Lee Justin Daho, Nguyen Amanda, Gibbs Chelsea E, Jin Zheyu Ruby, Wang Yuxuan, Moghadasi Aida, Wait Sarah J, Choi Hojun, Evitts Kira M, Asencio Anthony, Bremner Samantha B, Zuniga Shani, Chavan Vedant, Pranoto Inez K A, Williams C Andrew, Smith Annette, Moussavi-Harami Farid, Regnier Michael, Baker David, Young Jessica E, Mack David L, Nance Elizabeth, Boyle Patrick M, Berndt Andre
Molecular Engineering and Sciences Institute, University of Washington, Seattle, WA, USA.
Department of Bioengineering, University of Washington, Seattle, WA, USA.
Nat Chem Biol. 2025 Apr 28. doi: 10.1038/s41589-025-01891-7.
Monitoring HO dynamics in conjunction with key biological interactants is critical for elucidating the physiological outcome of cellular redox regulation. Optogenetic hydrogen peroxide sensor with HaloTag with JF635 (oROS-HT) allows fast and sensitive chemigenetic far-red HO imaging while overcoming drawbacks of existing red fluorescent HO indicators, including oxygen dependency, high pH sensitivity, photoartifacts and intracellular aggregation. The compatibility of oROS-HT with blue-green-shifted optical tools allows versatile optogenetic dissection of redox biology. In addition, targeted expression of oROS-HT and multiplexed HO imaging enables spatially resolved imaging of HO targeting the plasma membrane and neighboring cells. Here we present multiplexed use cases of oROS-HT with other green fluorescence reporters by capturing acute and real-time changes in HO with intracellular redox potential and Ca levels in response to auranofin, an inhibitor of antioxidative enzymes, via dual-color imaging. oROS-HT enables detailed insights into intricate intracellular and intercellular HO dynamics, along with their interactants, through spatially resolved, far-red HO imaging in real time.
结合关键生物相互作用分子监测HO动态对于阐明细胞氧化还原调节的生理结果至关重要。带有JF635 HaloTag的光遗传学过氧化氢传感器（oROS-HT）可实现快速、灵敏的化学遗传远红光HO成像，同时克服了现有红色荧光HO指示剂的缺点，包括对氧的依赖性、高pH敏感性、光伪影和细胞内聚集。oROS-HT与蓝绿移光学工具的兼容性使得氧化还原生物学的多功能光遗传学剖析成为可能。此外，oROS-HT的靶向表达和多重HO成像能够对靶向质膜和邻近细胞的HO进行空间分辨成像。在此，我们通过双色成像捕获抗氧化酶抑制剂金诺芬作用下HO随细胞内氧化还原电位和Ca水平的急性实时变化，展示了oROS-HT与其他绿色荧光报告分子的多重应用案例。oROS-HT能够通过实时空间分辨的远红光HO成像，深入了解复杂的细胞内和细胞间HO动态及其相互作用分子。
