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

Tal Gilboa, Chen Torfstein, Matyas Juhasz, Assaf Grunwald, Yuval Ebenstein, Elmar Weinhold, Amit Meller

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)8861-8870
Number of pages10
JournalACS Nano
Volume10
Issue number9
DOIs
StatePublished - 27 Sep 2016

Keywords

  • 5-methylcytosine
  • electro-optical sensing
  • epigenetic modifications
  • metyltransferase
  • single-molecule
  • solid-state nanopores

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