Write and Read: Harnessing Synthetic DNA Modifications for Nanopore Sequencing.

Area of Science:

• Genomics
• Molecular Biology
• Biotechnology

Background:

• Nanopore sequencing offers multiomic data capture from single DNA molecules.
• Nucleotide modifications generate distinct electrical signatures detectable by nanopore sequencing.
• Integrating genomic and epigenomic data requires advanced sequencing capabilities.

Purpose of the Study:

• To develop a versatile "write-and-read" framework for detecting chemically modified DNA bases using nanopore sequencing.
• To demonstrate the ability to distinguish between native and modified nucleotides based on their electrical fingerprints.
• To explore the potential for simultaneous multi-omic data acquisition from individual DNA molecules.

Main Methods:

• Chemo-enzymatic DNA labeling with synthetic tags to create predictable electrical fingerprints.
• DNA glucosylation to modify 5-hydroxymethylcytosine (5hmC) with glucose or glucose-azide adducts.
• Enzymatic alkylation to introduce azide residues onto adenine bases.

Main Results:

• Modified nucleotides (5hmC, N6-adenine) produced distinct and reproducible electrical shifts in nanopore sequencing.
• Successful direct detection of chemically altered nucleotides was achieved.
• Demonstrated bio-orthogonal DNA labeling for an expanded set of detectable moieties.

Conclusions:

• This framework enables direct detection of DNA modifications through unique electrical signatures.
• Programmable chemical modifications facilitate simultaneous multi-omic analysis on individual DNA molecules.
• This approach significantly advances genetic research and discovery by integrating sequence and modification data.