Nanopores are nanometre-sized holes in thin solid-state membranes. Ions flow through the pores if a voltage is applied across the membranes when they are immersed in an ionic solution. This ion flow constitutes an electric current.

If DNA molecules are present in the solution, they travel through the pores and block the flow of some of the ions. This results in a current drop (or blockade) that can be monitored using a very sensitive electronic set-up. The size of this current drop and how long it lasts can be used to glean information about the structural state of the DNA – for example, whether there are proteins bound to it or not. This is because DNA-bound proteins produce unique signals, observed as additional current blockades on top of the original blockade produced by the DNA molecule.

Blockade signal is even larger than usual

Long DNA molecules can become entangled, producing knots, but until now, there was no way to observe such knots. A team led by Cees Dekker of the Delft University of Technology has now succeeded in doing just this.

“We have found that as a knot passes through a nanopore, the blockade signal is even larger than usual because there are multiple DNA strands passing through the pore at the same time,” explains team member Calin Plesa. The researchers are able to detect the knots as spikes in the nanopore current as they pass through the pore thanks to a high-resolution measurement system made from high-concentration lithium chloride buffers.

Knots are smaller than 100 nm

“Knots are most prevalent in long DNA molecules and our approach allows us to determine how the number of knots scales with DNA lengths,” says Plesa. “We are also able to determine other properties such as the DNA knot’s size and its position on the molecule.”

From the time it takes for the knot to translocate through a nanopore, the team was also able to calculate that the majority of the knots are smaller than 100 nm. “This is surprisingly small and indicates that knots in DNA are remarkably tight,” says Dekker. Such small-sized knots were already theoretically predicted by Yitzhak Rabin and Alexander Grosberg in 2007 but never experimentally confirmed until now.

Important to take knots into account

“I think the presence of knots has been largely ignored in many nanopore applications because of the limited resolution of the measurements, which prevented us from observing them,” says Plesa. “But now we have shown that the knots are there it will be important to take them into account in applications such as detecting DNA proteins, particularly when probing long DNA lengths.”

Dekker adds that the study has been “gratifying”. “Ever since I started working on DNA translocation through nanopores over 15 years ago, I wondered why the DNA would traverse such a tiny pore in a nice head-to-tail fashion without encountering any knots,” he tells nanotechweb.org. “Imagine doing the same exercise with your garden hose as you pull it through a four-inch hole in the garden fence – I bet you get stuck with a knot. By systematically increasing the time resolution of our technique, we are now able to observe such knots and in doing so have actually learnt quite a bit about their basic properties.”

“Measuring fundamental aspect of DNA”

Adam Hall of Wake Forest University School of Medicine, who was not involved in this work, says that the new paper is “very interesting and thorough”.

“Long, duplex DNA has been the workhorse molecule in the solid-state nanopore field for 15 years, and so it is incredible to see that we are just now measuring fundamental aspects of it that we never noticed before,” he says. “I think this work opens the way to studying a novel concept with fundamental and translational importance. Knot formation is both an interesting polymer dynamics question and an issue that must be dealt with when it occurs in the cell, lest it gum up the proper functioning of the cellular machinery. Existing techniques like atomic force microscopy are limited because they require DNA to be surface bound, which can make interpretation challenging. This paper addresses the issue nicely by probing DNA in solution.

With so much attention on the application of nanopores strictly to DNA sequencing, it’s important to remember how many other important uses the system may have.”

The information gained during this study will also be important for any application involving long polymers, not only DNA molecules, adds Plesa.

The work is described in Nature Nanotechnology doi:10.1038/nnano.2016.153.

More from Dekker's group and the latest developments in DNA sequencing in the Nanotechnology focus collection.