P2-seq resolves topology-dependent DNA structural states and their long-range coupling in individual molecules
This paper introduces Permanganate Pore-Seq (P2-seq), a single-molecule sequencing method that provides direct evidence of long-range torsional coupling and competition between distant non-B DNA structures by mapping supercoiling-dependent conformational states at near-nucleotide resolution.
Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer
Inside every living cell, the genetic code is not a loose, straight string but a tightly wound, twisted ladder. This twisting, known as supercoiling, is a fundamental physical property of DNA that arises naturally as the cell's machinery reads and copies the genetic instructions. When the molecular motors that read DNA move along the strand, they twist the helix behind them, creating tension much like a rubber band that has been wound too tight. This tension is not just a passive byproduct; it is a powerful force that can reshape the DNA molecule itself. Under the right conditions, this stress can cause the double helix to unzip, flip inside out, or fold into complex, non-standard shapes. These alternative structures are thought to act as switches, turning genes on or off, yet for decades, scientists have struggled to see exactly how these shapes form, how they interact with one another, and how the physical stress is shared across a single molecule of DNA.
A team of researchers at the National Institutes of Health has now developed a new method to watch these events unfold on individual DNA molecules with unprecedented clarity. They created a technique called P2-seq, which combines a chemical stain that highlights open or unstable parts of the DNA with a powerful sequencing technology that reads long strands of genetic code in one go. By treating DNA with a chemical that marks only the bases that have temporarily separated from their partners, and then reading the entire molecule, the researchers could map exactly where the DNA was twisted, melted, or folded. This approach allowed them to see not just the average behavior of a crowd of DNA molecules, but the unique story of each individual strand, revealing how distant parts of the same molecule communicate and compete for the available physical stress.
The researchers began by taking circular DNA plasmids and carefully adjusting their level of twist, creating a range of molecules from relaxed to tightly wound. They then applied a chemical treatment that reacts specifically with thymine bases when they are exposed, which happens when the DNA strand opens up or forms a non-standard shape. Using a nanopore sequencer, they passed these treated molecules through tiny pores, measuring the electrical current as they passed. Because the chemical marks altered the current in a distinct way, the team could pinpoint exactly which bases had been modified. To make sense of the massive amount of raw data, they developed a new computational tool that compared the signals from treated DNA against untreated controls, allowing them to detect these subtle changes without needing to synthesize artificial reference samples.
What emerged from this high-resolution view was a dynamic landscape of structural changes. The team found that as the DNA became more twisted, specific regions would unzip or fold into shapes like cruciforms, which look like crosses. Crucially, they observed that these structural changes were not random or independent events. Instead, the researchers discovered a direct competition between distant parts of the same molecule. When one section of the DNA absorbed the twisting stress by forming a stable structure, it reduced the amount of stress available for other sections to change shape. For example, if a specific region far away from a folding site began to melt and unzip, it prevented a nearby cruciform structure from forming. This provided the first direct, single-molecule evidence that different DNA elements within the same topological domain are inversely coupled, fighting over a shared pool of physical energy.
The study also revealed that the DNA does not always relieve stress by forming large, distinct structures. In many cases, the researchers detected a more subtle, distributed form of stress where the double helix became slightly unstable across its entire length, rather than just at specific hotspots. This "backbone" reactivity increased as the overall twist increased, but it decreased when stable structures like cruciforms formed, suggesting that the molecule has two ways to handle tension: either by creating a few strong, localized shapes or by spreading the instability out over a wide area. The researchers were able to track how the balance between these two modes shifted as they added just a single extra twist to the DNA, showing that the molecule redistributes its stress in a non-linear, highly specific way.
By looking at individual molecules, the team was also able to catch rare, fleeting intermediates that are usually hidden in bulk measurements. They identified specific steps in the folding process of cruciform structures, seeing molecules caught in the act of forming a hairpin loop before it fully extruded. They found that the path a molecule takes to fold depends on the chemical environment, with different salt conditions favoring different folding routes. This level of detail allowed them to reconstruct the folding pathways and see that multiple structural states can coexist on the same molecule, depending on the precise amount of twist and the local sequence.
The findings suggest that DNA topology acts as a carrier of regulatory information, transmitting physical signals across long distances within a gene domain. The ability to see these interactions on a single-molecule level changes how scientists understand the mechanics of the genome. It shows that the physical state of DNA is not a static background but a dynamic, competitive system where distant elements influence each other directly. The new method, P2-seq, offers a way to map these structural states and their coupling with near-nucleotide precision, providing a framework to study how the physical shape of DNA influences its function in health and disease. While the experiments were conducted on circular DNA in a test tube, the principles of how stress is partitioned and shared are expected to apply to the complex, linear DNA found inside living cells, opening the door to understanding how the physical forces of the genome drive biological processes.
Drowning in papers in your field?
Get daily digests of the most novel papers matching your research keywords — with technical summaries, in your language.