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A dual-constriction biological nanopore resolves homonucleotide sequences with high fidelity

Journal Contribution - Journal Article

Single-molecule long-read DNA sequencing with biological nanopores is fast and high-throughput but suffers reduced accuracy in homonucleotide stretches. We now combine the CsgG nanopore with the 35-residue N-terminal region of its extracellular interaction partner CsgF to produce a dual-constriction pore with improved signal and base-calling accuracy for homopolymer regions. The electron cryo-microscopy structure of CsgG in complex with full-length CsgF shows that the 33 N-terminal residues of CsgF bind inside the β-barrel of the pore, forming a defined second constriction. In complexes of CsgG bound to a 35-residue CsgF constriction peptide, the second constriction is separated from the primary constriction by ~25 Å. We find that both constrictions contribute to electrical signal modulation during single-stranded DNA translocation. DNA sequencing using a prototype CsgG-CsgF protein pore with two constrictions improved single-read accuracy by 25 to 70% in homopolymers up to 9 nucleotides long.

Journal: Nat Biotechnol
ISSN: 1087-0156
Issue: 12
Volume: 38
Pages: 1415-1420
Publication year:2020
Keywords:nanopore, sequencing, curli, protein transport, functional amyloid, bacterial cell surface, outer membrane
CSS-citation score:2
Accessibility:Closed