UK-based startup Oxford NanoLabs plans to make sequencing by protein nanopores a reality. While many researchers believe this won’t be possible for at least another decade, the University of Oxford spinout maintains it could be achieved in as few as five years.
The company’s short-term goal is to develop handheld protein nanopore-based point-of-care diagnostics. This week, Oxford NanoLabs is at the Molecular Medicine Tri-Conference in San Francisco, where it will present during a session for early-stage companies and look for partners to develop and sell its tests.
Eventually, Oxford NanoLabs plans to focus on sequencing and “use the [technical] milestones we achieve to deliver improved products for partners to take forward in separate areas,” Spike Willcocks, the company’s director of business development, told In Sequence last week.
The company’s technology is based on research on pore-forming proteins, called porins, conducted by Hagan Bayley, a professor of chemical biology at the University of Oxford.
Bayley, who was a professor at Texas A&M University before he came to Oxford in 2003, has been studying these bacterial proteins, which punch holes in the membranes of their host cells. He has been engineering them genetically and chemically to turn them into biosensors for a variety of molecules. Specifically, he has focused on alpha-hemolysin, a pore made by Staphylococcus aureus.
In 2005, Bayley and Reza Ghadiri, a professor of chemistry at the Scripps Research Institute, jointly won a five-year, $4.2-million grant from the National Human Genome Research Institute to work on “single molecule DNA sequencing with engineered nanopores.”
In a biosensor, up to 20 self-assembling protein pores, each with a hole about two nanometers in diameter, sit in a lipid bilayer that spans a hole, thus sealing two aqueous chambers from each other. Researchers apply a voltage between the chambers so a current flows through the pores. Whenever a molecule from the solution, such as a protein or a piece of DNA, interacts with the inside, or near the entrance, of the pore, the current changes for a short time and to a certain degree, depending on the nature of the molecule. Each signal is recorded from a single pore, even though several pores can be present in each membrane.
The alpha-hemolysin pores can be engineered to carry a probe that is highly specific for a DNA sequence, among other things. A single base mismatch in such a sequence, Willcocks said, alters by an order of magnitude the duration the current gets blocked.
The system is also very sensitive: When controlled by diffusion, a nanomolar analyte solution results in one interaction per second with every pore, he said.
Willcocks believes it is possible to engineer this system for DNA sequencing in less than a decade. “The general view in the outside world is that nanopore sequencing is possible, but it’s the other side of 10 years,” he said. “Our view is that it’s more in the 5-to-10-year timeframe.”
Single-stranded DNA can pass through the pore, and researchers led by Daniel Branton at Harvard showed more than a decade ago that they can distinguish homopolymers of different nucleotides passing through an alpha-hemolysin pore by changes in the current block.
“That was the first indication that in principle some sort of sequencing is possible,” Willcocks said.
The key difference of Bayley’s work has been that he has been working with mutant forms of the protein. “Every one else’s alpha-hemolysin are always just natural, straight out of the bug,” Willcocks said.
The advantage of engineered protein nanopores over inorganic nanopores, Willcocks said, is that the former self-assemble, while it has been difficult to reproduce artificial nanopores with small holes. Hybrid systems that combine larger inorganic nanopores and protein pores are another possibility, and Oxford NanoLabs is working with Bayley’s group to investigate such an approach as well.
Scientists have raised several objections to sequencing with alpha-hemolysin, Willcocks said, but he believes none of them is valid.
“One of the things that’s always quoted against protein nanopores is that they are unstable,” he said, “but that’s actually not the case.” Once inserted in a lipid bilayer, they can be heated to over 95 degrees Celsius without falling apart. The lipid bilayer can also be further stabilized by a new method for creating it that is currently under development, Willcocks said. “That bilayer is incredibly stable; you can drop them on the floor and they don’t break.”
People have also pointed out that the DNA needs to be single-stranded to pass through the pore, requiring high temperatures to keep the strands apart, but Willcocks said enzymes can do the same job.
Another problem is to distinguish between individual bases. By chemically adding a cyclic molecule to the pore, Bayley was able to “see” the four bases, provided as single nucleoside monophosphates, in solution. Though this is still a long way from sequencing, Willcocks said, “it pointed out that as a system, it did have a potential to discriminate between the bases.”
Yet another issue is the length of the pore, which can accommodate up to 20 bases. “People said that these holes are too long. ‘How are you going to be able to tell which base in that hole is causing the change in current? Is it all of them?’” Willcocks said. But in a series of experiments, he said, Bayley showed that the change in current can indeed be traced to a single base.
“The general view in the outside world is that nanopore sequencing is possible, but it’s the other side of 10 years. Our view is that it’s more in the five-to-ten-year timeframe.”
A further problem is that at the measuring voltage, single-stranded DNA passes through the pore at about a million bases per second, too fast for the electronics to keep up. “No-one has solved that problem yet,” Willcocks admitted, but researchers at Harvard showed several years ago that a molecular motor, such as a helicase or a polymerase, can hold on to the DNA and slow it down several orders of magnitude.
“If you get to about a thousand [bases per second], the electronics will be able to cope,” he said. “There is ongoing research to find ways of, for example, bolting a helicase or a polymerase to the nanopore, and then seeing if you can feed the DNA strand into the hole.”
In principle, he said, there are two ways of harnessing alpha-hemolysin for sequencing, both of which Oxford NanoLabs is pursuing in collaboration with Bayley’s group. One is to use an exonuclease to clip off one base at a time from the DNA and feed the released nucleotides into the nanopore. “That’s an earlier, near-term goal,” Willcocks said. The concept of exonuclease sequencing is not new, he said, but using a nanopore as a readout system is novel.
The other way is to keep the single-stranded DNA intact and drive it through the pore. This will require not only slowing down the DNA but also controlling its “slightly chaotic” molecular motion as it travels through the pore, he said.
In order to sequence large amounts of DNA, the company is also working on microarrays of individually addressable electrodes.
While Oxford NanoLabs works to overcome the technical challenges of its methodology, it is developing near-term diagnostic applications for the protein pores.
Company researchers have already developed hand-held prototypes — electronic readers where users can slot in a plastic biochip containing the electrodes and dried-down reagents, membranes, and nanopores. After they inject the sample, the system self-assembles. So far, the researchers have demonstrated they can detect short strands of amplified DNA from viruses.
“Our goal is not to take a product through a clinical trial process, but to be able to demonstrate that we can create a clinically approvable prototype system with a manufacturing setup which would enable a partner to take that forward and scale it up,” Willcocks said.
Such a partner could be a diagnostics firm or an electronics company, such as Siemens or GE. “We are in discussions with a number of people at the moment,” he said, looking for a small number of partners for different applications.
Oxford NanoLabs was founded in 2005 with £490,000 ($960,000) in seed funding from technology commercialization company IP2IPO. It is housed in a science park just north of Oxford and has 17 employees.
Last summer, Oxford NanoLabs raised £7.5 million in a second financing round from a number of hedge funds. Earlier that year, the company also won a £165,000 research contract from the UK government’s Biotechnology and Biological Sciences Research Council to develop a nanopore biosensor for prion detection.Last fall, it received a £600,000 award, together with the University of Oxford and the company Nanotecture, to develop protein pore sensors of bioterror agents.