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Proof-of-Principle Study Supports Use of Bacterial Porin for Nanopore Sequencing

By Andrea Anderson

NEW YORK (GenomeWeb News) – In a paper set to appear online this week in the Proceedings of the National Academy of Sciences, researchers from the Universities of Washington and Alabama describe their preliminary findings on the use of a bacterial porin called MspA for nanopore sequencing.

The team engineered a specialized version of the Mycobacterium smegmatis porin A (MspA) for the proof-of-principle study, demonstrating that MspA-based protein channels could be used to distinguish between nucleotides in single-stranded DNA based on ion current signals. By tossing in DNA hairpins or bits of double-stranded DNA, researchers explained, they were able to slow DNA progression through the pores so that they could glean this nucleotide sequence.

"What we portray in this paper is the kernel of a possible new sequencing technique," senior author Jens Gundlach, a nanopore physics researcher at the University of Washington, told GenomeWeb Daily News. "We're now hoping that other people or industry may be interested in developing this further."

In general, nanopore sequencing refers to sequencing that involves channeling DNA through a tiny pore while applying an external voltage and measuring the ionic current across this pore.

"You form a tiny little hole and through this tiny little hole you drive DNA," Gundlach explained. "You do this by applying a voltage across it."

Because DNA is negatively charged, he added, it gets sucked through the hole, while ion current flows through the pore and reflects the charge of the DNA and the electrolytic solution it's sitting in. Measuring this current should theoretically reveal nucleotide sequence of DNA moving through the pore, Gundlach explained.

For the study, funded through the National Human Genome Research Institute $1,000 Genome grant program, researchers focused on developing protein-based nanopores.

But rather than creating pores using the alpha-hemolysin protein, which Gundlach called the "workhorse of ion channel studies," he and his co-workers decided to use a modified version of the porin A protein MspA, produced by M. smegmatis. Based on the short, narrow nature of the MspA pore, researchers suspected it would be ideal for nanopore sequencing applications.

"It's just short enough to accommodate one or two nucleotides," Gundlach said, noting that it's also possible to genetically engineer MspA to optimize the constriction zone for nanopore sequencing. "We can control it and engineer it at the few angstrom level."

For instance, he explained, the researchers designed a mutant version of the M. smegmatis protein MspA that has a neutral, uncharged constriction zone that allows charged single stranded DNA to move through it unfettered.

After expressing and extracting this protein from M. smegmatis, the team coerced them into pores in lipid bilayers, looking at whether they could discern DNA sequences using a nanopore sequencing approach.

"We show that the MspA constriction zone, indeed, can resolve single nucleotides within a strand of DNA," Gundlach said, noting that signals were very distinct for homopolymers, representing runs of identical nucleotides. When they tossed different individual nucleotides into these homopolymers, he added, it was possible to identify these nucleotides as well.

Once they'd characterized ion current and other patterns associated with nucleotide movement through the nanopore, the researchers further tweaked the approach by finding ways to ease single-stranded DNA through the pore at a rate allowing signals to be measured — first by introducing DNA hairpins.

"The DNA moves through there way too fast to even be able to resolve a single nucleotide," Gundlach noted. "When we hold it — for example, we prepare DNA that has a hairpin that holds it up for a few milliseconds or so — we can very cleanly read the current."

During this pause, he added, the nucleotides of interest get held within the constriction zone of the MspA pore.

Similarly, the team's subsequent experiments suggested that punctuating single-stranded DNA with double-stranded sequence — an approach dubbed "duplex interrupted" nanopore sequencing — can also slow nucleotide movement through the channel to aid sequencing, since double-stranded DNA tends to get stuck in the pore and can't feed through until dissociated.

"Our experiments demonstrate that the protein pore MspA has great potential to advance nanopore sequencing," the team concluded. "Using this concept, we provide proof of principle for a nanopore DNA sequencing technique that would use DNA converted to have double-stranded sections between analyte nucleotides."

Nevertheless, the researchers ultimately aim to develop an MspA-based nanopore sequencing strategy that can unravel sequence of DNA that has not been modified. To that end, Gundlach said the team is devising and testing other strategies for slowing single-stranded DNA movement through the channels.

"The ultimate goal of our research is still to use unmodified DNA," he said. "In the end, we want to provide a sequencing platform that's incredibly cheap and also easy to do."

The researchers have patented their approach, Gundlach noted, and hope to eventually see it become commercially available. He did not disclose which companies, if any, have expressed interest in the MspA-based method.

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