Structure-guided engineering of a fast genetically encoded sensor for real-time H 2 O 2 monitoring

• Published in: bioRxiv : the preprint server for biology
• Main Authors: Lee, Justin Daho, Won, Woojin, Kimball, Kandace, Wang, Yihan, Yeboah, Fred, Evitts, Kira M, Neiswanger, Carlie, Schattauer, Selena, Rappleye, Michael, Bremner, Samantha B, Chun, Changho, Smith, Netta, Mack, David L, Young, Jessica E, Justin Lee, C, Chavkin, Charles, Berndt, Andre
• Format: Journal Article
• Language: English
• Published: United States 04.02.2024
• DOI: 10.1101/2024.01.31.578117

Hydrogen Peroxide (H O ) is a central oxidant in redox biology due to its pleiotropic role in physiology and pathology. However, real-time monitoring of H O 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 or 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 O dynamics. We successfully tracked real-time transient and steady-state H O levels in diverse biological systems, including human stem cell-derived neurons and cardiomyocytes, primary neurons and astrocytes, and mouse neurons and astrocytes in ex vivo brain slices. These applications demonstrate oROS's capabilities to monitor H O as a secondary response to pharmacologically induced oxidative stress, G-protein coupled receptor (GPCR)-induced cell signaling, 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. oROS is a versatile tool, offering a window into the dynamic landscape of H O signaling. This advancement paves the way for a deeper understanding of redox physiology, with significant implications for diseases associated with oxidative stress, such as cancer, neurodegenerative disorders, and cardiovascular diseases.