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NorthShore Bio Develops Solid-State 'Tunable' Nanopore Chips for Sequencing-by-Degradation

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NorthShore Bio has created a scalable chip-based platform with solid-state "tunable" nanopores that it plans to develop for DNA and RNA sequencing applications using a sequencing-by-degradation approach.

Last week, the firm, which is based in the Portland, Oregon metropolitan area but uses lab space at the Institute for Systems Biology in Seattle, became a winner of this year's TiE50 annual awards program, which honors 50 technology startups selected from more than 1,000 entries.

The company, founded in 2009 as Lux Bio Group by CEO Jonathan DeHart and CSO Gordon Holt, a former R&D director of proteomics firm Oxford GlycoSciences, grew out of a biochip program funded by Intel that focused on silicon chip-based DNA microarrays. The firm originally considered setting up shop in Luxembourg but changed its name to NorthShore Bio after those plans changed.

Following the 2008 recession, funding from Intel was drying up, so Holt and DeHart spun the company out, largely self-funding it initially. In late 2011, NorthShore Bio raised an undisclosed amount of funding in a Series A round with the Oregon Angel Fund and the ISB. "People are surprised by what we've done with as much capital as we have," DeHart said. "We've been very capital-efficient so far."

At the moment, NSB, which has four full-time employees and is working with a number of outside contractors, is preparing for a multi-million dollar Series B round it hopes to close before the end of the year. That new funding will allow it to at least double its staff and to develop its platform, which has a variety of potential molecular sensing applications for nucleic acid sequencing.

At NSB, Holt and his colleagues developed methods to deposit and grow membranes across nanowells on a silicon chip that each carry electrodes on their side walls and bottom. They also found ways to tune the dimensions of the hole, or nanopore, that forms as the membrane closes in from the sides of a nanowell.

The electronically programmable deposition method is proprietary to NSB, which has filed several patents around the technology. In addition, it licensed a patent covering aspects of the work from an undisclosed large US national laboratory.

Holt explained that the membranes are sometimes called "synthetic metals" because they can carry electrical currents, and distribute current densities, in a similar way to metals. These properties allow NBS to program the deposition of the membranes, so they close uniformly inward across the nanowell, leaving a hole with a defined size.

For sequencing applications, the company is targeting pore dimensions of less than 20 nanometers in length and 10 nanometers in diameter, similar to the nanopores explored for sequencing by others.

"Using fairly standard laboratory electronics, it already is routine for NSB to form nanopores with dimensions and properties quite akin to solid-state and protein nanopores," Holt said. This is done by monitoring the formation of the pores in real time using a bias current between electrodes above and below the membrane.

The properties of the "synthetic metals" also allow the researchers to incorporate other materials toward the center of the membrane, he said, so they form a chemically distinct layer around the nanopore. This can be used, for example, to attach enzymes, such as nucleases, near the pore.

A key advantage of synthetic membranes over lipid bilayers, which most of the protein nanopore sequencing efforts use, is that they are very stable – an issue that Oxford Nanopore Technologies is reportedly grappling with in the development of its sequencing platform. "They can survive being carried around in your pocket," Holt said.

"The durability is a critical innovation, as well as the fact that they are manufactured by methods that are very amenable to high-throughput scaling," he added.

The company has already manufactured nanopores over several thousand nanowells at once, "and this just as easily could have been hundreds of thousands of wells," Holt said.

Right now, however, NSB works with isolated wells in order to characterize the parameters of a future product chip "such that each nanowell in a large array operates independently to manage its own nanopore synthesis and sequencing reaction monitoring.

"In short, we don't have much electronics on our chips right now − we're taking that approach because it reduces our cost of manufacturing the chips," he said. A later step will be to integrate the electronics into the bottom of the chip.

NSB plans to describe its nanopore chip platform in a peer-reviewed publication in the near future, Holt added.

Sequencing-by-Degradation

For sequencing, NSB is pursuing a sequencing-by-degradation approach, which is similar in principle to the exonuclease sequencing strategy Oxford Nanopore was exploring before it abandoned it in favor of DNA strand sequencing.

NSB's strategy uses a DNA or RNA nuclease, tethered close to the nanopore, that snips off individual nucleotides from a DNA strand, which then enter the pore and are monitored by measuring a change in ion current.

NSB is currently working on showing that it can distinguish between the four types of nucleotides, which researchers demonstrated four years ago for a modified protein nanopore (IS 2/24/2009).

According to Cees Decker, a nanopore researcher at Delft University of Technology in the Netherlands, no one has done this so far with solid-state nanopores. The company is not ready to speculate how long this partial proof-of-concept might take: "It could be any day, or a lot of days – all the moving parts are available to us now," Holt said.

To improve the distinction between different bases, he and his colleagues "have a number of knobs that we can turn," he said, such as changing the dimensions of the pore, switching the material lining of the nanopore, or changing the ion concentration.

If the pore misses one of the nucleotides, he said, the system might still be able to detect that using the electrodes in the nanowell, allowing the researchers to put a placeholder in the sequence.

If successful, DeHart said, the approach promises minimal sample prep, very long read length, high throughput and low cost because of the scalability of the chip.

NSB does not license any IP from Oxford Nanopore, which boasts an extensive patent portfolio related to both protein and solid-state nanopore sequencing. Holt said Oxford and many other companies are pursuing methods that are "foundationally different" from theirs, so NSB sees "no inhibition to our freedom to operate with the technologies and IP portfolio we already have in place."

While sequencing is NSB's focus right now, the firm might pursue other applications of its nanopore platform in the future, given sufficient funding.

For example, because the nanopores, or nanochannels, can be coated with different materials, the chip might be used as a nanoscale HPLC. It might also have applications in the pharmaceutical industry for testing membrane proteins, where the lipid bilayer and the protein would be inserted into the solid-state pore. The electronics for measuring the protein function would be the same, Holt said.

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