(By: Nanotechweb.org)

Researchers in the Netherlands, the US and Israel have succeeded in observing DNA knots as they pass through solid-state nanopores. The presence of such knots had been largely ignored in such studies until now, but the new work shows that it is important to take them into account in applications such as detecting DNA-bound proteins.

DNA molecule with a knot translocating through a nanopore

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.

Reference
Calin Plesa, Daniel Verschueren, Sergii Pud, Jaco van der Torre, Justus W. Ruitenberg, Menno J. Witteveen, Magnus P. Jonsson, Alexander Y. Grosberg, Yitzhak Rabin & Cees Dekker: Direct observation of DNA knots using a solid-state nanopore, Nature Nanotechnology 15 August 2016. DOI:10.1038/nnano.2016.153

Abstract
Long DNA molecules can self-entangle into knots. Experimental techniques for observing such DNA knots (primarily gel electrophoresis) are limited to bulk methods and circular molecules below 10 kilobase pairs in length. Here, we show that solid-state nanopores can be used to directly observe individual knots in both linear and circular single DNA molecules of arbitrary length. The DNA knots are observed as short spikes in the nanopore current traces of the traversing DNA molecules and their detection is dependent on a sufficiently high measurement resolution, which can be achieved using high-concentration LiCl buffers. We study the percentage of molecules with knots for DNA molecules of up to 166 kilobase pairs in length and find that the knotting occurrence rises with the length of the DNA molecule, consistent with a constant knotting probability per unit length. Our experimental data compare favourably with previous simulation-based predictions for long polymers. From the translocation time of the knot through the nanopore, we estimate that the majority of the DNA knots are tight, with remarkably small sizes below 100 nm. In the case of linear molecules, we also observe that knots are able to slide out on application of high driving forces (voltage).

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