Detection of DNA Methylation with Aerolysin Nanopore

Abstract

DNA methylation is an important epigenetic modification in mammals, which plays an essential role in regulating cell growth, gene expression and genetic diseases. Nanopore technique has been utilized as a rapid, precise and label-free method for detection of methylated DNA at single-molecule level. Recently, we found that wild-type (WT) aerolysin nanopore performed a higher current sensitivity for discrimination of oligonucleotides compared with the mostly used biological nanopores such as α-hemolysin. Herein, we explored the ability of WT aerolysin for identification of methylated cytosine (mC) in a single-strand DNA. It has shown that aerolysin nanopore could not only directly distinguished the mC from unmethylated cytosine (C) by different amplitude of residual currents, but also obtain a perfect separation (∼ 98%) in the mixture of C and mC sample at voltage of + 60 mV. The scatter plots clearly indicated that mC located at 14.7 pA while C was concentrated at 15.1 pA. The duration time of mC traverse aerolysin pore (6.68 ms) was longer than that of C (4.05 ms), which provided an additional parameter to improve the accuracy of separation. Furthermore, we measured the mC in real sample of human serum to explore the possibility of aerolysin nanopore for clinical diagnosis application. The statistical results revealed that mC could be obviously distinguished from C in such complex sample. Therefore, aerolysin protein pore will be a promising candidate for DNA methylation detection and apply to the relevant disease diagnosis in the future.