New research uncovers hidden DNA structures during nanopore sensing: plectonemes take centre stage

For decades, scientists believed that when DNA passed through nanopores – a powerful technique for analysing genetic material – complex electrical signals indicated the formation of knots. It was much like pulling a shoelace through a small hole: if the lace gets tangled, the movement becomes irregular. Researchers assumed the same applied to DNA and that any signal complexity was due to it getting knotted as it threaded through.

Only now, the new findings, published in Physical Review X, reveal that DNA doesn’t just get knotted (like the tangled shoelaces) due to disruptions in the electrical signal as it threads the pore during nanopore translocation. Rather, the researchers have revealed that these presumed knots are frequently plectonemes – a structure where the DNA twists around itself, like a twisted phone cord, rather than tying an actual knot.

“Our experiments showed that as DNA is pulled through the nanopore, the ionic flow inside twists the strand, accumulating torque and winding it into plectonemes, not just knots. This ‘hidden’ twisting structure has a distinctive, long-lasting fingerprint in the electrical signal, unlike the more transient signature of knots,” explained lead author Dr Fei Zheng from the Cavendish Laboratory.

The scientists used glass and silicon nitride nanopores to analyse DNA under a range of voltages and experimental conditions. They observed that “tangled” events – instances where multiple DNA strands simultaneously occupied the pore – were far too common to be explained by equilibrium knot formation alone. Instead, the excess of these events increased with voltage and DNA length, hinting at an unknown mechanism.

They discovered that these twists are driven by electroosmotic flow—a movement of water inside the nanopore that generates torque on the helical DNA molecule. As the strand spins, this torque is transmitted to sections of DNA outside the pore, causing them to coil up. Unlike knots, which are tightened by pulling forces and tend to be short-lived, plectonemes can grow larger and persist throughout translocation. To investigate further, the researchers simulated DNA under realistic forces and torques. The simulations confirmed that plectonemes are generated by the twisting motion imposed by the nanopore’s electroosmotic flow and that their formation depends on the DNA’s ability to propagate twist along its length.

Further, in a clever twist, the researchers engineered “nicked” DNA, molecules interrupted at precise intervals, which blocked twist propagation and drastically reduced plectoneme formation in their experiments. This has not only confirmed the structure’s role but also points to potential new ways to sense or even diagnose DNA damage using nanopores.

“What’s really powerful here is that we can now tell apart knots and plectonemes in the nanopore signal based on how long they last,” says Prof Ulrich F. Keyser, who is also the co-author of the paper.

“Knots pass through quickly, just like a quick bump, whereas plectonemes linger and create extended signals. This offers a path to richer, more nuanced readouts of DNA organisation, genomic integrity, and possibly damage.”

The implications go even further. In biophysics, these findings could deepen our understanding of DNA entanglements within cells, where plectonemes and knots regularly emerge through the action of enzymes, playing crucial roles in genome organisation and stability. For biosensors and diagnostics, the ability to control or detect these twist structures may open the door to a new generation of biosensors that are more sensitive to subtle DNA changes, potentially enabling the early detection of DNA damage in diseases.

“From the perspective of nanotechnology, the research highlights the power of nanopores, not only as sophisticated sensors but also as tools for manipulating biopolymers in novel ways,” concluded Keyser.