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

Hydrogen Peroxide (HO) is a central oxidant in redox biology due to its pleiotropic role in physiology and pathology. However, real-time monitoring of HO 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 HO dynamics. We successfully tracked real-time transient and steady-state HO levels in diverse biological systems, including human stem cell-derived neurons and cardiomyocytes, primary neurons and astrocytes, and mouse brain and . These applications demonstrate oROS's capabilities to monitor HO 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 HO signal which highlights redox-based mechanisms of GPCR regulation. oROS is a versatile tool, offering a window into the dynamic landscape of HO 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.