Abstract

Upon illumination, photosensitizer molecules produce reactive oxygen species that can be used for functional manipulation of living cells, including protein inactivation, targeted-damage introduction and cellular ablation. Photosensitizers used to date have been either exogenous, resulting in delivery and removal challenges, or genetically encoded proteins that form or bind a native photosensitizing molecule, resulting in a constitutively active photosensitizer inside the cell. We describe a genetically encoded fluorogen-activating protein (FAP) that binds a heavy atom−substituted fluorogenic dye, forming an 'on-demand' activated photosensitizer that produces singlet oxygen and fluorescence when activated with near-infrared light. This targeted and activated photosensitizer (TAPs) approach enables protein inactivation, targeted cell killing and rapid targeted lineage ablation in living larval and adult zebrafish. The near-infrared excitation and emission of this FAP-TAPs provides a new spectral range for photosensitizer proteins that could be useful for imaging, manipulation and cellular ablation deep within living organisms.

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Acknowledgements

This work was supported in part with funds from the US National Institutes of Health (NIH), Technology Centers for Networks and Pathways program (grant U54GM103529 to M.P.B., S.C.W., J.H. and Y.W.), NIH grant R01EB017268 (to J.H., Y.W., M.A.M., E.O., C.M.S., M.T. and M.P.B.) and NIH grant R21ES025606 (to M.P.B. and S.C.W.). We thank E. Kelley and C.J. Bakkenist for helpful discussion and guidance on establishing ROS involved in cellular toxicity; G. Daskivich for help in establishing zebrafish lines; and E. Drill, C.T. Wallace and M.A. Ross for help in larval zebrafish TUNEL imaging.

Author information

Affiliations

  1. Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA.

    • Jianjun He
    •  & Marcel P Bruchez
  2. Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA.

    • Yi Wang
    • , Lydia A Perkins
    •  & Marcel P Bruchez
  3. Department of Developmental Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

    • Maria A Missinato
    • , Ezenwa Onuoha
    •  & Michael Tsang
  4. Center for Biologic Imaging, Department of Cell Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

    • Simon C Watkins
    •  & Claudette M St Croix
  5. Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA.

    • Marcel P Bruchez

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Contributions

J.H. designed and performed experiments, analyzed data and wrote the paper. Y.W. provided cell culture, performed experiments and wrote the paper. E.O., M.A.M. and M.T. developed and provided zebrafish lines, designed and performed experiments and analyzed data. L.A.P. provided reagents. S.C.W. and C.M.C. performed experiments and analyzed data. M.P.B. designed experiments, analyzed data and wrote the paper.

Competing interests

M.P.B. is a founder in Sharp Edge Labs, a company applying the FAP-fluorogen technology commercially.

Corresponding author

Correspondence to Marcel P Bruchez.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–11, Supplementary Tables 1 and 2, and Supplementary Note

Videos

  1. 1.

    Change in cell morphology after photosensitization.

    The first frame is an image of FAP-TAPs fluorescence (640 channel) before illumination; the last frame was an image of EthD-1 fluorescence (560 channel, dead cell stain) and taken 30 minutes after illumination. Scale bar, 10 μm.

  2. 2.

    Fluorescence bleaching of FAP-TAPs on cell surface with 10 min of continuous illumination using a confocal microscope (40×, 640 nm, 0.43 W/cm2).

    Less than 30% of self-bleaching was observed in the first one minute of illumination, which was the typical duration for inducing sufficient cytotoxicity. Scale bar, 10 μm.

  3. 3.

    A Tg(myl7:MBIC5-mCer3) larvae labeled with 500 nM MG-ester at 72 h.p.f. (incubated for 3 h).

    Movies were taken with a confocal microscope, 20×, 640 nm, 100% laser power (640 channel in red).

  4. 4.

    Tg(myl7:MBIC5-mCer3) larvae treated with different conditions: MG-2I and 20-min illumination, MG-2I and 5-min illumination, MG-2I and no light, and MG-ester and 20-min illumination.

    Movies were taken right after illumination using a Canon camera (10×, 470/40 ex, 510/40 em, green).

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DOI

https://doi.org/10.1038/nmeth.3735