title: Single-Molecule DNA Methylation Quantification Using Electro-optical Sensing in Solid-State Nanopores.
abstract: 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 ∼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.
authors: Department of Biomedical Engineering, The Technion-Israel Institute of Technology , Haifa, 32000 Israel.
authors: Tal
authors: T
authors: Gilboa
authors: Department of Biomedical Engineering, The Technion-Israel Institute of Technology , Haifa, 32000 Israel.
authors: Chen
authors: C
authors: Torfstein
authors: Institute of Organic Chemistry, RWTH Aachen University , Landoltweg 1, D-52056 Aachen, Germany.
authors: Matyas
authors: M
authors: Juhasz
authors: Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv University , Tel Aviv, 6997801 Israel.
authors: Assaf
authors: A
authors: Grunwald
authors: Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv University , Tel Aviv, 6997801 Israel.
authors: Yuval
authors: Y
authors: Ebenstein
authors: Institute of Organic Chemistry, RWTH Aachen University , Landoltweg 1, D-52056 Aachen, Germany.
authors: Elmar
authors: E
authors: Weinhold
authors: Department of Biomedical Engineering, The Technion-Israel Institute of Technology , Haifa, 32000 Israel.|Department of Biomedical Engineering, Boston University , Boston, Massachusetts 02215, United States.
authors: Amit
authors: A
authors: Meller
mesh_terms: D004247:DNA / Q000032:analysis*; D019175:DNA Methylation; D058608:Nanopores; D036103:Nanotechnology; D016133:Polymerase Chain Reaction
chemical_list: D004247:DNA
keywords: 5-methylcytosine; electro-optical sensing; epigenetic modifications; metyltransferase; single-molecule; solid-state nanopores