A 6-nm ultra-photostable DNA FluoroCube for fluorescence imaging

Abstract

Photobleaching limits extended imaging of fluorescent biological samples. We developed DNA-based ‘FluoroCubes’ that are similar in size to the green fluorescent protein, have single-point attachment to proteins, have a ~54-fold higher photobleaching lifetime and emit ~43-fold more photons than single organic dyes. We demonstrate that DNA FluoroCubes provide outstanding tools for single-molecule imaging, allowing the tracking of single motor proteins for >800 steps with nanometer precision.

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Fig. 1: Design, assembly and photophysical properties of DNA FluoroCubes.
Fig. 2: Tracking steps of a single kinesin over more than 6 µm.

Data availability

Example raw datasets of DNA FluoroCubes with six dyes, single dye cubes (FluoroCubes with a single dye), one-dye dsDNA constructs, single, biotinylated dyes and compact cubes used to determine the photophysical properties are hosted on Zenodo: https://doi.org/10.5281/zenodo.3561024 (ref. 36). All other data files are available from the authors upon request.

Code availability

μManager acquisition and analysis software is available partly under the Berkeley Software Distribution license, partly under the GNU Lesser General Public License and development is hosted on GitHub at https://github.com/nicost/micro-manager. The latest version for MacOS and Windows can be downloaded here: https://micro-manager.org/wiki/Download%20Micro-Manager_Latest%20Release (v.2.0 gamma). The custom written Python code used to determine the total number of photons, the average number of photons per frame, and the half-life (photostability) for all fluorescent samples used in this study is hosted on Zenodo at https://doi.org/10.5281/zenodo.3629746 (ref. 37). All other code is available from the authors upon request.

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Acknowledgements

We are grateful to J. Sung (UCSF) for critical discussions of the manuscript. We thank D. Mullins (UCSF) for teaching us how to use the ISS K2 multifrequency fluorometer. We are thankful to Y.-W. Jun and Y. Zhao (UCSF) for teaching us how to use the Malvern Zetasizer ZS90. We thank L. Lavis (Janelia Research Campus) for the suggestion of comparing sulfonated versus nonsulfonated dyes and for providing the JF549 and JF646 dyes. We are thankful to S. Douglas (UCSF) and A.G. York (Calico Laboratories) for feedback on the manuscript after preprinting. A. Carter (MRC Laboratory of Molecular Biology) and E. Villa (UCSD) supplied the MATLAB script for step detection of kinesin. We acknowledge funding from the National Institutes of Health grant nos. R01GM097312 and 1R35GM118106 (to R.D.V. and S.N.), and the Howard Hughes Medical Institute (to R.D.V., S.N. and N.S.).

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Authors

Contributions

S.N., N.S. and R.D.V. designed the research. S.N. prepared samples, collected TIRF microscopy data and electron microscopy data and analyzed it. S.N., N.S. and R.D.V. wrote the article. All authors read and commented on the paper.

Corresponding author

Correspondence to Ronald D. Vale.

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The authors declare no competing interests.

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Peer review information Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–21, Tables 1–13 and Supplementary Protocol.

Reporting Summary

Supplementary Video 1

Single-molecule imaging of ATTO 488 six-dye FluoroCubes and ATTO 488 one-dye dsDNA. The movie shows surface-immobilized single molecules of ATTO 488 six-dye FluoroCubes and ATTO 488 one-dye dsDNA over ~50 min under continuous laser illumination. The graph shows the photostability of six-dye FluoroCubes and one-dye dsDNA. The survival rate was quantified by counting the percentage of probes in the ‘on’ state at any given time from 0 to 3,000 s. Time is in minutes. Each experiment was repeated five or four times with freshly assembled six-dye FluoroCubes or one-dye dsDNA, respectively, and on freshly prepared microscope slides. All acquired movies looked very similar and grave consistent results (see Fig. 1 l–n).

Supplementary Video 2

Single-molecule imaging of ATTO 565 six-dye FluoroCubes and ATTO 565 one-dye dsDNA. The movie shows surface-immobilized single molecules of ATTO 565 six-dye FluoroCubes and ATTO 565 one-dye dsDNA over ~50 min under continuous laser illumination. The graph shows the photostability of six-dye FluoroCubes and one-dye dsDNA. The survival rate was quantified by counting the percentage of probes in the ‘on’ state at any given time from 0 to 3,000 s. Time is in minutes. Each experiment was repeated five or four times with freshly assembled six-dye FluoroCubes or one-dye dsDNA, respectively, and on freshly prepared microscope slides. All acquired movies looked very similar and grave consistent results (see Fig. 1 l–n).

Supplementary Video 3

Single-molecule imaging of ATTO 647N six-dye FluoroCubes and ATTO 647N one-dye dsDNA The movie shows surface-immobilized single molecules of ATTO 647N six-dye FluorocCubes and ATTO 647N one-dye dsDNA over ~50 min under continuous laser illumination. The graph shows the photostability of six-dye FluoroCubes and one-dye dsDNA. The survival rate was quantified by counting the percentage of probes in the ‘on’ state at any given time from 0 to 3,000 s. Time is in minutes. Each experiment was repeated five or four times with freshly assembled six-dye FluoroCubes or one-dye dsDNA, respectively, and on freshly prepared microscope slides. All acquired movies looked very similar and grave consistent results (see Fig. 1l–n)

Supplementary Video 4

Single-molecule imaging of Cy3 six-dye FluoroCubes and Cy3 one-dye dsDNA; The movie shows surface-immobilized single molecules of Cy3 six-dye FluoroCubes and Cy3 one-dye dsDNA over ~50 min under continuous laser illumination. The graph shows the photostability of six-dye FluoroCubes and one-dye dsDNA. The survival rate was quantified by counting the percentage of probes in the ‘on’ state at any given time from 0 to 3,000 s. Time is in minutes. Each experiment was repeated five or four times with freshly ssembled six-dye FluoroCubes or one-dye dsDNA, respectively, and on freshly prepared microscope slides. All acquired movies looked very similar and grave consistent results (see Fig. 1l–n).

Supplementary Video 5

Single-molecule imaging of Cy5 six-dye FluoroCubes and Cy5 one-dye dsDNA The movie shows surface-immobilized single molecules of Cy5 six-dye FluoroCubes and Cy5 one-dye dsDNA over ~50 min under continuous laser illumination. The graph shows the photostability of six-dye FluoroCubes and one-dye dsDNA. The survival rate was quantified by counting the percentage of probes in the ‘on’ state at any given time from 0 to 3,000 s. Time is in minutes. Each experiment was repeated five or four times with freshly assembled six-dye FluoroCubes or one-dye dsDNA, respectively, and on freshly prepared microscope slides. All acquired movies looked very similar and grave consistent results (see Fig. 1l–n)

Supplementary Video 6

Single-molecule imaging of Cy3N six-dye FluoroCubes and Cy3N one-dye dsDNA The movie shows surface-immobilized single molecules of Cy3N six-dye FluoroCubes and Cy3N one-dye dsDNA over ~50 min under continuous laser illumination. The graph shows the photostability of six-dye FluoroCubes and one-dye dsDNA. The survival rate was quantified by counting the percentage of probes in the ‘on’ state at any given time from 0 to 3,000 s. Time is in minutes. Each experiment was repeated five or four times with freshly assembled six-dye FluoroCubes or one-dye dsDNA, respectively, and on freshly prepared microscope slides. All acquired movies looked very similar and grave consistent results (see Fig. 1l–n).

Supplementary Video 7

Movement of Kif1A along an axoneme The movie shows the movement of Kif1A along an axoneme. This particular molecule was tracked for the stepping trace shown in Fig. 2. Note that the axoneme is not visible. The movie plays with 30× real time. Time is in minutes. Scale bar is 1 μm. The imaging of kinesin stepping was repeated multiple times with very similar outcomes (see Supplementary Fig. 20). However, all traces were collected with motors from the same preparation but with freshly prepared microscope slides.

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Niekamp, S., Stuurman, N. & Vale, R.D. A 6-nm ultra-photostable DNA FluoroCube for fluorescence imaging. Nat Methods 17, 437–441 (2020). https://doi.org/10.1038/s41592-020-0782-3

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