P2-seq resolves topology-dependent DNA structural states and their long-range coupling in individual molecules

DNA supercoiling can be accommodated through changes in DNA conformation, from global duplex rearrangements to localized non-B DNA structures. These alternative states are predicted to compete for shared superhelical stress, producing long-range torsional coupling between distant DNA elements, but direct evidence for such coupling has been lacking. Here, we present Permanganate Pore-Seq (P2-seq), which integrates potassium permanganate footprinting with Oxford Nanopore native long-read sequencing to map supercoiling-dependent DNA conformations along individual molecules at near-nucleotide resolution. To detect permanganate oxidation products, we developed Rembo, a generalizable computational workflow based on raw signal comparison. Using P2-seq on supercoiled plasmids across a range of superhelical densities, we resolve multiple non-B DNA structural transitions and identify rare structural intermediates. Importantly, we find that distant non-B DNA structures within the same molecule are inversely coupled, providing direct single-molecule evidence of competition among alternative torsional sinks. P2-seq also detects distributed duplex destabilization associated with residual superhelical stress, enabling simultaneous analysis of localized and distributed torsional stress accommodation. Together, these results establish P2-seq as a platform for single-molecule, sequence-resolved analysis of topology-dependent DNA structural states and their coupling, providing a framework for studying how DNA topology shapes genome function.