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.

Multiplexed direct detection of barcoded protein reporters on a nanopore array

Abstract

Detection of specific proteins using nanopores is currently challenging. To address this challenge, we developed a collection of over twenty nanopore-addressable protein tags engineered as reporters (NanoporeTERs, or NTERs). NTERs are constructed with a secretion tag, folded domain and a nanopore-targeting C-terminal tail in which arbitrary peptide barcodes can be encoded. We demonstrate simultaneous detection of up to nine NTERs expressed in bacterial or human cells using MinION nanopore sensor arrays.

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: NTERs.
Fig. 2: Classification and multiplexed detection of NTER expression levels with a MinION.

Data availability

Data are available upon request and on can be found on github.com/uwmisl/NanoporeTERs.

Code availability

Codes are available upon request and can be found on github.com/uwmisl/NanoporeTERs. Custom MinION MinKNOW runscripts can also be obtained from Oxford Nanopore Technologies upon request.

References

  1. 1.

    Ghim, C. M., Lee, S. K., Takayama, S. & Mitchell, R. J. The art of reporter proteins in science: past, present and future applications. BMB Rep. 43, 451–460 (2010).

    CAS  Article  Google Scholar 

  2. 2.

    Rodriguez, E. A. et al. The growing and glowing toolbox of fluorescent and photoactive proteins. Trends Biochem. Sci 42, 111–129 (2017).

    CAS  Article  Google Scholar 

  3. 3.

    Martin, L., Che, A. & Endy, D. Gemini, a bifunctional enzymatic and fluorescent reporter of gene expression. PLoS ONE 4, e7569 (2009).

    Article  Google Scholar 

  4. 4.

    Parrello, D., Mustin, C., Brie, D., Miron, S. & Billard, P. Multicolor whole-cell bacterial sensing using a synchronous fluorescence spectroscopy-based approach. PLoS ONE 10, e0122848 (2015).

    Article  Google Scholar 

  5. 5.

    Shimo, T., Tachibana, K. & Obika, S. Construction of a tri-chromatic reporter cell line for the rapid and simple screening of splice-switching oligonucleotides targeting DMD exon 51 using high content screening. PLoS ONE 13, e0197373 (2018).

    Article  Google Scholar 

  6. 6.

    Wroblewska, A. et al. Protein barcodes enable high-dimensional single-cell CRISPR screens. Cell 175, 1141–1155 (2018).

    CAS  Article  Google Scholar 

  7. 7.

    He, W., Yuan, S., Zhong, W. H., Siddikee, M. A. & Dai, C. C. Application of genetically engineered microbial whole-cell biosensors for combined chemosensing. Appl. Microbiol. Biotechnol. 100, 1109–1119 (2016).

    CAS  Article  Google Scholar 

  8. 8.

    Nielsen, A. A. K. et al. Genetic circuit design automation. Science 352, aac7341 (2016).

    Article  Google Scholar 

  9. 9.

    Shi, W., Friedman, A. K. & Baker, L. A. Nanopore sensing. Anal. Chem. 89, 157–188 (2017).

    CAS  Article  Google Scholar 

  10. 10.

    Jain, M., Olsen, H. E., Paten, B. & Akeson, M. The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community. Genome Biol. 17, 239 (2016).

    Article  Google Scholar 

  11. 11.

    Garalde, D. R. et al. Highly parallel direct RNA sequencing on an array of nanopores. Nat. Methods 15, 201–206 (2018).

    CAS  Article  Google Scholar 

  12. 12.

    Nivala, J., Marks, D. B. & Akeson, M. Unfoldase-mediated protein translocation through an α-hemolysin nanopore. Nat. Biotechnol. 31, 247–250 (2013).

    CAS  Article  Google Scholar 

  13. 13.

    Nivala, J., Mulroney, L., Li, G., Schreiber, J. & Akeson, M. Discrimination among protein variants using an unfoldase-coupled nanopore. ACS Nano 8, 12365–12375 (2014).

    CAS  Article  Google Scholar 

  14. 14.

    Yim, H. H. & Villarejo, M. osmY, a new hyperosmotically inducible gene, encodes a periplasmic protein in Escherichia coli. J. Bacteriol. 174, 3637–3644 (1992).

    CAS  Article  Google Scholar 

  15. 15.

    Kotzsch, A. et al. A secretory system for bacterial production of high-profile protein targets. Protein Sci. 20, 597–609 (2011).

    CAS  Article  Google Scholar 

  16. 16.

    Goyal, P. et al. Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG. Nature 516, 250–253 (2014).

    CAS  Article  Google Scholar 

  17. 17.

    Taylor, S. S. et al. PKA: a portrait of protein kinase dynamics.Biochim. Biophys. Acta Proteins Proteom. 1697, 259–269 (2004).

    CAS  Article  Google Scholar 

  18. 18.

    Román, R. et al. Enhancing heterologous protein expression and secretion in HEK293 cells by means of combination of CMV promoter and IFNα2 signal peptide. J. Biotechnol. 239, 57–60 (2016).

    Article  Google Scholar 

  19. 19.

    Peroutka, R. J., Elshourbagy, N., Piech, T. & Butt, T. R. Enhanced protein expression in mammalian cells using engineered SUMO fusions: secreted phospholipase A 2. Protein Sci. 17, 1586–1595 (2008).

    CAS  Article  Google Scholar 

  20. 20.

    Gorochowski, T. E. et al. Genetic circuit characterization and debugging using RNA‐seq. Mol. Syst. Biol. 13, 952 (2017).

    Article  Google Scholar 

  21. 21.

    Gach, P. C. et al. A droplet microfluidic platform for automating genetic engineering. ACS Synth. Biol. 5, 426–433 (2016).

    CAS  Article  Google Scholar 

  22. 22.

    Chao, R., Mishra, S., Si, T. & Zhao, H. Engineering biological systems using automated biofoundries. Metab. Eng. 42, 98–108 (2017).

    Article  Google Scholar 

  23. 23.

    Madison, A. C. et al. Scalable device for automated microbial electroporation in a digital micro fluidic platform. ACS Synth. Biol. 6, 1701–1709 (2017).

    CAS  Article  Google Scholar 

  24. 24.

    Chen, Z. & Elowitz, E. B. Programmable protein circuit design. Cell 184, 2284–2301 (2021).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank additional members of the Molecular Information Systems Lab for helpful discussion and feedback on this work. The OsmY expression plasmid was generously provided by C. Bryan and L. Carter (Institute for Protein Design, University of Washington). We also thank A. Heron and R. Gutierrez (Oxford Nanopore Technologies) for providing the configurable MinION run script and discussions on its use, and M. Jain (UCSC) for a custom Matlab script that facilitated visualization of the raw MinION data. This work was supported in part by NSF EAGER Award no. 1841188 and NSF CCF Award no. 2006864 to L.C. and J.N., an NIH/NCI Cancer Center Support Grant (no. P30 CA015704) Pilot Award and NSF Award 2021552 to J.N. and a sponsored research agreement from Oxford Nanopore Technologies.

Author information

Affiliations

Authors

Contributions

N.C., K.Z., A.N. and N.B. performed wet laboratory experiments. K.Z. and K.D. developed the data analysis pipeline and performed computational analyses. Z.S. implemented the machine learning approach. N.B., K.S., L.C. and J.N. supervised the project. J.N. conceived and directed the project. All authors contributed to writing and editing of the manuscript.

Corresponding author

Correspondence to Jeff Nivala.

Ethics declarations

Competing interests

A provisional patent has been filed by the University of Washington covering aspects of this work (Patent Application no. 17/283,007). K.S. is an employee of Microsoft. J.N. is a consultant to Oxford Nanopore Technologies. The remaining authors declare no competing interests.

Additional information

Peer review information Nature Biotechnology thanks Yi-Tao Long, Meni Wanunu 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. 1–13, Notes and References.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Cardozo, N., Zhang, K., Doroschak, K. et al. Multiplexed direct detection of barcoded protein reporters on a nanopore array. Nat Biotechnol (2021). https://doi.org/10.1038/s41587-021-01002-6

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