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Infinite re-reading of single proteins at single-amino-acid resolution using nanopore sequencing

Henry Brinkerhoff, Albert S. W. Kang, Jingqian Liu, Aleksei Aksimentiev, Cees Dekker
doi: https://doi.org/10.1101/2021.07.13.452225
Henry Brinkerhoff
1Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, van der Maasweg 9, 2629 HZ Delft, The Netherlands
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Albert S. W. Kang
1Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, van der Maasweg 9, 2629 HZ Delft, The Netherlands
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Jingqian Liu
2Department of Physics, University of Illinois at Urbana—Champaign, Urbana, Illinois 61801, United States
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Aleksei Aksimentiev
2Department of Physics, University of Illinois at Urbana—Champaign, Urbana, Illinois 61801, United States
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Cees Dekker
1Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, van der Maasweg 9, 2629 HZ Delft, The Netherlands
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  • For correspondence: c.dekker@tudelft.nl
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Abstract

As identifying proteins is of paramount importance for cell biology and applications, it is of interest to develop a protein sequencer with the ultimate sensitivity of decoding individual proteins. Here, we demonstrate a nanopore-based single-molecule sequencing approach capable of reliably detecting single amino-acid substitutions within individual peptides. A peptide is linked to a DNA molecule that is pulled through the biological nanopore MspA by a DNA helicase in single amino-acid steps. The peptide sequence yields clear stepping ion current signals which allows to discriminate single-amino-acid substitutions in single reads. Molecular dynamics simulations show these signals to result from size exclusion and pore binding. Notably, we demonstrate the capability to ‘rewind’ peptide reads, obtaining indefinitely many independent reads of the same individual molecule, yielding virtually 100% read accuracy in variant identification, with an error rate less than 10−6. These proof-of-concept experiments constitute a promising basis for developing a single-molecule protein sequencer.

One-sentence summary This paper presents proof-of-concept experiments and simulations of a nanopore-based approach to sequencing individual proteins.

Competing Interest Statement

TU Delft has filed a patent application on technologies described herein, with H.B. and C.D. listed as inventors.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted July 14, 2021.
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Infinite re-reading of single proteins at single-amino-acid resolution using nanopore sequencing
Henry Brinkerhoff, Albert S. W. Kang, Jingqian Liu, Aleksei Aksimentiev, Cees Dekker
bioRxiv 2021.07.13.452225; doi: https://doi.org/10.1101/2021.07.13.452225
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Infinite re-reading of single proteins at single-amino-acid resolution using nanopore sequencing
Henry Brinkerhoff, Albert S. W. Kang, Jingqian Liu, Aleksei Aksimentiev, Cees Dekker
bioRxiv 2021.07.13.452225; doi: https://doi.org/10.1101/2021.07.13.452225

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