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Researchers Use Atomic Force Microscope to Elucidate and Control DNA Translocation through Nanopore

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NEW YORK (GenomeWeb) — Researchers from the University of Notre Dame have demonstrated the use of an atomic force microscope to control the translocation of single-stranded DNA through a solid-state nanopore.

According to senior author Gregory Timp, professor of electrical engineering and biological sciences at the University of Notre Dame, while a device that uses an atomic force microscope would ultimately not be ideal for a commercial system, the AFM yielded important insights into the kinetics of DNA translocation through a nanopore that the team will now be able to make use of in designing a system.

In addition, the researchers were able to overcome another hurdle in the field of solid-state nanopores ¬¬¬– designing a pore small enough for just one nucleotide to fit.

Reporting in ACS Nano this month, the team constructed nanopores between 1 nm and 3.5 nm in diameter on SiN membranes between 6 nm and 10 nm thick. They coupled biotinylated single-stranded DNA to an AFM, which served both to guide the DNA to the pore and also to control translocation through the pore.

One important improvement in the design of the nanopore was that the researchers were able to create pores small enough so that only one nucleotide would be present in a pore at once. Many efforts to develop solid-state nanopores have yielded pores that are either too wide, as in the case of other SiN nanopores, or too long, as in the case of carbon nanotubes, said Timp.

Typically, researchers constructing SiN nanopores use an ion beam to drill the pores; however, those can only make pores that are about 6 nm in diameter, explained Edward Nelson, first author of the study and a postdoc in Timp's lab at Notre Dame. Instead, the researchers used a focused electron beam, which can drill pores with diameters as small as 1 nm.

To build its system, the team started with standard silicon wafers with a layer of silicon nitride and used standard lithography techniques to develop a resist pattern on the back of the wafer. Next, they etch through the wafer to develop a membrane that is between 6 nm and 10 nm thick. Lasers are then used to scribe out a chip, and electron beams drill nanopores. The construct is then embedded into a microfluidic device.

Because of the way the membrane is constructed, the area where the pore is tends be a bit more flexible and thin, said Nelson, making it easier to find.

In the study, the researchers used ssDNA between 150 bases and 200 bases long attached to an AFM via streptavidin beads. The AFM physically moves the DNA to the pore, as opposed to other methods, which typically rely on diffusion.

The researchers then lower the tip of the AFM to the pore, after which the electric field pulls the DNA into the pore. Because the DNA remains attached to the AFM, the researchers control the translocation speed and can also use the AFM to measure the forces on the DNA.

In the paper, the researchers recorded translocation speeds of about 60 bases per second, which Timp said would be much too slow for a commercial device, but enabled the team to study the kinetics in greater detail.

The use of an AFM also enabled the team to measure both the force and the current, Nelson said. Thus far, most nanopore sequencing research has focused on measuring the current as a molecule passes through the nanopore, without studying other interactions and forces, Nelson said.

In the ACS Nano study, however, the team observed two types of translocation kinetics, which they dubbed "stick and slip" and "frictionless slide."

"We see a lot of events where DNA will enter the pore and stick," said Nelson. "When that happens, the velocity is not constant, so you can't know where you are [along the molecule]." A sufficient amount of force has to be applied to free the molecule.

The other event, frictionless slide, is when the "molecule slides effortlessly through the pore," which is ideal, said Timp.

The team tested the device on both homopolymer strands and heteropolymer strands, and in each case, observed both types of kinetic motion, indicating that the specific base, whether adenine, cytosine, guanine, or thymine, likely did not determine whether it would be sticky or not.

"Our guess is that it has to do with the DNA nonspecifically binding to hydrophobic patches either on the membrane or the nanopore itself," said Timp.

Nelson said that it will be important to improve the translocation kinetics.

"We want the molecule to slide through the pore frictionlessly all the time," Timp said, adding that the group has some ideas for removing the hydrophobic interactions, which should improve the kinetics.

In addition, Timp said that an AFM would not be used in any commercial device. The group wanted to use the AFM to study translocation kinetics, but because AFMs are temperature-, noise-, and vibration-sensitive, and also have a footprint of five to six square feet, they would only be suitable for research laboratories, Timp said.

Timp added that the group decided to use an AFM to control translocation after work led by Cees Dekker's group at Delft University of Technology in Holland demonstrated the feasibility of using a mechanical method to control translocation. Dekker's group has been working with optical tweezers, which Timp said his lab has also tried. However, while the optical tweezers are "a great tool for manipulating biologicals, the trouble is they do not have enough force," he said. Optical tweezers are limited to around 10 piconewtons of force, while AFMs can generate up to tens of nanonewtons, he said.

In the future though, Timp said that the group plans to try and control DNA translocation by just using the electric field and the mechanics of the molecule.

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