Thirty years ago, scientists discovered that nanopores could detect biological compounds. Yes, you read that correctly: thirty years ago. Only recently, the ripples have converged into a tidal wave of technological advancement, particularly in the field of DNA sequencing.
One could say nanopore DNA sequencing technology is en vogue, the latest fashion in lab bench equipment. Its importance is being proven over and over, from healthcare through to conservation and back again. The Ebola virus genome was sequenced in West Africa just 2 days after some of the samples were collected, conservation efforts of endangered species – such as the European eel – and species identification are aided by genome sequencing, and parts of the human genome can be rapidly sequenced to aid with personalised medicines and diagnosis.
noun: nanopore; plural noun: nanopores
a pore or cavity with dimensions of only a few nanometres.
Companies such as Oxford Nanopore Technologies are revolutionising the way in which lab bench sequencing is achieved. Their MinION computer accessory is little bigger than an external hard-drive yet has the ability to gather up to 20 gigabases of DNA sequence reads (hundreds of millions of base pairs) every 48 hours. This technology rests on the discovery by the Bayley group at the University of Oxford that passing a current across a membrane with a single α-hemolysin nanopore inserted generates a stable signal and molecules passing through the pore will disrupt the current reading in a predictable and measurable way. This gives each molecule an electrical “signature”, distinguishing divalent metal ions or each of the four DNA bases from one another despite their similar structures. The parallel sequencing of many strands of DNA at once by a large number of nanopores enables a high throughput and rapid sequencing. If you learn visually, have a look at this video on the Oxford Nanopore Technologies website about how their technology works.
Along with the technology and its inevitable improvement of scientific process, another key part of developing such equipment is the understanding of the vital processes within it. In 2010, the process of moving the polymer DNA through a nanopore was described by the Akeson group at the University of California in Santa Cruz. This year (2017) in August the dynamics of the DNA polymer translocating through an α-hemolysin pore were described by Ghosal, Ulrich and colleagues at the University of Cambridge. This incredible achievement will not only help comprehend this mechanism of polymer sequencing but will also contribute to understanding similar molecular motors and translocation systems. The applications to determining physical properties, such as the bending rigidity, of polymers at a molecular level make this an incredibly exciting technology for materials science as well.
Image: A double stranded DNA helix being unfolded and “read” through a nanopore embedded in a membrane. The electrical “fingerprint” trace represents individual base detection as the single strand is fed through the pore. http://sequencing.roche.com/en/technology-research/technology/nanopore-sequencing.html
A recent publication claims to have taken nanopore sequencing one step further into the realms of peptides and proteins. Understandably, the hype surrounding using proteins to reliably sequence DNA has led to a lot of excitement of applying this same principle to protein sequencing. However, this feat is much more difficult due to the greater variation in building blocks used to create proteins. There are 20 amino acids used in eukaryotic proteins, as opposed to just 4 bases in DNA (excluding any modifications to these bases), but there can also be some uncommon amino acids added – such as AIB in alamethicin, an antimicrobial peptide produced by the fungus Trichoderma viride.
Another issue to be overcome in protein sequencing is the folding of secondary, tertiary and even quaternary structure. Nivala and colleagues at UC, Santa Cruz attached ClpX to alpha-hemolysin to enable unfolding of the proteins before being threaded through the pore. ClpX is an AAA+ unfoldase which also forms a ring and pushes the unfolded peptides through the α-hemolysin protein in a ratchet-like fashion. An obvious issue with this particular system is if ClpX cannot unravel the protein, which means it can’t be sequenced by this method.
Other techniques have proven fruitful to research groups around the world. Researchers in the Abu Dhabi branch of NYU have used DNA scaffolds to template the assembly of α-hemolysin monomers. The ability to program the size of an α-hemolysin pore could allow the detection of particles of a greater range of sizes such as full sized and fully folded proteins. Each would give a unique signal for detection. A group at the University of California in San Diego are in the process of applying machine learning techniques to aid detection of a wider range of proteins by nanopores. And researchers at the University of Groningen in the Netherlands have patented a nanopore technology that can distinguish between proteins of between 1.3 kDa and 2.5 kDa which differ by just one amino acid. An exciting technology in itself but no doubt an important step in the path to protein sequencing.
Is it just a matter of time before peptide sequencing is as simple as adding your pure sample to a USB device and returning a few hours later to a sequence? I very much hope so! For the lab I work in, such technology would reduce dependence on sensitive and expensive mass spectrometry equipment which can break very easily. Passive systems (ie., “add sample and wait”) have much fewer “moving parts” for researchers to accidentally damage.
Have you heard about any other breakthroughs in nanopore sequencing technology? Get in touch in the comments and tell us about it!