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Universal, label-free, single-molecule visualization of DNA origami nanodevices across biological samples using origamiFISH

Nature Nanotechnology volume 19, pages 58–69 (2024)Cite this article

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Abstract

Structural DNA nanotechnology enables the fabrication of user-defined DNA origami nanostructures (DNs) for biological applications. However, the role of DN design during cellular interactions and subsequent biodistribution remain poorly understood. Current methods for tracking DN fates in situ, including fluorescent-dye labelling, suffer from low sensitivity and dye-induced artifacts. Here we present origamiFISH, a label-free and universal method for the single-molecule fluorescence detection of DNA origami nanostructures in cells and tissues. origamiFISH targets pan-DN scaffold sequences with hybridization chain reaction probes to achieve 1,000-fold signal amplification. We identify cell-type- and DN shape-specific spatiotemporal distribution patterns within a minute of uptake and at picomolar DN concentrations, 10,000× lower than field standards. We additionally optimize compatibility with immunofluorescence and tissue clearing to visualize DN distribution within tissue cryo-/vibratome sections, slice cultures and whole-mount organoids. Together, origamiFISH enables the accurate mapping of DN distribution across subcellular and tissue barriers for guiding the development of DN-based therapeutics.

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Fig. 1: Overview of origamiFISH protocol and analyses.
Fig. 2: origamiFISH enables highly sensitive imaging of DNs in situ.
Fig. 3: origamiFISH detects loss of DN stability in situ.
Fig. 4: origamiFISH reveals cell-type-specific, DN shape-specific and time-dependent uptake patterns of DNs.
Fig. 5: origamiFISH is compatible with immunohistochemistry.
Fig. 6: origamiFISH is robust across tissue models.

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Data availability

The data that support the methods and findings of this study are available within the article and its Supplementary Information. The raw imaging data are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

W.X.W. acknowledges the PRiME initiative for fellowship support. T.R.D. acknowledges the Barbara and Frank Milligan Graduate fellowship. Y.L. acknowledges NSERC Discovery Grant (RGPIN-2020-06345); Medicine by Design, Simons Foundation (479754); Brain Canada, Stem Cell Network (ECI-14); Can-GARD; and Brain & Behavior Research Foundation (29772). J.M. acknowledges Brain Canada (FL2021-BCF-Sickkids), Stem Cell Network (ECR-C4R1-4), NSERC Discovery Grant (RGPIN-2019-06938) and The Hospital for Sick Children. L.Y.T.C. acknowledges NSERC Discovery Grant (RGPIN-2020-05966), Medicine by Design (MbDNI-2019-01, MbDNI-2020-01) and the Canadian Foundation for Innovation (39262) for funding. We thank W. Shih for sharing the DN barrel design sequences. We thank members of the Chou lab for helpful comments on this paper. We thank P. Paroutis and the Hospital for Sick Children imaging facility for instrument support and guidance on light-sheet microscopy. We thank J. Lefebvre for the generous gift of antibodies, reagents and use of equipment.

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Authors and Affiliations

  1. Institute of Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada

    Wendy Xueyi Wang, Travis R. Douglas, Meghan Rothenbroker, Yu Sun & Leo Y. T. Chou

  2. Department of Neurosurgery, Guizhou Provincial People’s Hospital, Guiyang, China

    Haiwang Zhang

  3. Program in Neurosciences and Mental Health, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada

    Haiwang Zhang, Zhengping Jia & Julien Muffat

  4. Department of Physiology, University of Toronto, Toronto, Ontario, Canada

    Haiwang Zhang & Zhengping Jia

  5. Program in Developmental and Stem Cell Biology, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada

    Afrin Bhattacharya & Yun Li

  6. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

    Afrin Bhattacharya, Julien Muffat & Yun Li

  7. Department of Mechanical and Industrial Engineering, University of Toronto, Toronto, Ontario, Canada

    Wentian Tang & Yu Sun

  8. Department of Electrical and Computer Engineering, University of Toronto, Toronto, Ontario, Canada

    Yu Sun

  9. Department of Computer Science, University of Toronto, Toronto, Ontario, Canada

    Yu Sun

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Contributions

W.X.W. and L.Y.T.C. conceptualized the project and designed the experiments. W.X.W. performed the experiments with assistance from T.R.D., H.Z., A.B., M.R. and W.T. W.X.W. performed the analyses. Z.J., J.M., Y.L., Y.S. and L.Y.T.C. participated in the data interpretation and provided supervision. W.X.W. and L.Y.T.C. wrote the paper with input from all authors.

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Correspondence to Leo Y. T. Chou.

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Nature Nanotechnology thanks Amelie Heuer-Jungemann, Tania Patiño and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–11, Notes 1–4 and Methods.

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Supplementary Data 1

Supplementary Tables 1 and 2.

Supplementary Data 2

Raw data for Supplementary Figs. 4 and 8–10.

Supplementary Video 1

DN microinjection video.

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Source Data Fig. 2

Raw data for the graphs in Fig. 2.

Source Data Fig. 3

Raw data for the graphs in Fig. 3.

Source Data Fig. 4

Raw data for the graphs in Fig. 4.

Source Data Fig. 5

Raw data for the graphs in Fig. 5.

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Wang, W.X., Douglas, T.R., Zhang, H. et al. Universal, label-free, single-molecule visualization of DNA origami nanodevices across biological samples using origamiFISH. Nat. Nanotechnol. 19, 58–69 (2024). https://doi.org/10.1038/s41565-023-01449-5

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