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Electronic BioSciences Developing 'Flossing' Technique for Nanopore Sequencing


NEW YORK (GenomeWeb) – Electronic BioSciences is developing a DNA "flossing" technique that it plans to employ in a commercial nanopore sequencing instrument.

The firm, which has offices in both San Diego and Salt Lake City, launched a lipid bilayer platform called Nanopatch for electrophysiological measurements in 2013, but has the ultimate goal of developing a nanopore sequencing platform.

During a National Human Genome Research Institute-sponsored sequencing technology conference in San Diego last week, staff scientist Anna Schibel discussed the company's progress toward commercializing the device. She said the firm has optimized an alpha-hemolysin protein pore, has developed a flossing technique that increases accuracy, and is making use of DNA binding proteins to stabilize single-stranded DNA.

Schibel told GenomeWeb that the firm plans to finish demonstrating its proof of concept by the end of the summer, with a fully tested prototype available in two years, followed by early-access beta testing and commercialization. EBS is aiming for "high-accuracy, low-cost reads," Schibel said, but has not yet set a price.

The firm's core technology is a quartz nanopore membrane, which was developed by Eric Ervin, who is now the company's vice president of R&D, when he was at the University of Utah in Henry White's laboratory. EBS licenses the technology from the University of Utah and it forms the basis of the company's Nanopatch device.

A lipid bilayer can be put across the hole and an alpha-hemolysin pore is inserted. According to Schibel, the advantages of this setup are that the glass nanopore membrane has low noise and high bandwidth. In addition, the bilayer formation and pore insertion are fast and can be automated.

Last year, the company described progress it made in developing varieties of alpha-hemolysin pores with mutations that would make it more amenable to sequencing. It had tested approximately 400 variations engineered to have different properties, such as creating a single sensing zone within the pore, or increasing the contrast between nucleotides.

One challenge with nanopore sequencing is that the signal produced as DNA passes through the pore's constriction site is based not on just one single nucleotide, but several, making it difficult to get single-base resolution. In Oxford Nanopore's MinIon device, for instance, the current generated is the function of several bases in the pore. Oxford Nanopore has developed bioinformatics to translate those k-mers into reads, but some researchers think that a pore that is able to produce a unique signal for each single base would be more accurate.

At last week's conference, Schibel discussed one particular pore that she said has been engineered to have a "semi-single sensing zone" and has better contrast between bases. For instance, compared to wild-type alpha-hemolysin, it has a seven-fold higher signal-to-noise ratio.

A second technique that the company has been developing is a method to thread DNA back and forth through the pore to obtain multiple reads of the same strand. Schibel said that the company accomplishes this flossing technique by using changes in the voltage bias. A constant high AC bias of about 150 millivolts monitors pore conductance, while changing the DC bias controls DNA motion. DC bias is flipped from -120 millivolts as DNA moves in the forward direction to 50 millivolts for reverse translocation.

The DNA currently moves through the pore at approximately 20 microseconds per base, but Schibel said that EBS is working on slowing that rate five-fold to 100 microseconds per base, which it is doing through its use of single-stranded DNA binding proteins.

Single-stranded DNA binding proteins not only help slow translocation, but also prevent the DNA from forming secondary structures that may skew the readout from the pore or clog the pore.

In nanopore sequencing schemes like the MinIon, a polymerase is used to unzip double-stranded DNA, so that it goes through the nanopore as a single strand. EBS is taking a different approach, forgoing the use of polymerase in its device, and is starting with single-stranded DNA. However, ssDNA tends to twist and fold. The bases form hydrogen bonds with each other, creating large secondary structures that block the nanopore or impact the signal.

To get around this problem, the firm is using single-stranded DNA binding proteins (SSBs), which attach to the DNA strand, preventing it from binding to itself and forming secondary structures.

Prior to threading the DNA through the pore, it is capped on its 5' end with streptavidin, which immobilizes the ssDNA after the 3' end enters the pore. The DNA strand translocates through the pore, and on the other side of the pore, an SSB binds to it, preventing secondary structures from forming. Once the DNA molecule passes through in the forward direction, the DC current bias is lowered and the DNA goes back in the reverse direction.

EBS has screened a number of SSBs to find one with a high affinity for ssDNA that also slows translocation rate. Schibel said the team has tested bacteria, viral and mutant SSBs and has so far identified three that met its criteria of a five-fold slower translocation rate and high affinity for DNA.

Now, she said, the firm is working on "matching up a combination of an SSB and protein pore to optimize resolution," by screening the three SSBs against its suite of alpha-hemolysin pores under its flossing conditions.

In addition, she said, the company is working on "tuning" the flossing strategy. For instance, one facet it is working on is figuring out the best method for reversing translocation. One technique it is testing is a sequence-specific trigger, so when the pore encountered a specific series of bases, it would automatically trigger the change in DC current to reverse translocation.