PT  - JOURNAL ARTICLE
AU  - Henry Brinkerhoff
AU  - Albert S. W. Kang
AU  - Jingqian Liu
AU  - Aleksei Aksimentiev
AU  - Cees Dekker
TI  - Infinite re-reading of single proteins at single-amino-acid resolution using nanopore sequencing
AID  - 10.1101/2021.07.13.452225
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.07.13.452225
4099  - http://biorxiv.org/content/early/2021/07/14/2021.07.13.452225.short
4100  - http://biorxiv.org/content/early/2021/07/14/2021.07.13.452225.full
AB  - 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 StatementTU Delft has filed a patent application on technologies described herein, with H.B. and C.D. listed as inventors.