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Translocation of linearized full-length proteins through an engineered nanopore under opposing electrophoretic force

Nature Biotechnology (2023)Cite this article

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Abstract

Nanopores have recently been used to identify and fingerprint proteins. However, because proteins, unlike DNA, do not have a uniform charge, the electrophoretic force cannot in general be used to translocate or linearize them. Here we show that the introduction of sets of charges in the lumen of the CytK nanopore spaced by ~1 nm creates an electroosmotic flow that induces the unidirectional transport of unstructured natural polypeptides against a strong electrophoretic force. Molecular dynamics simulations indicate that this electroosmotic-dominated force has a strength of ~20 pN at −100 mV, which is similar to the electric force on single-stranded DNA. Unfolded polypeptides produce current signatures as they traverse the nanopore, which may be used to identify proteins. This approach can be used to translocate and stretch proteins for enzymatic and non-enzymatic protein identification and sequencing.

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Fig. 1: Engineering and EOF.
Fig. 2: Translocation of unfolded substrates using engineered nanopores.
Fig. 3: EOF and force on peptide estimated by MD.
Fig. 4: Translocation of unfolded proteins across 2E-4D-CytK nanopores.

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Data availability

Data and the setup files for the simulations can be found at https://doi.org/10.5281/zenodo.8123395. Source data are provided with this paper.

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Acknowledgements

A.S. and G.M. acknowledge NWO-VICI grant 192.068 and NIH grant 1086554. M.C. and B.M.d.R acknowledge supercomputer time provided through the s1178 Production Grant from the Swiss National Supercomputing Centre (CSCS). M.C and B.M.d.R thank G. Di Muccio for useful discussions.

Author information

Authors and Affiliations

  1. Groningen Biomolecular Sciences & Biotechnology Institute, University of Groningen, Groningen, The Netherlands

    Adina Sauciuc, Matthijs Jonathan Tadema & Giovanni Maglia

  2. Department of Biology, University of Rome Tor Vergata, Rome, Italy

    Blasco Morozzo della Rocca

  3. Department of Industrial Engineering, University of Rome Tor Vergata, Rome, Italy

    Mauro Chinappi

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  1. Adina Sauciuc
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Contributions

G.M. and A.S. designed the research. A.S. carried out the experimental research. M.T. made the electrostatic maps of the nanopores. B.M.d.R. and M.C. carried out the simulations. All authors contributed to the writing of the manuscript and approved the final version.

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Correspondence to Giovanni Maglia.

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Competing interests

The authors declare no competing interests. G.M. is a founder, director and shareholder of Portal Biotech Limited, a company engaged in the development of nanopore technologies. This work was not supported by Portal Biotech Limited.

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Nature Biotechnology thanks Henry Brinkerhoff 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. 1–28 and Supplementary Tables 1–7

Reporting Summary

Supplementary Tables

This file contains the following tables: Supplementary Table 1: oligonucleotides (primers) used to generate the mutant CytK nanopores; Supplementary Table 2: oligonucleotides (primers) to generate the aerolysin mutant; Supplementary Table 3: oligonucleotides (primers) to generate the substrates; and Supplementary Table 4: the chemicals and reagents used in this study and their corresponding suppliers and catalog/article numbers.

Supplementary Data

Source data for Supplementary Fig. 28. Unprocessed SDS-PAGE of the substrates (proteins that were translocated across the nanopores).

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Sauciuc, A., Morozzo della Rocca, B., Tadema, M.J. et al. Translocation of linearized full-length proteins through an engineered nanopore under opposing electrophoretic force. Nat Biotechnol (2023). https://doi.org/10.1038/s41587-023-01954-x

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