NEW YORK (GenomeWeb) – Researchers from the University of Oxford have used DNA nanostructure scaffolds to build custom peptide nanopores.
Detailed in a paper published last month in Nature Nanotechnology, the approach could enable construction of larger, more uniform nanopores for applications including DNA and protein detection, said Hagan Bayley, professor of chemical biology at Oxford and senior author on the study.
Bayley is also the co-founder of the nanopore-based sequencing firm Oxford Nanopore, though the company was not involved in the study.
Currently, nanopore-based molecular detection is focused around using these complexes to sequence nucleic acids, with Oxford Nanopore being the leading firm pursuing this work. As nanopore-based nucleic acid sequencing has become feasible, researchers, including Bayley, have begun exploring use of nanopores for detecting other molecules, most notably proteins.
"People are beginning to turn to looking at other biopolymers," Bayley said, adding that his group is particularly interested in using nanopores to look at different protein isoforms, including post-translationally modified forms and alternative splice forms.
"And to do that, we would preferably have newly designed nanopores that were built specifically for looking at proteins, which, obviously, is very different from looking at DNA," he said.
A major goal, Bayley said, is to make wider pores than those currently used by Oxford Nanopore for its nucleic acid sequencing. While wider nanopores do exist in nature, they are not uniform enough to be effective as biosensors, he said.
Wider nanopores could also prove useful for DNA sequencing, Bayley said, noting that they could allow for sequencing of double-stranded DNA, which is easier to manipulate than the single-stranded molecules currently used in nanopore-based sequencing. He and his co-authors also noted that this could allow for the "mapping of epigenetic markers on long strands of double-stranded DNA."
"There are proteins like streptolysin O from Streptococci that are very large pores [consisting of] 14 or so subunits," he said. "But when you look at them carefully, they will contain 14 [subunits] plus or minus, say, five subunits. So they have a distribution of subunits, and we'd like to be able to control that [subunit number] exactly in order to make the uniform pores that are required for sensing."
To do this, Bayley and his colleagues have turned to the use of synthetic peptides, which can be arranged into membrane-spanning nanopores of the desired size. However, some sort of scaffolding is required to get these peptide components to assemble the desired structure.
"If you want to bring eight [peptide] helices together, that is quite hard to do without some kind of scaffold," Bayley said. "You can get just kind of a mess of different structures."
DNA scaffolds, he added, "can be beautifully designed using existing software. And it's a good way to bring peptides and other molecules together to form complex structures. So we thought that we would use DNA nanostructures as the scaffold for assembling these [peptide] components."
In the Nature Nanopore study, the researchers combined DNA scaffolding with peptides from the protein Wza, a polysaccharide transporter, finding that with the scaffolding, the Wza peptides consistently formed nanopores composed of eight peptide helices. Any fewer than eight helices and the nanopores don't come together, Bayley noted.
"But if we have more than eight, they still come together as eight, and presumably the [extra] helices are excluded," he said.
The ultimate goal, Bayley said, is to be able to reproducibly arrange a set number of peptide helices "around a central axis in a pre-designed order. That is where we are heading."
Mark Akeson, a nanopore researcher and professor of biomolecular engineering at the University of California, Santa Cruz, said he saw Bayley's approach attractive in that it could allow for "self-assembly of engineered nanoscale structures that can be stable at ambient temperature."
This is in contrast to custom solid-state pores fabricated by milling and etching that "erode under ambient conditions," he said.
Akeson, who was not involved in the Nature Nanotechnology study, noted that while the process described by Bayley and his colleagues is still in the early stages, "given time [it] could bear fruit." He added that nanopore-based DNA sequencing took roughly 20 years to implement.
He suggested, though, that one challenge for Bayley's group would be to "generate structures that outperform naturally occurring protein nanopores that are modified by site-directed mutagenesis to optimize their stability and sensitivity."
He cited the example of Oxford Nanopore's work with the naturally occurring CsgG pore, which the company has re-engineered to optimize for nucleic acid sequencing.
Bayley said that he saw the synthetic nature of the process as an advantage that could provide his group's approach with additional flexibility.
"We're not using expression in bacteria or other cells [to generate the peptide components]," he said. "We're just using synthetic DNA and synthetic peptides to build these structures. So we're not restricted to the 20 natural amino acids. We can use any amino acid side chains. In fact, we can introduce into these structures [components] that aren't amino acids at all. So we have huge versatility."
This versatility could allow the researchers to tailor the structure of their nanopores to particular types of proteins being targeted by an analysis," Bayley added.
"You can envisage chips that contain 20 or 50 different nanopores that would allow you to more readily distinguish between the myriad proteins that are in cells," he said. "As you know, there are around 20,000 genes for proteins. But it doesn't end there, because there are literally millions of different modified forms of proteins in cells, either through post-translational modification or alternative splicing. The analysis of proteins from cells is going to be a very difficult task."
"Of course, the idea of sequencing [nucleic acids] with pores was seemingly somewhat crazy at the beginning, too, but it was done," he added.
And, indeed, progress is being made on the protein front by Bayley's group and others. For instance, Bayley and his colleagues have demonstrated the ability of nanopore sensors to distinguish between differentially phosphorylated protein forms. Akeson and his colleagues, meanwhile, have devised a method for driving unfolded proteins through a model α-hemolysin nanopore and using it to distinguish between different sequence-dependent features on the proteins.
Last year, researchers at the University of Groningen demonstrated the ability of Fragaceatoxin C (FraC) nanopores to identify peptide and protein biomarkers in simple mixtures and to distinguish between polypeptides differing by as little as a single amino acid.
Bayley said he expects nanopores would initially find a use for targeted protein analyses wherein researchers would use some form of upfront enrichment to isolate a specific protein and then use nanopore analysis to study the different variants of that protein in a particular sample.
He added that because nanopore sequencing is a single-molecule technique, it could potentially be used for single-cell studies.
"I think [nanopore protein sequencing] will see stepwise progress, initially looking at individual important proteins, then looking at the proteins in a substantial tissue sample and counting the different forms of the many different proteins in the sample, and then eventually pushing it down to the single-cell level," he said.
Regarding the DNA scaffold work specifically, Bayley said he and his colleagues are now working to develop new scaffolds and identify new peptides that will form pores of varying sizes "so that we can really vary the diameter of the [pores] at will."