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.

具有生物纳米孔的单分子长读DNA测序速度快，通量高，但在同核苷酸序列中准确性降低。现在，我们将CsgG纳米孔与其细胞外相互作用伴侣CsgF的35个残基的N末端区域结合起来，以产生双缩孔，提高均聚物区域的信号和碱基检出准确度。CsgG与全长CsgF的复合物的电子冷冻显微镜结构表明，CsgF的33个N末端残基结合在孔的β-桶内，形成了确定的第二颈缩。在与35个残基的CsgF缩窄肽结合的CsgG的复合物中，第二个缩窄与主要的缩窄分开约25Å。我们发现这两个收缩有助于单链DNA易位期间的电信号调制。