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

Area of Science:

• Epigenetics and Molecular Biology
• Biosensing and Nanotechnology

Background:

• Epigenetic markers like 5-methylcytosine are vital for gene regulation, with altered DNA methylation implicated in cancer.
• Existing single-molecule detection primarily targets hypermethylation, leaving a gap in sensing hypomethylation crucial for oncogene activity.
• Hypomethylation of oncogenes during cancer development presents a significant biosensing challenge.

Purpose of the Study:

• To develop a novel labeling and single-molecule quantification method for unmethylated cytosine-guanine dinucleotides (CpGs).
• To enable direct detection of hypomethylated sites in long DNA molecules without PCR or bisulfite conversion.
• To establish a foundation for sequence-specific targeting of clinically relevant hypomethylated genomic regions.

Main Methods:

• A single-step covalent coupling of DNA with synthetic cofactor analogues using DNA methyltransferases (MTases).
• Molecule-by-molecule electro-optical nanopore detection and quantification using single or multiple colors.
• Analysis of approximately 10 kbp long double-stranded DNA.

Main Results:

• Developed a method to quantify the number of unmethylated CpGs in target DNA sequences at the single-molecule level.
• Demonstrated the ability to analyze long DNA molecules (∼10 kbp) without PCR amplification or bisulfite conversion.
• Successfully expanded the technique to two colors for sensing multiple DNA MTases via orthogonal labeling.

Conclusions:

• The developed method provides a calibrated scale for direct quantification of unmethylated CpGs in individual DNA molecules.
• This technique circumvents common limitations of PCR amplification and bisulfite conversion for DNA methylation analysis.
• The proof-of-principle study enables sequence-specific, direct targeting of clinically relevant hypomethylated sites, advancing cancer diagnostics.