Abstract
Nanopore sensing is a powerful single-molecule method for DNA and protein sequencing. Recent studies have demonstrated that aerolysin exhibits a high sensitivity for single-molecule detection. However, the lack of the atomic resolution structure of aerolysin pore has hindered the understanding of its sensing capabilities. Herein, we integrate nanopore experimental results and molecular simulations based on a recent pore structural model to precisely map the sensing spots of this toxin for ssDNA translocation. Rationally probing ssDNA length and composition upon pore translocation provides new important insights for molecular determinants of the aerolysin nanopore. Computational and experimental results reveal two critical sensing spots (R220, K238) generating two constriction points along the pore lumen. Taking advantage of the sensing spots, all four nucleobases, cytosine methylation and oxidation of guanine can be clearly identified in a mixture sample. The results provide evidence for the potential of aerolysin as a nanosensor for DNA sequencing.
Highlights
Nanopore sensing is a powerful single-molecule method for DNA and protein sequencing
We found that these current values presented a strong dependence with the length of oligonucleotides for dAn shorter than 14 bases (n
We present an analysis of single-strand DNA (ssDNA) translocation across aerolysin biological pores, which is based on both experimental and computational analysis, and allows us to unveil novel properties of aerolysin-oligonucleotide interplay
Summary
Nanopore sensing is a powerful single-molecule method for DNA and protein sequencing. We integrate nanopore experimental results and molecular simulations based on a recent pore structural model to precisely map the sensing spots of this toxin for ssDNA translocation. Aerolysin pore models have been recently proposed based on near-atomic resolution cryo-electron microscopy (EM) structures (3.9–4.5 Å) of its mutated prepore and quasi-pore states, along with a 7.9-Å resolution model of the wild-type pore state[24] This structural analysis revealed a heptameric pore architecture featuring a very long (~10 nm) membrane spanning the β-barrel pore channel, which resembles the one found for anthrax toxin[25] in dimensions, and is much longer than the α-hemolysin pore[3]. By rationally probing ssDNA length and composition upon pore translocation, we gained new important molecular insights that which could help the future design of aerolysin variants with improved capabilities for sequencing
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