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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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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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.
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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Supplementary Figs. 1–28 and Supplementary Tables 1–7
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.
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