Single Molecular Anchor-Assisted Nanopore Profiling of DNA Abasic Site Reactivity Dynamics

DNA repair intermediates such as abasic (AP) sites are among the most abundant and reactive DNA lesions. Despite their central roles in repair fidelity and as therapeutic targets, their transient dynamics and covalent reactivity remain difficult to probe with existing methods. Here, we introduce a molecular anchor-assisted strategy that immobilizes AP-containing ssDNA within the α-hemolysin (α-HL) nanopore, enabling real-time monitoring of its reactivity dynamics. Vitamin B12 is covalently attached to thiol-modified ssDNA through a cobalt-sulfur bond, serving as a molecular anchor to tether ssDNA inside the α-HL nanopore. The resulting B12-anchored ssDNA produces uniform current blockages of defined amplitude. This approach allows high-resolution discrimination of the four canonical nucleobases and dynamic tracking of AP site formation. By recording the reactions of hydroxylamine compounds with AP sites, we discovered that different substituent groups on hydroxylamines modulate their reactivity toward AP sites. Furthermore, this B12-anchored ssDNA strategy functions as a single-molecule reactor, enabling the capture of transient chemical structural changes at AP sites and direct observation of their reaction processes with nucleophilic agents at the single-molecule level. The results demonstrate that nanopore-based single-molecule profiling enables detailed kinetic and mechanistic studies of reactive DNA intermediates, which are otherwise difficult to monitor, thereby highlighting its potential for molecular diagnostics and targeted therapeutic development.