Dynamics of a DNA Mismatch Site Held in Confinement Discriminate Epigenetic Modifications of Cytosine

The identification and discrimination of four epigenetic modifications to cytosine in the proposed active demethylation cycle is demonstrated at the single-molecule level, without the need for chemical pretreatment or labeling. The wild-type protein nanopore α-hemolysin is used to capture individual DNA duplexes containing a single cytosine-cytosine mismatch. The mismatch is held at the latch constriction of α-hemolysin, which is used to monitor the kinetics of base-flipping at the mismatch site. Base-flipping and the subsequent interactions between the DNA and the protein are dramatically altered when one of the cytosine bases is replaced with methyl-, hydroxymethyl-, formyl-, or carboxylcytosine. As well as providing a route to single-molecule analysis of important epigenetic markers in DNA, our results provide important insights into how the introduction of biologically relevant, but poorly understood, modifications to cytosine affect the local conformational dynamics of a DNA duplex in a confined environment.