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


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.

Authors: Sander E. Van der Verren, Nani Van Gerven, Wim Jonckheere, Richard Hambley, Pratik Singh, John Kilgour, Michael Jordan, E. Jayne Wallace, Lakmal Jayasinghe, Han Remaut

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