Ultra-fast genetically encoded sensor for precise real-time monitoring of physiological and pathophysiological peroxide dynamics.

Hydrogen Peroxide (H 2 O 2 ) is a central oxidant in redox biology due to its pleiotropic role in physiology and pathology. However, real-time monitoring of H 2 O 2 in living cells and tissues remains a challenge. We address this gap with the development of an optogenetic hydRogen perOxide Sensor (oROS), leveraging the bacterial peroxide binding domain OxyR. Previously engineered OxyR-based fluorescent peroxide sensors lack the necessary sensitivity and response speed for effective real-time monitoring. By structurally redesigning the fusion of Escherichia coli (E. coli) ecOxyR with a circularly permutated green fluorescent protein (cpGFP), we created a novel, green-fluorescent peroxide sensor oROS-G. oROS-G exhibits high sensitivity and fast on-and-off kinetics, ideal for monitoring intracellular H 2 O 2 dynamics. We successfully tracked real-time transient and steady-state H 2 O 2 levels in diverse biological systems, including human stem cell-derived neurons and cardiomyocytes, primary neurons and astrocytes, and mouse brain ex vivo and in vivo . These applications demonstrate oROS's capabilities to monitor H 2 O 2 as a secondary response to pharmacologically induced oxidative stress and when adapting to varying metabolic stress. We showcased the increased oxidative stress in astrocytes via Aβ-putriscine-MAOB axis, highlighting the sensor's relevance in validating neurodegenerative disease models. Lastly, we demonstrated acute opioid-induced generation of H 2 O 2 signal in vivo which highlights redox-based mechanisms of GPCR regulation. oROS is a versatile tool, offering a window into the dynamic landscape of H 2 O 2 signaling. This advancement paves the way for a deeper understanding of redox physiology, with significant implications for understanding diseases associated with oxidative stress, such as cancer, neurodegenerative, and cardiovascular diseases.