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Monitoring in real time and far-red imaging of H2O2 dynamics with subcellular resolution

Nature Chemical Biology (2025)Cite this article

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A preprint version of the article is available at bioRxiv.

Abstract

Monitoring H2O2 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-HT635) allows fast and sensitive chemigenetic far-red H2O2 imaging while overcoming drawbacks of existing red fluorescent H2O2 indicators, including oxygen dependency, high pH sensitivity, photoartifacts and intracellular aggregation. The compatibility of oROS-HT635 with blue-green-shifted optical tools allows versatile optogenetic dissection of redox biology. In addition, targeted expression of oROS-HT635 and multiplexed H2O2 imaging enables spatially resolved imaging of H2O2 targeting the plasma membrane and neighboring cells. Here we present multiplexed use cases of oROS-HT635 with other green fluorescence reporters by capturing acute and real-time changes in H2O2 with intracellular redox potential and Ca2+ levels in response to auranofin, an inhibitor of antioxidative enzymes, via dual-color imaging. oROS-HT635 enables detailed insights into intricate intracellular and intercellular H2O2 dynamics, along with their interactants, through spatially resolved, far-red H2O2 imaging in real time.

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Fig. 1: Structure-guided engineering of oROS-HT635: bright far-red optogenetic sensor for H2O2.
Fig. 2: Characterization of oROS-HT635: a bright, sensitive and fast H2O2 sensor.
Fig. 3: Biophysical properties and versatility of oROS-HT635 under varying environmental conditions.
Fig. 4: Multiparametric analysis of the acute effect of auranofin on H2O2, redox potential and Ca2+.
Fig. 5: Modeled interplay between ICaL and SERCA reveals oxidative stress-induced Ca2+ dysregulation.
Fig. 6: Spatiotemporally resolved imaging of H2O2.

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Data availability

The complete minimal raw dataset from the experiments, representative images, downstream analysis and visualizations generated for this article are available on figshare at https://doi.org/10.6084/m9.figshare.28306691 (ref. 96). Plasmids for oROS-HT and its loss-of-function (C199S) and subcellular targeting variants described in this paper are available through Addgene at pC1-lifeact-oROS-HT (216420), pC1-IMS-oROS-HT (216419), pC1-dmito-oROS-HT (216418), pC1-oROS-HT-CaaX (216417), pDisplay-oROS-HT (216416), AAV2_CAG_oROS-HT(C199S)_WPRE (216415), AAV2_CAG_oROS-HT_WPRE (216414), pC1_oROS-HT (216413) and pC1_oROS-HT_LF(C199S) (216412). We will also provide plasmids upon request. The study accessed the PDB database (1I6A, 1I69 and 6U2M) for structural analysis. Source data are provided with this paper.

Code availability

Source code for simulation, data extraction, analysis and visualization is available on figshare at https://doi.org/10.6084/m9.figshare.28306691 (ref. 96).

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Acknowledgements

J.D.L. was supported by 1F31DA056121-01A1 and the ISCRM Fellowship. A.B. was supported by the Brain Research Foundation, UW Royalty Research Fund, UW ISCRM IPA, NIGMS R01 GM139850-01, P30 DA048736-01-Pilot, NIMH RF1MH130391, NINDS U01NS128537, NIDA R21DA051193 and the McKnight Foundation’s Technologies in Neuroscience Award. S.J.W. was supported by the National Science Foundation DGE-2140004 and the Herbold Foundation. K.M.E. was supported by T32AG066574. A.A. was supported by the National Institute of General Medical Sciences grant RM1 GM131981, the National Institute of Arthritis and Musculoskeletal and Skin Diseases grant P30 AR074990, American Heart Association supplement grant AHA872208 and BCTP-NIH–NIBIB-5T32EB032787-02. We would like to thank the Janelia Materials program from Howard Hughes Medical Institute Janelia Research Campus for generous sharing of their Janelia Fluors essential for this study. This research received additional support from the Lynn and Mike Garvey Imaging Core, the UW NAPE Center, ISCRM Shared Equipment and Leica Center of Excellence for Cellular Imaging in Fred Hutch Cancer Research Center (H. West-Foyle and L. Schroeder). We also want to thank R. Moon for his support.

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Author notes

  1. These authors contributed equally: Chelsea E. Gibbs, Zheyu Ruby Jin, Yuxuan Wang.

Authors and Affiliations

  1. Molecular Engineering and Sciences Institute, University of Washington, Seattle, WA, USA

    Justin Daho Lee, Sarah J. Wait, Elizabeth Nance & Andre Berndt

  2. Department of Bioengineering, University of Washington, Seattle, WA, USA

    Justin Daho Lee, Amanda Nguyen, Chelsea E. Gibbs, Yuxuan Wang, Aida Moghadasi, Sarah J. Wait, Kira M. Evitts, Anthony Asencio, Samantha B. Bremner, Shani Zuniga, Vedant Chavan, Michael Regnier, David L. Mack, Elizabeth Nance, Patrick M. Boyle & Andre Berndt

  3. Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA

    Justin Daho Lee, Sarah J. Wait, Kira M. Evitts, Anthony Asencio, Inez K. A. Pranoto, C. Andrew Williams, Annette Smith, Farid Moussavi-Harami, Michael Regnier, Jessica E. Young, David L. Mack, Patrick M. Boyle & Andre Berndt

  4. Department of Chemical Engineering, University of Washington, Seattle, WA, USA

    Zheyu Ruby Jin & Elizabeth Nance

  5. Department of Biochemistry, University of Washington, Seattle, WA, USA

    Hojun Choi & David Baker

  6. Institute for Protein Design, University of Washington, Seattle, WA, USA

    Hojun Choi & David Baker

  7. Center for Translational Muscle Research, University of Washington, Seattle, WA, USA

    Anthony Asencio, Farid Moussavi-Harami, Michael Regnier, Patrick M. Boyle & Andre Berndt

  8. Center for Cardiovascular Biology, University of Washington, Seattle, WA, USA

    Anthony Asencio, Farid Moussavi-Harami, Michael Regnier & Patrick M. Boyle

  9. Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA

    Inez K. A. Pranoto, C. Andrew Williams, Farid Moussavi-Harami & Jessica E. Young

  10. Division of Cardiology, University of Washington, Seattle, WA, USA

    Farid Moussavi-Harami

  11. Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA

    David Baker

  12. Department of Rehabilitation Medicine, University of Washington, Seattle, WA, USA

    David L. Mack

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  1. Justin Daho Lee
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Contributions

J.D.L. and A.B. conceived the study design and oROS-HT635 engineering strategies, performed experiments, and analyzed the data. J.D.L, A.N., S.Z. and A.S. cloned and screened the sensor variants. J.D.L., Y.W., A.M. and V.C. constructed constructs for chemigenetic/optogenetic demonstration of subcellular H2O2 and performed analysis. J.D.L, S.J.W. and H.C. performed purified protein assays and analysis. J.D.L., C.E.G. and P.M.B. performed in silico hiPS cell-CM simulations and analysis. J.D.L. and Z.R.J. performed maturation studies under hypoxia, and brain slice imaging and analysis. K.M.E., A.A., S.B.B., I.K.A.P. and C.A.W. differentiated and prepared hiPS cell-derived cells. A.B., P.M.B., E.N. D.L.M., J.E.Y., D.B., M.R. and F.M.-H. supervised the preparation of materials, experiments, analyses and paper writing.

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Correspondence to Andre Berndt.

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Supplementary information

Supplementary Information

oROS-HT amino acid sequence, Supplementary Table 1 and Figs. 1–11.

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Lee, J.D., Nguyen, A., Gibbs, C.E. et al. Monitoring in real time and far-red imaging of H2O2 dynamics with subcellular resolution. Nat Chem Biol (2025). https://doi.org/10.1038/s41589-025-01891-7

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