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Solid road to DNA sequencing future

A trio of Beckman Institute researchers are spearheading the effort at the University of Illinois to bring low-cost, accurate, full genome sequencing of human DNA to the world. Jean-Pierre Leburton, Aleksei Aksimentiev, and Rashid Bashir are leading the effort, funded by a recent agreement signed with Oxford Nanopore Technologies, Ltd.

Published on Feb. 4, 2013

The recent agreement between the University of Illinois and the British firm Oxford Nanopore Technologies, Ltd. may someday be considered historic, in terms of the impact the research could potentially have on the worlds of business, medicine, and science. And if the research behind the agreement delivers the Holy Grail of genomic medicine — low-cost, accurate, full genome sequencing of human DNA — it will be thanks to solid state nanopore technology developed by researchers from the Beckman Institute.

Oxford Nanopore Technologies (ONT) announced agreements in January with American and British universities to license DNA sequencing technology and to fund future research. The co-Principal Investigators (PIs) on the project at the University of Illinois are Beckman researchers Jean-Pierre Leburton, Aleksei Aksimentiev, and Rashid Bashir.

The Illinois technology has its origins in a white paper proposal to the National Science Foundation (NSF) in 2002 that included Leburton and Beckman colleague Klaus Schulten titled “A Nanometer-scale Gene Chip,” as well as a 2007 paper by Leburton reporting on a semiconductor membrane that could be used for sequencing.

Several Beckman and Illinois researchers have contributed over the years in developing solid state nanopore technology, but Leburton, Aksimentiev, and Bashir will lead this effort, funded by ONT for developing the DNA sequencing method. Their mission is to create what has been a long sought-after goal in genomics research: low-cost, fast, reliable, and highly accurate sequencing of a person’s whole genome.

It is often referred to as personal genomics, and predicted to be an essential facet of a healthcare future focused around personalized medicine. If the research achieves its goal, the impact will be felt in both the business and medical worlds. Estimates of the potential profits for the business that is first in what has been a race to produce the technology are in the billions of dollars.

Current methods have reduced sequencing costs greatly, in some cases to below $5,000, with promises of less than $1,000 in 2013. Bashir, who is Director of the Micro and Nanotechnology Laboratory where fabrication work on the nanopore sensor will be taking place, said a DNA sequencer could perhaps someday be done for less than $100. That would make the technology available on a worldwide scale.

“Something like this can have very broad applications, being able to sequence DNA at a very low cost,” Bashir said. “So it really brings this area of personalized medicine to the forefront and to reality. There is actually nothing that we know of in history that has been cost-reduced so much. About 10 years ago the cost of sequencing the entire human genome was about a billion dollars.”

The Technology

The Illinois technology being developed potentially has numerous advantages over other methods, including that it is solid state, multilayer, and does not require splitting the DNA into two strands, or denaturing, and amplifying the DNA molecule, as happens in current methods. The basic design is a solid-state, multilayer semiconductor membrane that uses nanopores about the diameter of a single DNA molecule (roughly a billionth of a meter wide). Using electric fields, single DNA molecules are passed through the nanopore, and a detector reads the sequence.

“So a DNA molecule is electrically charged, and you put this membrane in fluid with DNA on one side,” Bashir said. “And as long as there is one pore and DNA is driven through that pore like spaghetti, the molecule unwinds, uncoils, and eventually one end finds the nanopore and it transfers through.”

The white paper proposal in 2002 (which led to NSF funding the project) by Leburton, Schulten, and others called for a “revolutionary type of silicon integrated circuit that incorporates Metal-Oxide-Semiconductor (MOS) technology with an on-chip nanometer-scale mechanism for probing the electrical activity of DNA.”

One of the unique aspects of the method being developed is that it offers electrical tunability. It goes back to a design Leburton and his group reported on in 2007 of a semiconductor membrane — built from silicon layers — that can be used to regulate the flow of charged ions and molecules, thus enabling control of the pore electrically.

“It provides electrical tunability of the membrane, which people at that time hadn’t thought about,” Leburton said. “The second fact is the multilayer structure, where each layer can be energized differently. The membrane can also be made of different materials and with different voltage forces. That is unique.”

Leburton is from the Department of Electrical and Computer Engineering (ECE), while Bashir is faculty in ECE and Bioengineering, and Aksimentiev is faculty in the Department of Physics. As to their roles in this project, Leburton is the theorist who also does computer device modeling, while Bashir is an experimentalist who will be fabricating the technology, and Aksimentiev does computer modeling that has included breakthrough molecular dynamics simulations.

Aksimentiev uses supercomputers and specially developed programs tailored for nanopore simulations that have enabled the creation in silico of the intricate dance of a DNA strand making its way through a nanopore. In 2008 he led a team that used several supercomputers, including those at Oak Ridge National Laboratory, to simulate the shape of DNA going through a single nanopore. He said the work using molecular dynamics simulations to model the process of DNA translocation through a nanopore dates back a decade.

“Our modeling approach permits obtaining detailed and precise information about nanopore systems that are not accessible through experiments,” Aksimentiev said. “My role in the nanopore project at Illinois has been in exploring various nanopore systems for applications in nano-biotechnology, which include but is not limited to designing and refining new approaches for biosensing with nanopores, and providing microscopic interpretation to the results of experimental measurements.”

The Agreement

Oxford Nanopore Technologies is well-known in the business of genomic technology, and licenses its products to biotechnology giant Illumina. ONT has developed both biological and solid state nanopore sensors, and announced a plan to produce a “strand sequencing” DNA sequencer the size of a memory stick by the end of the year. It does not yet offer DNA sequencing for humans, which requires a higher threshold for accuracy than sequencing for animals or crops.

The research and licensing agreements are separate. The sponsored research agreement is funding the research of the three co-PIs, who are integrating their research in this area. The licensing agreement is for a package of patents and patent applications.

Several Illinois researchers have been working on nanopore sensor technology, and there had been enough success in the area for the University to create a proposal seeking an outside partner or partners to help develop the technology, and license the patents through the Office of Technology Management (OTM) at Illinois.

ONT, which has worked with the other universities such as Harvard and Oxford, turned out to be that partner. Steve Wille is associate director and senior technology manager at OTM. He said the fact that Illinois can do both modeling and experimental testing of the design made the technology attractive to ONT.

“The inventors had both modeled and experimentally demonstrated a very difficult, very important invention that can have tremendous con-sequences for health-care,” Wille said. “It is great that UIUC has three PIs working in different technology areas, which together can help create an extremely useful device for healthcare.”

In addition to the three co-PIs, the list of Beckman faculty members working on nanopore sensor technology includes Schulten and Narayana Aluru, and former faculty member Greg Timp. Bashir said several researchers on campus have contributed greatly to the presence of Illinois as a leader in solid state nanopore technology research.

“At the U. of I. we have great expertise in this area beyond these three people,” Bashir said. “It’s unlikely you would find that many people working on nanopores at any other place.”

Research Lines

If successful, the solid state nanopore they are developing will be something completely different in the field of biomedicine.

“The basic idea is that the nanopore becomes a tool to analyze single DNA molecules one molecule at a time,” Bashir said. “The Holy Grail is to be able to take the DNA from a cell and chop it into fragments, and sequence each fragment directly without having to amplify the molecule. Or take the entire molecule and sequence it directly without having to amplify it at all.

“The nanopore offers a possibility of ‘can I conceptually take a molecule, pass it through the pore, and, as it’s passing through, get the sequence directly from that single molecule?’ And that would certainly be revolutionary.”

Leburton said his idea for the multilayer membrane was based on the way nature uses biological membranes, and could be used for other types of sensing, although this agreement is only for DNA sequencing.

“What we try to do is actually mimic, with semiconductor nanotechnology, the functionality of a human membrane,” he said. “The human cell is actually a biological machine, and the interaction of this human cell with the outside is through this membrane. The membrane has a pore so it can exchange with the outside a certain amount of biological or biochemical information.

“So what we propose in this patent is replacing whatever this bi-lipid layer, this membrane, is with a semiconductor membrane that provides even more functionality. Now we not only have the ability to make multilayer structures, so each layer would have a specific function, but we can also energize with particular biases or current going through it. So we make each layer sensitive to any kind of biochemical agent that would go through the pore.”

Leburton said that could enable a broad range of applications, for example, in diagnostics, dialysis, and drug delivery.

“I don’t think people have understood that yet, because it is at the crossroads between semiconductor nanotechnology and biology,” Leburton said.

That crossroads is where the next generation of biomedical instruments will be coming from. The potential of what their technology — if it is successfully developed and commercialized — can do for medicine in the future is something that excites all three researchers.

“For me the theoretical part is interesting as a physicist but also I wish to find applications of my work,” Leburton said. “This is one that will certainly be very beneficial for humanity and society.”

“I’m excited about seeing the impact of my research efforts in everyday life and the well-being of people,” Aksimentiev said.

Bashir mentioned lower costs, including less than $100, as one of the biggest boosts for medicine, on a grand scale.

“I think clearly this is going to have an impact worldwide in all areas of health and medicine,” Bashir said. “If the cost comes down this low you can truly have genetic-based information, molecular information to understand heterogeneity of disease, for early detection, to look at all sorts of things like plants, bacteria, and other microbe organisms. Sequencing is not just for humans. Sequencing is for any biological entity.

“The cost of sequencing coming down is going to have a broad impact in agriculture, animal diseases, all sorts of things. For example when you have an outbreak of a certain virus, or a certain pathogen, if you can isolate it and sequence it from individuals, it definitely gives you so much more information, which strain it is, which exact variation. Right now it is relatively expensive. Something like this would make it very cheap and very easy to do.”