How does DNA travel through a nanopore? Head to tail, say researchers at Brown University. They found that when a DNA strand is captured and pulled through a nanopore, it’s much more likely to start the journey at one of its ends, rather than being grabbed somewhere in the middle and pulled through in a folded configuration.
“We think this is an important advance for understanding how DNA molecules interact with these nanopores,” says Derek Stein, assistant professor of physics at Brown, who performed the research with graduate students Mirna Mihovilivic and Nick Haggerty. “If you want to do sequencing or some other analysis, you want the molecule going through the pore head to tail.”
In nanopore sequencing, a hole only a few billionths of a meter wide is poked in a barrier separating two pools of salt water. An electric current is applied across the hole, which occasionally attracts a DNA molecule floating in the water. When that happens, the molecule is whipped through the pore in a fraction of a second. Scientists can then use sensors on the pore or other means to identify nucleotide bases.
The technology is advancing quickly, but there are still basic questions about how molecules behave at the moment they’re captured and before.
“What the molecules were doing before they’re captured was a mystery and a matter of speculation,” Stein said. “And we’d like to know because if you’re trying to engineer something to control that molecule—to get it to do what you want it to do—you need to know what it’s up to.”
In their experiments, the researchers tracked over 1,000 instances of a molecule zipping through a nanopore 8 nm in diameter. An applied voltage generated a current that varied according to how much the pore was blocked. The time course of the ionic current indicated whether single strands, multiple strands, or multiple folds of the same strand were passing through, or whether the molecule was a fragment or a damaged strand. By looking at differences in the current, Stein and his team could count how many molecules went through head first and how many started somewhere in the middle.
The researchers found that the probability for a strand to be pulled into the pore at a particular location increased rapidly close to the ends of the polymer. Previously, researchers had assumed that the likelihood of capture was constant along the chain.
The researchers compare their DNA findings to the theory of Jell-O. Jell-O is a polymer network—a mass of squiggly polymer strands that attach to each other at random junctions. In water, DNA molecules are jumbled up in random squiggles much like the gelatin molecules in Jell-O.
“There’s some powerful theory that describes how many ways the polymers in Jell-O can arrange and attach themselves,” Stein said. “That turns out to be perfectly applicable to the problem of where these DNA molecules get captured by a nanopore.”
When applied to DNA, the Jell-O theory predicts that if you were to count up all the possible configurations of a DNA strand at the moment of capture, you would find that there are more configurations in which it is captured by its end, compared to other points along the strand.
This measure of all the possible configurations—a measure of the molecule’s entropy—is all that’s needed to explain why DNA tends to go head first. Some scientists had speculated that perhaps strands would be less likely to go through by the middle because folding them in half would require extra energy. But that folding energy appears not to matter at all. As Stein puts it, “The number of ways that a molecule can find itself with its head sticking in the pore is simply larger than the number of ways it can find itself with the middle touching the pore.”
The paper, published in the journal Physical Review Letters, is called “Statistics of DNA Capture by a Solid-State Nanopore”.