Researchers use virus to reveal nanopore physics

06/25/2014

Nanopores may one day lead a revolution in DNA sequencing. By sliding DNA molecules one at a time through tiny holes in a thin membrane, it may be possible to decode long stretches of DNA at lightning speeds.

Scientists, however, have not quite figured out the physics of how polymer strands like DNA interact with nanopores. Now, with the help of a particular type of virus, researchers from Brown University have shed new light on this nanoscale physics. The findings might not only help in the development of nanopore devices for DNA sequencing, they could also lead to a new way of detecting dangerous pathogens.

The concept behind nanopore sequencing is fairly simple. A hole just a few billionths of a meter wide is poked in a membrane separating two pools of salty water. An electric current is applied to the system, which occasionally snares a charged DNA strand and whips it through the pore — a phenomenon called translocation. When a molecule translocates, it causes detectable variations in the electric current across the pore. By looking carefully at those variations in current, scientists may be able to distinguish individual nucleotides – the A's, C's, G's and T's coded in DNA molecules.

Despite advances in the field, surprisingly little is known about the basic physics involved when polymers interact with nanopores. That is partly because of the complexities involved in studying DNA. In solution, DNA molecules form balls of random squiggles, which make understanding their physical behavior extremely difficult.

For example, the factors governing the speed of DNA translocation are not well understood. Sometimes molecules zip through a pore quickly; other times they slither more slowly, and nobody completely understands why. One possible explanation is that the squiggly configuration of DNA causes each molecule to experience differences in drag as they are pulled through the water toward the pore. "If a molecule is crumpled up next to the pore, it has a shorter distance to travel and experiences less drag," said Angus McMullen, a physics graduate student at Brown and the study's lead author. "But if it is stretched out then it would feel drag along the whole length and that would cause it to go slower."

The drag effect is impossible to isolate experimentally using DNA, but the virus McMullen and his colleagues studied offered a solution. The researchers looked at fd, a harmless virus that infects e. coli bacteria. Two things make the virus an ideal candidate for study with nanpores. First, fd viruses are all identical clones of each other. Second, unlike squiggly DNA, fd virus is a stiff, rod-like molecule. Because the virus does not curl up like DNA does, the effect of drag on each one should be essentially the same every time.

With drag eliminated as a source of variation in translocation speed, the researchers expected that the only source of variation would be the effect of thermal motion. The tiny virus molecules constantly bump up against the water molecules in which they are immersed. A few random thermal kicks from the rear would speed the virus up as it goes through the pore. A few kicks from the front would slow it down. The experiments showed that while thermal motion explained much of the variation in translocation speed, it did not explain it all. Much to the researchers' surprise, they found another source of variation that increased when the voltage across the pore was increased.

"It has been predicted that depending on where an object is inside the pore, it might be pulled harder or weaker," McMullen said. "If it is in the center of the pore, it pulls a little bit weaker than if it is right on the edge. That has been predicted, but never experimentally verified. This could be evidence of that happening, but we are still doing follow up work." A better understanding of translocation speed could improve the accuracy of nanopore sequencing, McMullen says. It would also be helpful in the crucial task of measuring the length of DNA strands. "If you can predict the translocation speed," McMullen said, "then you can easily get the length of the DNA from how long its translocation was."

In addition to shedding light on basic physics, the work might also have another application. While the fd virus itself is harmless, the bacteria it infects – e. coli – is not. Based on this work, it might be possible to build a nanopore device for detecting the presence of fd, and by proxy, e. coli. Other dangerous viruses – Ebola and Marburg among them – share the same rod-like structure as fd.

COMPAMED.de; Source: Brown University