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Nanopore Approach Shows Promise for Protein Detection


NEW YORK (GenomeWeb) – Researchers at Syracuse University have developed a nanopore-based approach to detecting protein-protein interactions.

Described in a study published this week in Nature Biotechnology, the approach uses engineered protein nanopores to detect protein-protein interactions and could ultimately be used for larger-scale proteomic experiments, said Liviu Movileanu, professor of physics at Syracuse and senior author on the study.

The sensing platform consists of a protein nanopore with a receptor protein attached via a hexapeptide tether. When target proteins in a sample bind to the receptor it causes a change in the electric current across the nanopore, allowing researchers to detect protein binding events.

In the Nature Biotechnology study, Movileanu and his co-author Avinash Kumar Thakur, a graduate student at Syracuse, demonstrated the ability of the system to detect binding between the protein receptor RNase barnase (Bn) and the protein barstar (Bs), which is known to inhibit Bn activity.

They also demonstrated that the system could distinguish between a high-affinity binding partner (Bs) and a low-affinity one (D39A Bs, a variant with a much lower binding affinity for Bn). Additionally, the researchers showed it could detect Bn-Bs binding in the presence of fetal bovine serum, suggesting that the sensor could be effective in complex biological samples.

Key to the method is the use of single polypeptide nanopores, Movileanu said, noting that this allows for a high level of control over the structure of the pore.

"With a single polypeptide, you have absolute atomic resolution [of the structure]," he said. "You can control it genetically. Once you know how it folds, it's easy."

He said that he has been working on the development of single-polypeptide nanopore sensors for roughly a decade. Over that time he has received $3.8 million in funding for the work from the National Institute of General Medical Sciences, including a four-year, $1.2 million grant announced in September.

While the Nature Biotechnology paper focused on detection of a small set of protein-protein interactions, Movileanu said he believed the technology has potentially broad applications given its apparent ability to detect proteins in complex samples.

He noted that he and his colleagues are currently working on a follow-up paper focused on testing the capabilities of the sensor in serum.

"We're working to show that this single polypeptide nanopore with a receptor tethered at the opening can work in the very hard conditions of biological fluids," he said. "I think that with some adaptation, you will be able to use this sensor in complex biofluids including cell lysates and tissue biopsies, which could let you, for instance, look at protein-protein interactions occurring under oncogenic conditions. You could develop biomarker assays where you could look at whether some proteins bind more strongly to [a given receptor] under oncogenic conditions.

Movileanu said he also envisions building nanopore arrays functionalized with different protein binding agents that could be used for large-scale proteomic analyses. He said that while antibodies are probably too large to use with his lab's nanopores, they are experimenting with other smaller affinity reagents like nanobodies, which consist of smaller antibody fragments.

This notion is similar to an approach published in 2012 by researchers at the Technical University of Munich, who used solid-state nanopores functionalized with various proteins to detect binding to those proteins.

Also similar was work published that same year in the Journal of the American Chemical Society by Oxford Nanopore researchers and company founder Hagan Bayley that used αHL protein nanopores linked to aptamers for protein detection. In experiments using thrombin-binding aptamers, the researchers wrote that they were able to quantify nanomolar concentrations of thrombin, suggesting that "aptamer-based nanopores have the potential to be integrated into arrays for the parallel detection of multiple analytes."

Since then, however, much of Bayley's nanopore protein detection work — and much of the work in the nanopore protein detection field more generally — has focused on approaches that involve translocating unfolded proteins through a nanopore and sequencing them in much the same way as is done with nucleic acids. Using such an approach, he and his colleagues have used nanopore sensors to distinguish between differentially phosphorylated protein forms.

Mark Akeson, a nanopore researcher and professor of biomolecular engineering at the University of California, Santa Cruz, has taken a similar approach, devising 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.

Researchers at the University of Groningen have 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.

These experiments have typically been done using simple mixtures of target analytes, however, which makes the ability of Movileanu's system to potentially detect proteins in complex samples interesting.

In addition to further testing the capabilities of the method in samples like serum, he said he and his collaborators are investigating whether they could use it to study enzymes like kinases.

"We're trying to tether [to the nanopore] enzymes like kinases so that we can see how they interact with their [substrates] and how those interactions are modulated by, for instance, protein co-factors," he said.

Movileanu said that he has filed patents on the technology but does not have any firm commercialization plans at the moment. He said that he would like to collaborate with an outside company on developing protein detection applications using the method, but added that he is still looking for "the right problem and the right company."