Simultaneous Dual-Site Identification of 5^mC/8^oG in DNA Triplex Using a Nanopore Sensor

DNA triplex participates in delivering site-specific epigenetic modifications critical for the regulation of gene expression. Among these marks, 5^mC with 8^oG functions comprehensively on gene expression. Recently, few research studies have emphasized the necessity of incorporation detection of 5^mC with 8^oG using one DNA triplex at the same time. Herein, DNA triplex structure was designed and tailored for the site-specific identification of 5^mC with 8^oG by means of nanopore electroanalysis. The identification was associated with the distinguishable current modulation types caused by DNA unzipping through the nanopore in an electrical field. Results demonstrated that the epigenetic modification proximity to the latch zone or constriction area of the nanopore enables differentiation of modification series at single nucleotide resolution in one DNA triplex, at both physiological and mildly acidic environment. In addition, our nanopore method enables the kinetic and thermodynamic studies to calculate the free energy of modified DNA triplex with applied potentials. Gibbs' energy provided the direct evidence that the DNA triplex with these epigenetic modifications is more stable in acidic environment. Considering modified DNA functions significantly in gene expression, the presented method may provide future opportunities to understand incorporating epigenetic mechanisms of many dysregulated biological processes on the basis of accurate detection.