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Fabricated Nanopores Could Prove Useful for Protein Detection

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NEW YORK – Two independent research teams have published novel protein nanopore designs that could prove useful for applications including protein detection and sequencing.

The efforts, one led by scientists at the Tokyo University of Agriculture and Technology (TUAT) and detailed in a paper published in Nature Nanotechnology, and the other led by researchers at the University of Groningen and presented in a study published in Nature Chemistry, aim to devise fabricated nanopores with new properties and functionalities suited to various biosensing purposes.

Over the last decade, nanopore sequencing of nucleic acids has become a reality, with UK-based Oxford Nanopore bringing several such platforms to market. A number of researchers have also been working on nanopore-based sequencing of proteins, and if that proves technically feasible, it could enable single-molecule level detection and, potentially, de novo sequencing of peptides and proteins, along with detection of post-translational modifications.

Nanopore-based protein sequencing has proved a hard problem, however, facing challenges ranging from the difficulty of moving peptides through pores in a consistent fashion to that of correlating the signals produced by that movement to the presence of a particular amino acid. Additionally, naturally occurring nanopores are not necessarily well-suited to protein detection, as they may not provide the ideal pore size or chemical properties.

This has led some researchers to pursue the development of artificial nanopores that can be designed to have properties that make them amenable to protein analysis. University of Notre Dame Professor Gregory Timp, for instance, has been exploring the use of semiconductor technology to produce synthetic nanopores and sub-nanopores (pores with sub-nanometer diameters) for protein detection.

Several years ago, researchers including Hagan Bayley, professor of chemical biology at the University of Oxford and a cofounder of Oxford Nanopore, demonstrated the use of DNA nanostructure scaffolds to build custom peptide nanopores. (Oxford Nanopore was not involved in this work.) In 2019, a team led by scientists at Aarhus University published a study in Nature Communications using DNA to create nanopores that could be used for biosensing applications including protein detection. This year, a team including Bayley as well as researchers from the University of Bristol described a process for constructing nanopores. Also this year, University of Washington researchers detailed a process for computationally designing transmembrane β-barrel proteins that could be used in custom nanopore construction.

In their work published this week, the TUAT researchers detailed the design of a pore-forming peptide named SV28 that forms stable nanopores in bilayer lipid membranes. The group's initial design for the SV28 nanopore produced pores of various sizes, ranging from 1.7 nm to 6.3 nm in diameter. Introducing the glycine-kink mutation allowed the researchers to control the size of the pore, consistently producing nanopores with a diameter of 1.7 nm. This consistency will prove useful for biomolecule detection, said Ryuji Kawano, a professor at TUAT and senior author on the study.

Kawano said he and his colleagues are now trying to further refine their ability to control the size of their nanopores with goal of developing them to detect molecules including miRNAs, peptides and unfolded proteins, and some small molecules like allergens.

With regard to protein detection, he noted that there are two basic approaches, each of which calls for different nanopore designs. In one approach, researchers would try to detect folded proteins. Kawano said he expects that fabricated solid-state nanopores would likely be used for this application given that most biological nanopores aren't large enough to fit the typical unfolded protein.

In the case of unfolded proteins, Kawano said the ideal nanopore would have a diameter of around 1 nm or smaller to account for the small diameter of these molecules.

In their work, the Groningen researchers combined a β-barrel nanopore sensor with a 20S proteasome from the archaean Thermoplasma acidophilum, producing a piece of molecular machinery that enabled them to unfold and fragment proteins and translocate them across the pore.

Giovanni Maglia, associate professor of chemical biology at the University of Groningen and senior author on the study, said he took inspiration for the machinery from nature's habit of combining biological elements in various ways.

"To my eyes nature appears to take different [molecular] components and put them together just out of serendipity," he said. "And evolution then optimizes it. This kind of swapping of elements happens randomly and then maybe one out of 1,000 works and eventually gets optimized."

He noted, however, that developing the combined nanopore-proteosome machinery proved difficult.

"We tested like tens of different constructs and had to optimize every step," he said, noting that a number of unexpected problems reared their heads. For instance, proteases in the E. coli system used to express the different components also worked to degrade the components.

"There isn't really a blueprint for how to do this," he said. "There was a lot of trial and error."

To use the system for protein detection, Maglia said the researchers could either chop the proteins into fragments using the proteosome and then identify different peptides based on their signal as they pass through the pore or pass the unfolded protein through the pore and sequence its amino acids.

Maglia noted that he and his colleagues demonstrated the former approach in a study published in October in Nature Communications, indicating that a protein fingerprinting approach similar to that used by mass spectrometry-based proteomics could be possible.

He added, though, that the nanopore-proteosome complex would require a number of refinements before it could be used in such a way. For instance, the proteosome would need to be engineered to cut target proteins at specific amino acids. Additionally, the researchers would need to induce the peptides to flow across the nanopore, by, for instance, manipulating the pH of the solution to ensure the peptides have a uniform charge or by using other forces like electroosmotic flow.

"All these things have been shown to be possible individually, but now we need to put them together," he said. "That can be challenging. It's not straightforward that because the individual pieces work separately they will work together. It requires lots of engineering and lots of tweaking."

Unfolding and passing a protein through the pore and reading its amino acid sequencing is, in theory, a more straightforward approach, Maglia said, but it also has its difficulties.

In the Nature Chemistry study the researchers were able to use their nanopore-proteosome complex to unfold and translocate proteins, but they were not able to collect signal that could detect the different amino acids.

"We think that is because the amino acids are too small [for the pore used]," Maglia said. "We probably need to exchange the pore to make it small enough for us to start seeing the individual [amino acids] in the polypeptide."

Another challenge is pulling the protein through the pore at a consistent pace to allow for detection of individual amino acids. Earlier this month, a team led by researchers at Delft University of Technology demonstrated that DNA helicases could potentially provide this kind of consistent movement as well as the ability to pass a peptide sequence through a pore multiple times to improve analysis.

"I think there is a huge range of ways such a system could work," Maglia said. "I think that at every single step we are learning a lot, even if it is still not perfect."

He said that he and colleagues several months ago launched a start-up called Portal Biotech to commercialize aspects of his lab's nanopore research.

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