DESCRIPTION
A chemo-enzymatic platform that actively encodes new biological information into DNA before nanopore sequencing — enabling simultaneous genetic and epigenomic readout from a single molecule, in a single run. By installing synthetic base modifications at targeted sites, the platform converts epigenetic events (5-hydroxymethylcytosine, chromatin accessibility, DNA damage) into distinct, machine-readable electrical signals without bisulfite conversion or antibody-based enrichment.

Figure A:  This plot shows an illustration of our write-and-read paradigm: (A) write: chemoenzymatic conversion of designated genomic bases with synthetic epigenetic modifications. (B) Nanopore sequencing of the modified strands. (C) Read: detection of modified signal using modified-basecalling algorithms.

UNMET NEED
Current epigenomic profiling methods are constrained by the existing alphabet of nucleobases and their naturaly occurring modification:

• Native nanopore sequencing passively detects naturally occurring modifications but cannot be programmed to target custom epigenetic events
• No existing platform supports active encoding of biological signals into DNA for downstream electrical readout

OUR SOLUTION
The write-and-read framework operates in three steps:

• WRITE: Protein binding sites, chromatin-accessible residues, or DNA-damage sites are labeled with synthetic chemical adducts via enzymatic reactions.
• SEQUENCE: Labeled DNA is sequenced on a standard Oxford Nanopore Technology (ONT) platform — no hardware modifications required
• READ: Custom basecalling algorithms decode the unique ionic current signatures of synthetic modifications, distinguishing them from natural bases and from each other

Validated in ACS Nano (2025): Synthetic modifications produce reproducible, position-specific current shifts across cytosine and adenine modification systems.

UNIQUE ADVANTAGES

• Single-molecule resolution — no averaging across bulk populations; each DNA molecule carries full epigenomic information
• Multi-omic in one run — genetic sequence and up to 5 epigenetic layers captured simultaneously from the same molecule
• Hardware-agnostic — runs on standard ONT MinION/PromethION; no new instruments required
• No bisulfite — preserves long reads essential for structural variant analysis and repeat regions
• Programmable and modular — any enzyme with specific base-modification activity can be integrated as a new write channel

Figure B. A schematic illustration of a single-pass multiomic experiment demonstrating how nanopore sequencing can simultaneously detect multiple epigenetic and chromatin features. The diagram shows the process in four stages: (A) Nuclear chromatin is composed of genomic DNA, carrying native 5mC (red) and 5hmC (green) modifications, and associated with a plethora of DNA-binding proteins such as nucleosomes and transcription factors. (B) Exposed genomic DNA is labeled within permeabilized nuclei to mark protein footprints by utilizing the high frequency of Adenine, which provides high-resolution mapping. Transcription factor binding sites are marked with N3-A tags (purple) via antibody-mediated proximity labeling, while chromatin accessibility is marked by 6 mA tags (yellow). (C) The DNA is extracted for nanopore sequencing, during which it is stripped from histones, transcription factors, and other bound proteins, maintaining both synthetic and natural modifications. (D) After sequencing and basecalling, the resulting data enable the simultaneous analysis of 5mC, 5hmC, chromatin accessibility, and transcription factor binding sites using standard bioinformatics tools.

COMPETITIVE ADVANTAGES
Compared to existing methods:

POTENTIAL APPLICATIONS

• Oncology and liquid biopsy — detecting cancer-specific signatures from minimal cfDNA
• Epigenetic drug development — profiling chromatin modification agents or DNMT/TET inhibitor effects at single-molecule resolution for target identification
• Chromatin accessibility mapping — encoding open chromatin and transcription-factor binding events alongside methylation
• Clinical diagnostics — relevant for FFPE tissue; enables retrospective epigenomic studies on archived clinical samples
• Personalized medicine — multi-omic profiling from minimal biopsy material for treatment stratification and monitoring

PATENTS
DETECTION OF BASE MODIFICATIONS BY ENHANCING ELECTRICAL CONTRAST IN NANOPORES, 63/394,002, National Phase

REFERENCES

1. Bertocchi U. et al. “Write and Read: Harnessing Synthetic DNA Modifications for Nanopore Sequencing.” ACS Nano (2025). DOI: 10.1021/acsnano.5c12530
2. Margalit S. et al. “Long-Read Structural and Epigenetic Profiling of a Kidney Tumor-Matched Sample with Nanopore Sequencing and Optical Genome Mapping.” NAR Genomics & Bioinformatics (2025).
3. Nifker G. et al. “Dam Assisted Fluorescent Tagging of Chromatin Accessibility (DAFCA) for Optical Genome Mapping.” ACS Nano (2023).
4. Detinis Zur T. et al. “Single-Molecule Toxicogenomics: Optical Genome Mapping of DNA-Damage in Nanochannel Arrays.” DNA Repair (2025).