Single Molecule Investigations of the Interaction of Ag+ with Single Cytosine, Methylcytosine and Hydroxymethylcytosine-Cytosine Mismatches in a Nanopore

Abstract

Cytosine (C) modifications such as 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) are important epigenetic markers associated with gene expression 1 and tumorigenesis 2, 3. However, bisulfite conversion, the gold standard methodology for mC mapping can not distinguish mC and hmC bases. Recent studies have demonstrated hmC detection via peptide recognizing 4, enzymes 5, fluorescence 6 and hmC-specific antibodies 7, nevertheless, a method for directly discriminating C, mC and hmC bases without labeling, modification and amplification is still missing. Here we show that the DNA duplex containing single cytosine-cytosine (C-C), cytosine-methylcytosine (C-mC) and cytosine-hydroxymethylcytosine (C-hmC) mismatches can be discriminated by their interactions with Ag+ inside the alpha-hemolysin nanopore. Molecular dynamics simulations reveal that the paring of a C-C mismatch through hydrogen bond results in a binding site for cations, such as K+ and Ag+. Cytosine modifications such as mC and hmC disrupted both the hydrogen bonds, which subsequently disrupts Ag+ binding. As a result, these modifications can be distinguished in the nanopore by differences in the stability of DNA-Ag+ complexes.