Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

You are viewing this page in draft mode.

Super-resolution RNA imaging using a rhodamine-binding aptamer with fast exchange kinetics

Abstract

Overcoming limitations of previous fluorescent light-up RNA aptamers for super-resolution imaging, we present RhoBAST, an aptamer that binds a fluorogenic rhodamine dye with fast association and dissociation kinetics. Its intermittent fluorescence emission enables single-molecule localization microscopy with a resolution not limited by photobleaching. We use RhoBAST to image subcellular structures of RNA in live and fixed cells with about 10-nm localization precision and a high signal-to-noise ratio.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Directed evolution and characterization of RhoBAST.
Fig. 2: Confocal, epifluorescence and SMLM with the RhoBAST:TMR-DN system.

Data availability

The data that support the findings of this study are available upon reasonable request from the corresponding authors. Aptamer sequences used for imaging ROIs are available in the Supplementary Information.

References

  1. 1.

    Sigal, Y. M., Zhou, R. & Zhuang, X. Science 361, 880–887 (2018).

    CAS  Article  Google Scholar 

  2. 2.

    Schmidt, A., Gao, G., Little, S. R., Jalihal, A. P. & Walter, N. G. Wiley Interdiscip. Rev. RNA 11, e1587 (2020).

  3. 3.

    Su, Y. & Hammond, M. C. Curr. Opin. Biotechnol. 63, 157–166 (2020).

    CAS  Article  Google Scholar 

  4. 4.

    Wirth, R., Gao, P., Nienhaus, G. U., Sunbul, M. & Jäschke, A. J. Am. Chem. Soc. 141, 7562 (2019).

    CAS  Article  Google Scholar 

  5. 5.

    Chen, X. et al. Nat. Biotechnol. 37, 1287–1293 (2019).

    CAS  Article  Google Scholar 

  6. 6.

    Sunbul, M. & Jäschke, A. Angew. Chem. Int. Ed. 52, 13401–13404 (2013).

    CAS  Article  Google Scholar 

  7. 7.

    Arora, A., Sunbul, M. & Jäschke, A. Nucleic Acids Res. 43, e144 (2015).

    PubMed  PubMed Central  Google Scholar 

  8. 8.

    Braselmann, E. et al. Nat. Chem. Biol. 14, 964–971 (2018).

    CAS  Article  Google Scholar 

  9. 9.

    Sunbul, M. & Jäschke, A. Nucleic Acids Res. 46, e110 (2018).

    Article  Google Scholar 

  10. 10.

    Li, Y., Ishitsuka, Y., Hedde, P. N. & Nienhaus, G. U. ACS Nano 7, 5207–5214 (2013).

    CAS  Article  Google Scholar 

  11. 11.

    Filonov, G. S., Moon, J. D., Svensen, N. & Jaffrey, S. R. J. Am. Chem. Soc. 136, 16299–16308 (2014).

    CAS  Article  Google Scholar 

  12. 12.

    Song, W. et al. Nat. Chem. Biol. 13, 1187–1194 (2017).

    CAS  Article  Google Scholar 

  13. 13.

    Autour, A. et al. Nat. Commun. 9, 656 (2018).

    Article  Google Scholar 

  14. 14.

    Schnitzbauer, J., Strauss, M. T., Schlichthaerle, T., Schueder, F. & Jungmann, R. Nat. Protoc. 12, 1198–1228 (2017).

    CAS  Article  Google Scholar 

  15. 15.

    Litke, J. L. & Jaffrey, S. R. Nat. Biotechnol. 37, 667–675 (2019).

    CAS  Article  Google Scholar 

  16. 16.

    Kim, H. & Jaffrey, S. R. Cell Chem. Biol. 26, 1725–1731 e1726 (2019).

    CAS  Article  Google Scholar 

  17. 17.

    Hagerman, R. J. & Hagerman, P. Nat. Rev. Neurol. 12, 403–412 (2016).

    CAS  Article  Google Scholar 

  18. 18.

    Fox, A. H., Nakagawa, S., Hirose, T. & Bond, C. S. Trends Biochem. Sci. 43, 124–135 (2018).

    CAS  Article  Google Scholar 

  19. 19.

    Steinberg, R., Knupffer, L., Origi, A., Asti, R. & Koch, H. G. FEMS Microbiol. Lett. 365 (2018).

  20. 20.

    Wu, Y. & Shroff, H. Nat. Methods 15, 1011–1019 (2018).

    CAS  Article  Google Scholar 

  21. 21.

    Strack, R. L., Disney, M. D. & Jaffrey, S. R. Nat. Methods 10, 1219–1224 (2013).

    CAS  Article  Google Scholar 

  22. 22.

    Grimm, J. B. et al. Nat. Methods 14, 987–994 (2017).

    CAS  Article  Google Scholar 

  23. 23.

    Bajar, B. T. et al. Sci. Rep. 6, 20889 (2016).

    CAS  Article  Google Scholar 

  24. 24.

    Schindelin, J. et al. Nat. Methods 9, 676–682 (2012).

    CAS  Article  Google Scholar 

  25. 25.

    Manz, C. et al. Nat. Chem. Biol. 13, 1172–1178 (2017).

    CAS  Article  Google Scholar 

  26. 26.

    Li, Y., Shang, L. & Nienhaus, G. U. Nanoscale 8, 7423–7429 (2016).

    CAS  Article  Google Scholar 

  27. 27.

    Ober, R. J., Ram, S. & Ward, E. S. Biophys. J. 86, 1185–1200 (2004).

    CAS  Article  Google Scholar 

  28. 28.

    Deschout, H. et al. Nat. Methods 11, 253–266 (2014).

    CAS  Article  Google Scholar 

  29. 29.

    Guizar-Sicairos, M., Thurman, S. T. & Fienup, J. R. Opt. Lett. 33, 156–158 (2008).

    Article  Google Scholar 

  30. 30.

    Fedorov, A. et al. Magn. Reson. Imaging 30, 1323–1341 (2012).

    Article  Google Scholar 

Download references

Acknowledgements

M.S. and A.J. were supported by the Deutsche Forschungsgemeinschaft (DFG grant no. Ja794/11) and G.U.N. by the Helmholtz Association (Program Science and Technology of Nanosystems) and the DFG (GRK 2039). We thank the Nikon Imaging Center, Heidelberg for granting access to their facilities, U. Engel for technical advice in fluorescence microscopy and M. Mayer and L. Rohland for assistance with stopped-flow measurements. We gratefully acknowledge R. Ma and A. Kobitski for technical support with SMLM experiments and analysis. We thank BASF SE for kindly providing Lutensol AT50.

Author information

Affiliations

Authors

Contributions

M.S., G.U.N. and A.J. designed the study. A.M. and M.S. evolved RhoBAST, and M.S., D.E. and F.G. characterized RhoBAST’s photophysical properties. M.S. created all plasmid constructs and strains and carried out confocal and SIM microscopy. J.L. carried out SMLM experiments and analyzed the data. J.L. and B.H. developed the assay for single-molecule binding kinetics and performed the experiments and analysis. M.S., K.N., G.U.N. and A.J. supervised the work. M.S. wrote the first draft, and all authors contributed to reviewing, editing and providing additional text for the manuscript.

Corresponding authors

Correspondence to Murat Sunbul, G. Ulrich Nienhaus or Andres Jäschke.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Biotechnology thanks Don Lamb and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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, Supplementary Tables 1–5 and Supplementary Notes 1–3

Reporting Summary

Supplementary Video 1

Fluorescence decay of Pepper and RhoBAST

Supplementary Video 2

3D CGG repeat-containing FMR1-GFP mRNA aggregates

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Sunbul, M., Lackner, J., Martin, A. et al. Super-resolution RNA imaging using a rhodamine-binding aptamer with fast exchange kinetics. Nat Biotechnol 39, 686–690 (2021). https://doi.org/10.1038/s41587-020-00794-3

Download citation

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing