Big advances from tiny technology
Is a game-changing device in DNA sequencing about to revolutionise the business of decoding genes?
15 May 2012
It was only from 2007 when next-generation sequencers brought the time and cost of deciphering the genetic code from weeks and millions of dollars to days and tens of thousands. Now a new wave of electronic sensing devices based around bacterial nanopores – tiny holes in the membranes of microbes – look poised to answer biological questions in real time and bring a substantially lower cost 'pay as you go' model to sequencing.
The technology, some of which is hand-held and can plug into a computer like a memory stick, was announced by Oxford Nanopore Technologies (ONT) to a mix of astonishment and excitement (ref 1, ref 2, ref 3). If the technology delivers as expected, it could, finally, usher in the long-awaited era of personalised medicine by making analysis extremely rapid, cost-effective and, critically, results are obtained in real time, a significant advance on present technologies.
"It is truly amazing," says ONT founder Hagan Bayley, Professor of Chemical Biology at the University of Oxford. "It is amazing in terms of sequencing technology, but also in terms of single molecule detection – many other molecules can be detected this way."
ONT was founded at the University of Oxford in 2005. Since 2006, Bayley has utilised BBSRC-funded training grants to make critical discoveries that have ultimately contributed to the company's success. And DNA is only part of the story – the technology provides a platform that can also detect proteins, sugars, health-related biomarkers and be used in a host of on-the-spot chemical diagnostic applications in the laboratory, clinic and the environment.
The path of discovery often meanders like a river. Bayley says that ONT was not founded as a DNA sequencing firm, but as a company interested in developing single molecule sensing technology for metabolites, sugars, calcium ions and other compounds of biochemical interest. "We saw it as a platform technology," says Bayley. "But quite soon it became obvious that its application in third-generation DNA sequencing was an area with enormous potential."
Bayley's sensors are based on protein nanopores – tiny channels that span membranes to control what goes in and out of a cell – which are partly blocked by analytes (molecules of interest). Over the years, he's worked mainly on nanopores from the bacteria Staphylococcus aureus. The nanopore in question, the alpha-haemolysin pore, is a toxin secreted naturally by the bacterium, which forms the cornerstone of ONT's sensing and DNA-sequencing technology (ref 4).
An electric current is applied across the pore, and when analytes bind at a site engineered within the pore, they cause a tell-tale change in the current, which is specific to the compound passing through. Hence, the order of the individual base units of DNA – cytosine, adenine, guanine and thymine – can be recorded electronically as a strand of DNA passes though the pore.
BBSRC-funded Training Grants played an important part demonstrating that biological nanopores could differentiate between the individual bases of the genetic code (ref 5, ref 6) – a key aspect of the DNA sequencing methods developed by ONT.
"What I found out during my DPhil was that protein nanopores were sufficiently sensitive to be able to discriminate between single nucleotide differences in DNA strands when they were immobilized within the pore," says Dr David Stoddart, who was funded by BBSRC as a student and now works for ONT. "And that base discrimination can be influenced by the amino acid composition of the nanopore."
Further work, also funded by BBSRC Training Grants, showed that there was more than one point in the nanopore which registers DNA bases (ref 7) – improving sequencing accuracy. Bayley says that by having multiple points of recognition and a more complex algorithm to look at current levels, you can get more information than using a single point.
Later, BBSRC-funded student Emma Wallace was able to demonstrate that the nanopore-sensor system could also record epigenetic changes to DNA (ref 8). This is important because while the genetic code is a set of instructions for making proteins, there is more to the story than just the sequence of DNA bases. DNA is altered by enzymatic modifications that affect gene expression in organisms – picking up these molecular nuances is critical if the information in DNA is to be utilised in a way that is clinically useful on an individual basis.
These were key papers demonstrating that you could identify the four different bases (ref 9). "Furthermore, it is very important to be able to map epigenetic modifications on genomic DNA," says Bayley. "Quite critical to this was involvement of graduate students who were sponsored by these [BBSRC] training grants."
BBSRC also funded a colleague Bayley, Dr Mark Wallace, to investigate the fundamental bioscience behind how the S. aureus nanopore is assembled (ref 10). "This is not work that impinges directly on sensing, but cutting-edge research on how nanopores are put together," says Bayley. See 'More on pores'.
Bayley estimates that the nanopore system is one thousand times faster than present second-generation machines, and the results are delivered in real-time, not after the machine has completed numerous relatively slow sequencing cycles, which take days depending on the size of the genome.
"Techniques such as Solexa Sequencing are overkill for small sequencing jobs, such as a virus carried by a passenger at an airport," Bayley explains. "To get the results in a minute, you cannot use an Illumina sequencer." However, Bayley cautions that the parallel processing abilities (simultaneously decoding of around a billion DNA strands) of second-generation sequencers means they are not as yet obsolete, especially for larger genomes.
So was there a "eureka!" moment when he realised that nanopores could be used in such a way? In fact, others had proposed nanopore sequencing, but there was no practical technique to do it. "It was more like Jules Verne saying 'I'm going to fly a rocket to the moon' but there was no feasible way to do that at the time," says Bayley.
Progress in the nanopore sensor field was very slow from the mid-1990s to 2005-6, but the US National Institutes of Health $1000 genome project served as a catalyst. "That got a lot more people involved, including us," says Bayley. "Then in 2006 we had a big paper showing that bases could be identified by a nanopore. That was a huge stimulus to the field and the company."
The future has arrived
Bayley and his company have set their sights beyond the realms of DNA to sense metal ions, drugs, biochemical markers almost anything in an aqueous solution.. "Oxford Nanopore was formed to develop a sensing platform, and this takes us into limitless territory," says Bayley. "There's far more to it than genomics. The same sensing platform, similar pores, similar chips, similar readers, and similar software for analysis can be used for a very wide variety of analytes."
This means that not only can the technology be used for speedy clinical diagnosis in a laboratory or healthcare centre, but that there are applications in environmental monitoring, as well as defence and security.
To this end, work continues on nanopores under a BBSRC CASE Studentship training grant. "The main aim of this project is to exploit our knowledge of structure-function relationships in bacterial membrane proteins via advanced molecular simulations to provide novel possible designs for selective nanopores," says Professor Mark Sansom, Head of the Department of Biochemistry, University of Oxford. "It is early days, but the project should allow us to use computer simulations to aid the design of protein nanopores with modified DNA selectivity."
That should feed back into the DNA sequencing work, but Sansom says that his group's approach is general, and is not restricted to in silico [computer] design of protein nanopores. "We have also started to explore the design of novel nanopores and then 'embedding' them in non-biological nanopores such as carbon nanotubes."
This is because besides sensing and sequencing there are potentially many applications for nanopores and carbon nanotubes in, for example, clean biotechnology. "There has been interest in developing desalination technologies based on carbon nanotubes," says Sansom.
Microelectrical engineering and synthetic biology are two other areas where nanopores and carbon nanotubes could meet. In fact, Sansom thinks many other aspects of industrial biotechnology could exploit this general approach. "There is considerable interest in designing (and realising) synthetic protocells for clean biotechnology, and nanopores will be essential to allow the controlled import and export of materials to and from such protocells."
More on pores – Dr Mark Wallace
Under your BBSRC funding, what did you learn about how the Staphylococcus aureus nanopore?
The results were pretty surprising. The S. aureus nano pore (alpha-hemolysin, or aHL) is a heptamer made of seven individual proteins randomly diffusing about on the cell surface before coming together to form a donut that then punches a hole through the cell membrane.
But rather than coming together slowly as one, then two, then three subunits, as you might naively expect, aHL appears to assemble very rapidly – going from isolated proteins to an intact pore in less than a few milliseconds.
How does knowledge of nanopore structure relate to DNA sequencing?
We know that aHL can be used to measure current blocks and sequence DNA, but we are reliant on the limitations of this particular nanopore protein. Perhaps other pore-forming proteins would give us better base resolution, or allow us to measure the transport of larger or smaller biomolecules.
There are other pores out there with both fewer and many more subunits than aHL. If we understood how aHL builds itself, we might be able to design new biological nanopores with different properties. My work has helped us understand how aHL works in nature.
What other impacts could studying nanopore structure and assembly have?
They're important to understand in their own right. Other nanopores (or to be less fashionable, other pore-forming proteins) are an integral component of our immune system. They also play an important role in a many serious infections including pneumonia, MRSA, and even anthrax! Figuring out the role that these pores play in biology is also clearly important.
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- Stochastic sensing of organic analytes by a pore-forming protein containing a molecular adapter
- Single nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore
- Analysis of single nucleic acid molecules with protein nanopores
- Multiple base-recognition sites in a biological nanopore: two heads are better than one
- Identification of epigenetic DNA modifications with a protein nanopore
- Nucleobase recognition in ssDNA at the central constriction of the alpha-hemolysin pore
- Rapid Assembly of a Multimeric Membrane Protein Pore