Selective and Sensitive Detection of Methylcytosine by Aerolysin Nanopore under Serum Condition

Abstract

Detection of DNA methylation in real human serum is of great importance to push the development of clinical research and early diagnosis of human diseases. Herein, taking advantage of stable pore structure of aerolysin in a harsh environment, we distinguish methylated cytosine from cytosine using aerolysin nanopore in human serum. Since wild-type (WT) aerolysin enables high sensitivity detection of DNA, the subtle difference between methylated cytosine and cytosine could be measured directly without any specific designs. Methylated cytosine induced a population of I/I = 0.53 while cytosine was focused on I/I = 0.56. The dwell time of methylated cytosine (5.3 ± 0.1 ms) was much longer than that of cytosine (3.9 ± 0.1 ms), which improves the accuracy for the discrimination of the two oligomers. Moreover, the pore-membrane system could remain stable for more than 2 h and achieve the detection of methylated cytosine with zero-background signal in the presence of serum. Additionally, event frequency of methylated cytosine is in correspondence with the relative concentration and facilitate the quantification of methylation.

A General Strategy of Aerolysin Nanopore Detection for Oligonucleotides with the Secondary Structure

“…Next, the voltage‐dependent studies were conducted which revealed that the duration of Teln decreased exponentially with the applied voltage ranging from +120 to +180 mV ( Figure a). These results were consistent with our previous researches and confirmed the translocation of Teln through the aerolysin nanopore. The free energy barrier is associated with the transport of oligonucleotides.…”

“…Next, the voltage-dependent studies were conducted which revealed that the duration of Teln decreased exponentially with the applied voltage ranging from +120 to +180 mV (Figure 3a). These results were consistent with our previous researches [8,[26][27][28] Small 2018, 14, 1704520 Figure 2. Translocation of unfolded Teln (n = 1, 2, 3, 4, and 5) through an aerolysin nanopore in MgCl 2 solution.…”

Membrane protein-based biosensors

“…The technologies presented above show the future potential for the identification of epigenetic modification or single nucleotide polymorphism on DNA using the nanopore-nucleic acid interaction [52,53,61,62]. In addition, the nanopore coupled with a complementary DNA would facilitate the early detection of pathogenic DNA, shown in the detection of the anthrax-related DNA of Bacillus anthracis [63].…”

The aerolysin nanopore: from peptidomic to genomic applications

“…26 This study demonstrated the possibility that ARP systems could be utilized for highly sensitive and specific detection of single base variations, including the identification of gene mutations and base modifications. Later, Long’s group 27 reported that an ARP system could discriminate methylcytosine (mC) from C (5′-CAAA-3′ vs. 5′-mCAAA-3′) in serum. The translocation frequency of methylated cytosine correlated with its relative concentration (Fig.…”