizable spectral characteristics, enabling it to be employed alongside other biosensors within the same cell, many of which are based on YFP or GFP. Our findings revealed that, in tandem with red-shifted fluorogens, HyPerFAST can detect H2O2 in the nanomolar range, independently of pH, with a second- order reaction rate constant of ~104 M-1s-1. Furthermore, we demonstrated its capability to detect H2O2 in mammalian cells.
Original languageEnglish
Publication statusPublished - 23 Aug 2023


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