Single-Molecule DNA Methylation Quantification Using Electro-optical Sensing in Solid-State Nanopores

Detection of epigenetic markers, including 5-methylcytosine, is crucial due to their role in gene expression regulation and due to the mounting evidence of aberrant DNA methylation patterns in cancer biogenesis. Single-molecule methods to date have primarily been focused on hypermethylation detection; however, many oncogenes are hypomethylated during cancer development, presenting an important unmet biosensing challenge To this end, we have developed a labeling and single-molecule quantification method for multiple unmethylated cytosine guanine dinucleotides (CpGs). Our method involves a single-step covalent coupling of DNA with synthetic cofactor analogues using DNA methyltransferases (MTases) followed by molecule-by-molecule electro-optical nanopore detection and quantification with single or multiple colors. This sensing method yields a calibrated scale to directly quantify the number of unmethylated CpGs in the target sequences of each DNA molecule. Importantly, our method can be used to analyze similar to 10 kbp long double-stranded DNA while circumventing PCR amplification or bisulfite conversion. Expanding this technique to use two colors, as demonstrated here; would enable sensing of multiple DNA MTases through orthogonal labeling/sensing of unmethylated CpGs (or other epigenetic modifications) associated with specific recognition sites. Our proof-of-principle study may permit sequence-specific, direct targeting of clinically relevant hypomethylated sites in the genome.