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BU Team Uses Lasers to Control DNA Translocation Through Solid-State Nanopore

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Researchers at Boston University have found that laser light can control the speed at which DNA translocates through a solid-state nanopore. Additionally, directing a laser beam at a solid-state nanopore also enables very small proteins to be distinguished and helps to unclog blocked nanopores, increasing their lifespan.

The team, led by Amit Meller, an associate professor of biomedical engineering and physics at Boston University, described the phenomena in a recent study in Nature Nanotechnology.

Boston University holds the intellectual property for the method and Meller told In Sequence that the researchers are interested in licensing out the technique to other nanopore sequencing groups.

Meller said that the researchers made the discovery almost by accident. "It was something we did not anticipate," he said.

He has been working on nanopore sequencing technology using solid-state nanopores. "One of the phenomena we observed was [that] whenever we shone a low-intensity laser directly focused at the silicon nitride nanopore, we observed a big jump in the ionic current that flows through the pore."

Then, when the researchers shone the light on the nanopore during DNA translocation, they noticed that translocation slowed down "quite significantly," Meller said. "It was a very cool finding that we made kind of without expecting it."

The ability to control DNA translocation through nanopores is one key challenge to successful sequencing DNA using nanopore technology. Protein nanopores have made more headway in solving the challenging using an enzyme to ratchet DNA through a nanopore, but controlling translocation through solid-state nanopores has been more challenging.

Now, in the Nature Nanotechnology study, Meller and his colleagues demonstrated on multiple nanopores of varying diameters that focusing a laser light on the pore could slow translocation speed of double-stranded DNA by more than an order of magnitude.

In the study, the researchers were able to slow translocation down to an average rate of around 12 microseconds per nucleotide, around 10 times slower than without the laser.

According to Meller, to sequence single-stranded DNA through a solid-state nanopore, the speed would ideally be even slightly slower — closer to 100 microseconds per nucleotide — but, he said, "we are getting there."

To slow down DNA even further, he said his group is now working on tuning the sensitivity of the pores to light. Meller said that his group observed that when they shone the light on the pore, current flow through the pore increased. They figured out that the increase in current was due to making the surface of the pore more negatively charged, which previous research has shown increases the flow of positive ions across the surface of the pore. Those positive ions carry water molecules, which slow down the flow of DNA.

Now that the group has shown that this electroosmotic effect slows down DNA translocation, he said that fine-tuning the pore itself to make it even more reactive to light will increase that effect, slowing down translocation even more.

Shining light on a nanopore has another beneficial effect aside from slowing down DNA translocation. The light will also clear out blocked nanopores, Meller said. Solid-state nanopores in particular, have a tendency to become clogged with air bubbles or nano-sized particles, Meller said. Sometimes, electrical pulses will clear the pore, but that is not always very effective, he said.

However, the team demonstrated that laser light will clear the blocked pore without damaging it, and thereby increase the lifespan of the pore from hours to days or weeks.

Aside from sequencing applications, Meller's group demonstrated that the pore is effective at sensing small proteins. Typically, small proteins translocate through solid-state nanopores too quickly to be detected. However, when the team investigated the pore's ability to detect ubiquitin, a representative small protein with a diameter of 4 nanometers, they found that by shining the laser onto the pore, the protein slowed enough to be detected.

Without the light, ubiquitin passing through a 5-nanometer pore "yielded sporadic, brief downward spikes," the authors wrote. However, neither these current spikes, nor the dwell time of ubiquitin in the pore yielded a clear enough signal to accurately determine that the molecule was ubiquitin.

When the laser light was shone on the pore, translocation time increased by "at least two orders of magnitude, allowing full characterization of both amplitude and dwell times for ubiquitin translocations," the authors wrote.

"The optical effect was so significant that we could see 200-fold or more slowing down just by illuminating the pore with a tiny application of light," Meller added.

Another advantage of the technique is that it is "completely orthogonal to the readout," Meller said, and "doesn't interfere with the electronic readout."

Additionally, the technique "is not limited to any one specific nanopore sequencing technology." Meller, who is also cofounder and acting CTO of startup Noblegen Biosciences, said that his company would likely not use the laser technology because Noblegen uses optical detection, so the effect may not be needed.

Nevertheless, he said he has been "talking with some partners who might be interested in a license."

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