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Making Strides Toward $1K Genome, ASU Team Aims for Nanopore Sequencing Device by 2015


By Monica Heger

Under the National Human Genome Research Institute "$1,000 Genome" program, Stuart Lindsay's group at Arizona State University has made significant strides in nanopore sequencing and expects to have a prototype by the end of 2012 and a demonstration device by 2015.

Lindsay's group in August received a $4.1 million, four-year NHGRI grant to support further development of the technology, one of nine grants totaling $14.5 million that the agency awarded in the latest round of funding under the $1,000 Genome program (IS 8/23/2011).

The grant was the latest in a string awarded to Lindsay's team under the initiative, following a three-year grant worth $868,000 awarded last year; a one-year $370,000 grant in 2008; a three-year, $877,000 grant in 2007; and an exploratory grant in 2004.

Lindsay's group also recently signed a licensing agreement with Roche, which plans to incorporate the team's readout technology with IBM's DNA transistor technology (IS 10/11/2011).

Lindsay, who directs the Center for Single Molecule Biophysics at the Biodesign Institute at Arizona State, said that the prototype he is developing under the latest NHGRI grant would be simpler than the device developed by IBM. Rather than a commercial instrument that could be produced en masse, the prototype will be a one-off device whose function could be used to inform the construction of a commercial device.

His group has been developing a "recognition tunneling" method to read bases as DNA is passed through a nanopore. The technique uses recognition molecules attached to electrodes to temporarily trap each base and provide distinct electronic signatures for all four bases and 5-methyl C (IS 2/16/2010).

The group has recently developed a "third-generation reader molecule," which is "working very nicely," Lindsay added. He did not elaborate on the improvements because he expects to publish a paper soon.

Before publication, the team is first optimizing the readout software to handle the complex signal generated by the system, which Lindsay described as a "complicated train of pulses."

This data often appears to be something that "looks like a burst of noise," but is sometimes an actual signal that cannot be interpreted manually, he said, adding that the new software will be designed to call bases from these complicated reads.

While Lindsay has shown that his recognition tunneling technology does in fact elicit a unique signal for each of the four bases, combining the readout with a device through which DNA will translocate has proven tricky.

As such, a large amount of the NHGRI grant money is going to Oak Ridge National Laboratory where researchers are using computer simulations to gain insight into the process, Lindsay told In Sequence.

Specifically, the ORNL researchers are doing computer simulations to better understand the physics of what is going on inside the tunnel gap as DNA and solvent are translocated through a pore.

Predrag Krstic, a senior research staff member in the physics division at ORNL who is working with Lindsay's team, said that his group is simulating the processes on several different types of conducting nanopores — including gold-plated nanopores, graphene nanopores, and carbon nanotubes — to try and determine which one has the optimal characteristics.

So far, said Krstic, the group ASU experiment has made the most headway with gold, which will be "our main and first line" for implementing the method, he told IS.

Within the next calendar year, Krstic expects to have the "first prototype instrument, which will then need to be optimized." By 2015, the goal is to have a working instrument, he said.

The gold nanopore, functionalized with molecular probes that act as the readers, sits atop a silicon-based semiconductor structure, explained Krstic. This type of probe has already been demonstrated to work in recognition tunneling, said Krstic, and now it is a matter of understanding reading the DNA as it is translocated "through the nanopore when it is functionalized with readers and subject to the thermal fluctuations of the water environment."

Lindsay added that initial simulations were done assuming a vacuum and a temperature near-zero degrees Kelvin, but the latest technology will operate in an aqueous environment, where "water places a huge role in signal and readout," presenting additional challenges.

There are several major hurdles that will need to be overcome, including preventing the DNA from sticking to the nanopore. DNA is negatively charged, so electrolytes in the aqueous environment may screen the charge if positive ions bind to the DNA, which could partially or fully suppress the interaction with the applied electric field, preventing the DNA molecule from translocating through the pore.

Determining the optimum pore and the "intensity of the electric field that will allow DNA to translocate" are some of the simulations that Krstic's group is working on.

Additionally, Krstic said, the structure of the pore itself will create different binding times between the DNA and the pore as it passes through. The goal is to have a binding time that is long enough to read an individual base, but not so long as to make the process arduous, or to confound the signal.

Have topics you'd like to see covered by In Sequence? Contact the editor at mheger [at] genomeweb [.] com.