Identification of Essential Sensitive Regions of the Aerolysin Nanopore for Single Oligonucleotide Analysis

The aerolysin nanopore channel is one of the confined spaces for single molecule analysis which displays high spatial and temporal resolution for the discrimination of single nucleotides, identification of DNA base modification, and analyzing the structural transition of DNAs. However, to overcome the challenge of achieving the ultimate goal of the widespread real analytical application, it is urgent to probe the sensing regions of the aerolysin to further improve the sensitivity. In this paper, we explore the sensing regions of the aerolysin nanopore by a series of well-designed mutant nanopore experiments combined with molecular dynamics simulations-based electrostatic analysis. The positively charged lumen-exposed Lys-238, identified as one of the key sensing sites due to the presence of a deep valley in the electrostatic potentials, was replaced by different charged and sized amino acids. The results show that the translocation time of oligonucleotides through the nanopore can be readily modulated by the choice of the target amino acid at the 238 site. In particular, a 7-fold slower translocation at a voltage bias of +120 mV is observed with respect to the wild-type aerolysin, which provides a high resolution for methylated cytosine discrimination. We further determine that both the electrostatic properties and geometrical structure of the aerolysin nanopore are crucial to its sensing ability. These insights open ways for rationally designing the sensing mechanism of the aerolysin nanopore, thus providing a novel paradigm for nanopore sensing.