Optogenetic Methods for Spatiotemporally Resolved Observation of H2O2 in Biological Systems

Abstract

While hydrogen peroxide (H2O2) is widely recognized as a key redox signaling molecule essential for normal cellular functions, its supraphysiological accumulation is associated with the pathogenesis and progression of various diseases—including atherosclerosis, Duchenne muscular dystrophy (DMD), Alzheimer’s disease, and cancer—making it a primary target of antioxidative therapeutics. Due to its significant physiological role, precise understanding of peroxide dynamics at both intracellular and intercellular levels is crucial for effective and safe therapeutic discovery. Developing tools that enable site-specific and real-time detection of H2O2 in cells and model organisms is therefore essential.Guided by experimental and computational structural analyses, we engineered oROS, a multicolor fluorescence sensor suite for highly sensitive, real-time, in situ detection of H2O2. From human stem cell-derived models to animal models, oROS sensors demonstrate robust functionality in clinically relevant systems for studying peroxide biology. For instance, oROS elucidated the therapeutic efficacy of a putative antioxidative agent for Alzheimer’s disease and detected H2O2 signals in vivo to validate the NADPH oxidase-dependent opioid receptor inactivation mechanism in a systemic context. Furthermore, its multiplexed use with calcium and redox potential indicators enabled time-locked monitoring of H2O2 in relation to its key interactants. Lastly, oROS sensors were targeted to various subcellular compartments, including microdomains near the inner and outer plasma membranes, providing unprecedented precision in monitoring membrane H2O2 topology. We envision that oROS and its applications will stimulate new questions and discoveries in redox biology and medicine.