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

## TL;DR

This paper introduces a new method to detect synthetic DNA modifications using nanopore sequencing, allowing for the simultaneous analysis of multiple molecular features on individual DNA molecules.

## Contribution

The paper introduces a 'write-and-read' framework using synthetic DNA modifications to generate detectable electrical signals during nanopore sequencing.

## Key findings

- DNA glucosylation at 5-hydroxymethylcytosine produces distinct electrical signals detectable by nanopore sequencing.
- Enzymatic alkylation of adenines with azide residues results in characteristic nanopore signal shifts.
- The method allows for bio-orthogonal labeling, expanding the detectable DNA alphabet.

## Abstract

An exciting feature of nanopore sequencing is its ability
to record
multiomic information on the same sequenced DNA molecule. Well-trained
models allow the detection of nucleotide-specific molecular signatures
through changes in ionic current as DNA molecules translocate through
the nanopore. Thus, naturally occurring DNA modifications, such as
DNA methylation and hydroxymethylation, may be recorded simultaneously
with the genetic sequence. Additional genomic information, such as
chromatin state or the locations of bound transcription factors, may
also be recorded if their locations are chemically encoded into the
DNA. Here, we present a versatile “write-and-read” framework,
where chemo-enzymatic DNA labeling with unnatural synthetic tags results
in predictable electrical fingerprints in nanopore sequencing. As
a proof-of-concept, we explore a DNA glucosylation approach that selectively
modifies 5-hydroxymethylcytosine (5hmC) with glucose or glucose-azide
adducts. We demonstrate that these modifications generate distinct
and reproducible electrical shifts, enabling the direct detection
of chemically altered nucleotides. We further demonstrate that enzymatic
alkylation, such as the enzymatic transfer of azide residues to the
N6 position of adenines, also produces characteristic nanopore signal
shifts relative to the native adenine and 6-methyladenine. Beyond
direct nucleotide detection, this approach enables bio-orthogonal
DNA labeling, enabling an extended alphabet of sequence-specific detectable
moieties. The future use of programmable chemical modifications for
simultaneous analysis of multiple omics features on individual molecules
can significantly advance genetic research and discovery.