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Different Salt Slows Down DNA in Nanopore, Dutch Team Finds


By Julia Karow

This article was originally published Jan. 23.

One of the biggest challenges with DNA nanopore sequencing is that DNA zooms through the pore too fast to take measurements with single-base resolution — a hurdle that has so far limited the approach.

Researchers have been working on various ways to put the brakes on the molecule — including molecular motors, a "DNA transistor", and adjustments to the voltage, solvent viscosity, and temperature — but now a team of scientists in the Netherlands has found a surprisingly simple way to slow down DNA translocation: to use a different salt in the testing solution.

In a paper published online in Nano Letters this month, the researchers, led by Cees Dekker at the Kavli Institute of Nanoscience at Delft University of Technology, reported that they can slow down the DNA about 10-fold by replacing potassium chloride, which is commonly used in nanopore sequencing experiments, with lithium chloride at a high concentration.

Though experts in the field say this is not enough to reduce the speed of DNA sufficiently for sequencing purposes, in combination with other approaches, it could prove useful for DNA nanopore experiments.

"If people had done this 10 years ago, lithium chloride would be the standard solvent for doing experiments [with nanopores], and not potassium chloride, which everybody uses" today, said Stefan Kowalczyk, a postdoc in Dekker's lab and the first author of the paper.

He and his colleagues found the effect accidentally while trying out different solvents for DNA nanopore experiments and analyzing their data.

For their studies, they used solid-state silicon nitride membranes, about 15 to 20 nanometers in diameter, and either double-stranded or circular single-stranded DNA. As they changed the salt solution from potassium chloride to sodium chloride and then to lithium chloride, the translocation time of the DNA increased significantly, and higher concentrations of lithium chloride slowed the molecule even further.

"It was quite surprising to us, because intuitively, we would not expect any differences between these different ions because they all have a charge of plus one," said Kowalczyk. "We repeated the experiment many times, and we saw this every time."

To better understand the effect, they asked Alek Aksimentiev's group at the University of Illinois at Urbana-Champaign to perform molecular dynamics simulations of the experiments. They found that the lithium ions, being smaller, come closer to the DNA backbone and form stronger bonds that last longer, Kowalczyk said. That decreases the effective charge of the DNA, so in an electric field, it experiences a smaller force and takes a longer time to move through a nanopore.

So far, the researchers have not found any drawbacks of using lithium instead of potassium in their nanopore experiments, and the effect should be independent of the type of nanopore used, since it relies solely on interactions of the salt with the DNA, and not with the pore.

Kowalczyk and his colleagues are now using the lithium chloride solution in DNA studies with a graphene nanopore, though no results are in yet. "I'm not sure if we can really sequence the DNA, but at least we can increase the readout accuracy by a lot," he said. Also, the slow-down effect of lithium could be used in combination with "other tricks" to reduce the DNA's speed, such as a decrease in temperature or voltage, he added.

According to Kowalczyk, there is no IP protection for the discovery. While at least two academic groups are already using it, he does not know if any nanopore sequencing company might have adopted it. Oxford Nanopore Technologies declined to comment on whether it is exploiting the effect for its sequencing technology.

Two academic researchers who are also working on nanopore sequencing agreed that the lithium effect is not big enough to solve the problem of slowing down the DNA. "The translocation speed per nucleotide is still way too fast to extract sequence information," said one of the researchers, who asked to remain anonymous because his comment is negative.

According to the other researcher, who also asked not to be identified, there are other methods to slow down DNA translocation sufficiently for sequencing, for example molecular motors such as polymerases, which can reduce the speed by a thousandfold.

A little over a year ago, for example, Mark Akeson's group at the University of California, Santa Cruz, demonstrated that they can use a polymerase as a motor that drives DNA through a nanopore in a slow and controlled fashion (IS 9/28/2010). Oxford Nanopore licenses technologies developed in Akeson's lab, though it has not yet revealed details about its platform.

Have topics you'd like to see covered in In Sequence? Contact the editor at jkarow [at] genomeweb [.] com.

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