By Kevin Davies
February 22, 2009 | In an important paper published online this week in the journal Nature Nanotechnology, scientists at Oxford Nanopore Technologies present the first proof-of-principle of their label-free next-generation sequencing system.
The paper*, by James Clarke and colleagues, shows that an engineered nanopore complex can discriminate between the four bases of DNA (A, C, T and G) with remarkable specificity, and can even identify methylated C residues (“the fifth base”) for good measure. The senior author of the report is Hagan Bayley, Oxford University chemistry professor and co-founder of Oxford Nanopore, who has been leading research into nanopores as detectors for sequencing and other applications for over a decade.
There is immense appeal in a DNA sequencing technology that dispenses with fluorescently tagged molecules, CCD cameras, and expensive microscopes, and thus has the makings of an affordable, real-time, label-free sequencing system. The Oxford Nanopore Nature Nanotechnology paper does not go so far as describing a prototype device or even reading the sequence of a DNA molecule. But it does is show that Oxford scientists have engineered the alpha-hemolysin nanopore to the point that it can efficiently discriminate between the four natural bases of DNA at speeds suitable for high-throughput applications.
Nanopores have shown considerable potential for DNA sequencing for more than a decade, but single-stranded DNA molecules slide through the nanopore much too quickly to recognize individual bases. Taking a different tack, Bayley’s group showed a few years ago that it could partially identify individual bases as they dropped through a nanopore by the degree to which they impeded the current flowing across the membrane housing the nanopore.
Building upon that work, Clarke and coworkers genetically engineered the alpha-hemolysin to find the best location to covalently attach a molecule of cyclodextrin -- a kind of washer that sits in the middle of the pore. The result is a molecular machine – a ring-like assembly of seven subunits, one of which has been modified to clamp the cyclodextrin -- that recognizes virtually every base passing through the pore.
By altering variables such as salt concentration, pH and temperature, Clarke and colleagues found conditions that allowed them to resolve the four bases to an accuracy of about 99.8%. The slight difference in size of the four bases results in a discrete blockage of the pore, which is read as a reproducible dip in the applied current from a baseline of around 60 picoamps to anywhere from 20-40 picoamps. In the current system, bases can be identified as they flow through the nanopore at a rate of about 25 per second.
Clarke and colleagues conclude that they have “solve[d] the key technical problems required for real-time, high-resolution nucleoside monophosphate detection.” They go on to state: “These advances represent the realization of a complete nanopore base detector, which, when combined with a compatible exonuclease DNA processing system, will provide the basis of a complete nanopore sequencer.”
That indeed is the next crucial step for Oxford Nanopore – to couple an exonuclease enzyme to the nanopore so that single bases are cleaved off a template DNA strand and their identity read off instantaneously as they funnel through the nanopore. Clark and colleagues conclude the Nature Nanotechnology paper by showing that the nanopore does indeed work in salt conditions favoring exonuclease activity and can detect bases cleaved off DNA strands in solution by the enzyme.
Oxford Nanopore was founded in 2005 and is located on the outskirts of Oxford, UK. Last month, the company announced an $18-million sales and distribution deal with Illumina for its exonuclease-based sequencing technology.
Editor’s note: Oxford Nanopore’s James Clarke will be presenting a poster on this work at CHI’s Next-Generation Sequencing conference, March 17-19, in San Diego.
*FURTHER READING: Clarke, J. et al. “Continuous base identification for single-molecule nanopore DNA sequencing.” Nature Nanotech. 2009.