A lithium-ion-active aerolysin nanopore for effectively trapping long single-stranded DNA† †Electronic supplementary information (ESI) available: Protocol for aerolysin nanopore experiment, molecular dynamics simulations, histograms of the event frequencies and durations, and supporting figures. See DOI: 10.1039/c8sc03927e

By developing lithium-ion-active aerolysin, for the first time we have achieved aerolysin detection of single-stranded DNA longer than 100 nt.
Wild-type aerolysin (AeL) nanopores allow direct single nucleotide discrimination of very short oligonucleotides (≤10 nt) without labelling, which shows great potential for DNA sensing. To achieve real applications, one major obstacle of AeL is its poor capture ability of long single-stranded DNA (ssDNA, >10 nt). Here, we have proposed a novel and robust strategy for the electrostatic focusing of long ssDNA into a lithium-chloride (LiCl)-active AeL. By using this method, for the first time we have demonstrated AeL detection of ssDNA longer than 100 nt. Due to screening more negative charges, LiCl improves AeL capture ability of long ssDNA (i.e. 60 nt) by 2.63- to 10.23-fold compared to KCl. Further calculations and molecular dynamics simulations revealed that strong binding between Li+ and the negatively charged residue neutralized the AeL, leading to a reduction in the energy barrier for ssDNA capture. These findings facilitate the future high-throughput applications of AeL in genetic and epigenetic diagnostics.