Write and read: Harnessing Synthetic DNA Modifications for Nanopore Sequencing

Nanopore sequencing is enabled by detecting nucleotide-specific molecular signatures through changes in ionic current as DNA strands translocate through the pore. Well-trained models allow the detection of naturally occurring DNA modifications such as DNA methylation. Here, we present a versatile “write-and-read” framework, where chemo-enzymatic DNA labeling with unnatural synthetic tags results in predictable electrical fingerprints in na-nopore sequencing. We first explore a DNA glucosylation approach that selectively modifies 5-hydroxymethylcyto-sine (5hmC) with glucose or glucose-azide adducts via T4 β-glucosyltransferase (BGT) and examine the impact on nanopore current signals. We demonstrate that these modifications generate distinct and reproducible electrical shifts, allowing for direct detection of chemically altered nucleotides. We further show that enzymatic alkylation may write and read additional species. 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 introduces new possibilities for bio-orthogonal DNA labeling, enabling an extended alphabet of sequence-specific detectable moieties. This method expands the capabilities of nanopore-based single-molecule sensing by integrating real-time electrical detection with programmable chemical modifications.