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Polymerase Controls DNA Translocation Through Pore in Another Step Toward Nanopore Sequencing

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By Monica Heger

Overcoming a major hurdle of nanopore sequencing, researchers from the University of California, Santa Cruz, have demonstrated that they can control the rate of translocation of a single strand of DNA through a protein nanopore.

In a study published this week in Nature Nanotechnology, the researchers demonstrated that DNA translocation through an alpha hemolysin nanopore can be controlled by a DNA polymerase positioned at the edge of the pore, which initiates replication of the single strand of DNA and thus moves it through the pore only when the strand is captured in the pore and a certain voltage is applied.

"The polymerase works as a motor, moving the DNA through the pore in a controlled fashion," said Jens Gundlach, a biophysicist from the University of Washington, whose group has been working on building protein nanopores from Mycobacterium smegmatis porin A (IS 8/24/2010).

The key to the work was applying voltage to first capture the DNA and the DNA polymerase in the nanopore, and then lowering the voltage to initiate the polymerase action, said Mark Akeson, professor of biomolecular engineering at UC Santa Cruz and senior author of the paper. The initial voltage also served to make the DNA accessible to the polymerase.

"The idea is that a polymerase moves DNA through a nanopore sensor in a controlled and systematic way at one-nucleotide precision," he said.

Akeson and his team showed that at 180 millivolts, the single-stranded DNA and the DNA polymerase would be captured by the nanopore. The team then quickly lowered the voltage to 80 millivolts, which initiated polymerase activity, allowing the DNA to move through the pore, but keeping the polymerase, which was too bulky to pass through the pore, at the orifice.

Normally at that voltage the DNA would rapidly thread through the pore, but the enzyme slows down the translocation. The polymerase is essentially, working against the direction that the DNA naturally wants to go, said Gundlach. It "puts up a little bit of force and pulls these nucleotides through one at a time."

Each nucleotide was held in the pore for about 17 milliseconds, long enough to identify the base, said Gundlach. Without the polymerase, each nucleotide would translocate through the pore in several microseconds.

Oxford Nanopore, which is working toward commercializing a nanopore sequencing device, has licensed technology developed from Akeson's lab, and Akeson also sits on the company's technical advisory board. The company has focused mainly on exonuclease sequencing, where enzymes mediate the translocation of individual bases through a nanopore. The Nature Nanotechnology paper demonstrates an advance in strand sequencing, which the company has also invested in through its collaborations with other labs.

An Oxford Nanopore spokesperson told In Sequence that while exonuclease sequencing would continue to be the company's primary focus, it was also devoting some internal resources toward advancing strand sequencing, and many of its collaborators, including Akeson's lab, were making significant progress in this area.

The company declined to provide specifics as to what portion of its R&D was devoted to each method, although it did say that a large portion went to "common elements" of the platform such as instrumentation, fluidics, and integration of the nanopore constructs and chip.

Oxford Nanopore's CEO Gordon Sanghera wrote in an e-mail that the current work is important because it demonstrates a method for passing a DNA strand through a nanopore in a controlled manner. "This control is one of the key problems that has challenged nanopore researchers for many years."

Akeson said that the next step of the research will be to test different enzymes. In the current work, the tension from the applied electric field caused the enzyme to dissociate from the DNA after only several bases passed through the pore.

"It was surprising because in solution, the enzyme can add thousands of nucleotides," said Akeson. He said the team is now trying a host of other enzymes, some that are commercially available, and some that are not.

Additionally, Akeson said he thought the technique would work with other types of nanopores, such as the MspA nanopore being developed by Gundlach's lab or even solid-state graphene nanopores.

Coupling the technique with the MspA pore could be particularly useful, said Gundlach. The two main problems of nanopore sequencing are the "exquisite recognition of single nucleotides and slowing down the velocity of DNA," Gundlach said.

While Akeson's work solves the problem of slowing down the DNA, the MspA pore has the ability to distinguish individual nucleotides, so "the combination of the two is a perfect match," Gundlach said.

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