Four years ago, the NHGRI launched an initiative to fund innovative research aimed at sequencing the entire human genome for $1,000. This would represent a tremendous savings from the current price of several hundred thousand dollars. Hagan Bayley, Ph.D., an expert in membrane protein engineering at the University of Oxford in U.K., was one of the grant recipients.
Dr. Bayley founded Oxford NanoLabs in 2005 to develop a technology that identifies individual molecules using nanopores. One use of this platform is to detect nucleotides as DNA strands are pulled through nanopores. This technology also can be applied to rapid, cost-effective, and highly sensitive kits for diagnosing human diseases and bioterrorism agents.
In May 2008, the company changed its name to Oxford Nanopore Technologies. “When we set the company up three years ago, many people did not know what a nanopore was,” says Spike Willcocks, Ph.D., director of business development. Now the potential of nanopores, which form naturally in some proteins, has become better known to people working in DNA sequencing and genomics.
The first reference human genome was sequenced in 2003 by the Sanger chain termination method. This complex process requires DNA amplification, fluorescent tags, and capillary electrophoresis to identify bases. The fixed costs of the labor, reagents, and analytical instruments limit the usefulness of this method for screening large numbers of people to make personalized medicine affordable.
Protein nanopores are an alternative to all that. A few thousand nanopores running in parallel would scale up the nanopore sequencing process, eliminating the costs of reagents and complex steps, according to Dr. Willcocks, thereby bringing the $1,000 price tag closer to reality.
Leveraging the Nanopore Concept
Protein nanopores occur naturally in proteins secreted by bacteria, such as alpha-hemolysin made by Staphylococcus. Dr. Bayley’s academic laboratory investigates how alpha-hemolysin lodges in a cell membrane, where it forms a barrel-shaped nanotube made up of intertwining polypeptide strands. Bacteria make nanopores to punch holes in cell membranes to obtain nutrients from or secrete toxins into cells. The hole passing through the middle of the tube is 1 to 2 nanometers in diameter. Once formed, the nanopores are remarkably stable and withstand high heat.
Dr. Bayley’s group discovered genetic and chemical ways to alter nanopore proteins to bind and interact with molecules without destroying the nanopores. “Their specificity is amazing,” Dr. Willcocks states. For example, nanopores can be designed to tell the difference between the right- and left-hand forms of thalidomide and ibuprofen.
Dr. Bayley applies the technology to recognize individual DNA bases by changes in electrical current as they pass through the opening of the nanopore and transiently bind to the inner surface. Because DNA bases are negatively charged, they can be electrophoretically driven through the nanopore structure. As they pass through, the bases block the flow of ionic charges, resulting in changes in electrical conductance. Each base generates different electrical currents, providing “a highly exquisite measurement of biological molecules,” Dr. Willcocks comments.
Potentially, the linear sequence of DNA can be measured from the sequential changes in conductance as a DNA strand moves through a nanopore. This type of single-molecule sequencing is reagent-free and rapid. “If you can move from chemical-based measurements to physical-based measurements, you can greatly reduce costs,” Dr. Willcocks reports.
The potential advantage of nanopores is that they can provide measurement of DNA at the molecular level without the time and cost of reagents, amplification, and labeling. The only processing step needed is the extraction of pure DNA from biological samples, and several companies sell kits for doing this.
DNA, however, moves naturally through nanopores incredibly fast—one million bases per second. Consequently, the resolution is poor, and single bases cannot be detected. Scientists at Oxford Nanopore Technologies are working to overcome this problem by using exonucleases bound to nanopores to slow the flow of DNA and better identify the four bases in DNA.
Exonucleases chop DNA into individual bases. When coupled with nanopores, exonucleases “can grab DNA and clip off one base at a time before passing through the detector,” Dr. Willcocks explains. Integrating exonucleases into nanopores reduces the flow to a rate of 10 to 100 bases per second. So far, scientists at the company have successfully coupled exonucleases to protein nanopores. “No one else has done this to date,” according to Dr. Willcocks.
Although using exonnucleases optimizes base detection, a single nanopore would be much too slow for sequencing work. So the research team is creating arrays of nanopores that work in parallel to detect thousands of bases per second. They place protein nanopores in lipid bilayers, set in individual microwells on a silicon chip, with electrodes placed across the bilayer. The technology has been scaled up to arrays containing more than 100 microwells running in parallel. “In theory, we should be able to combine thousands of nanopores together in a powerful platform,” Dr. Willcocks points out.
The same nanopore technology can be applied to the highly sensitive detection of a wide range of molecules including infectious agents and biomarkers for disease. Oxford Nanopore Technologies is in the early stage of developing a hand-held device to rapidly test for influenza including the lethal H5N1 strain. The nanopores are engineered to bind a complementary strand of RNA from the influenza virus, and the binding site can be designed to specifically detect particular mutants.
Another nanoprobe is being designed for Alzheimer’s disease, based on a newly discovered biomarker associated with neurodegeneration. In addition, the company received funding from the U.K. government to develop an anthrax detection kit based on DNA or protein biomarkers. “The nanopore platform can be readily applied to many different biomarkers,” notes Dr. Willcocks.