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DNA and Proteins Can Be Sensed by This De Novo-Designed Nanopore

This new bottom-up design strategy could be integrated into the creation of molecular machines and robots, potentially advancing nanopore technology.

Biological nanopores are generally created by pore-forming proteins that function to detect certain molecules. These channels have been widely explored for potential use in low-cost, speedy DNA sequencing, nanopore mass spectroscopy, and the detection of peptides, proteins, and various small molecules. However, because these nanopores are difficult to identify, their applications have been limited. Moreover, there is a limited range of applicable target molecules since the selectivity of nanopore sensing is highly dependent on the chemical properties, pore size, and variations in the size of natural pore-forming proteins.

As a solution to these problems, researchers in Japan have recently developed a novel bottom-up strategy to synthesise peptides that can form artificial nanopores to identify and enable single molecule-sorting of genetic material in a lipid membrane. Unlike naturally-occurring nanopores, artificially designed nanopores could be tailored to accommodate a range of target molecule sizes and accurately detect their targets.

“Nanopore sensing is a powerful tool for label-free, single-molecule detection,” said corresponding author Ryuji Kawano, professor at Tokyo University of Agriculture and Technology. “This is the first time that DNA and polypeptides were sensed using a de novo-designed nanopore.”

Built “from scratch,” Kawano explained that their de novo-designed nanopores can potentially mimic natural proteins and their ability to detect specific proteins. Kawano also noted that they can be engineered to act as artificial molecular machines to detect a wide range of molecules, which would be useful to clarify how protein structure determines function.

“The folded structure of proteins is determined by their linear polypeptide sequence and gives rise to specific protein functionality,” Kawano noted. “The unique primary structure is the result of structural evolution such as the mutation and selection of amino acid residues over time. To reveal the relationship between this primary information and protein structure is one of the ultimate goals of science.”

In designing their artificial protein nanopore, the researchers first had to consider the process of pore insertion into the lipid membrane. In natural systems, membrane proteins are inserted into the cell membrane with the help of chaperones or via endoplasmic reticulum export. Another way is to use short peptides, such as the 35-amino-acid-long, α-helical barrelled peptide.

To create molecule-detecting nanopores that are suited for practical applications, Kawano and colleagues designed a peptide with a β-hairpin structure which they called SV28. They specifically chose to focus on β-barrel peptides because the transmembrane region of most biological nanopores has a β-barrel structure. The newly designed peptide has two arms of amino acids bent at a sharp angle and specific charges at the terminus.

By applying a voltage, the orientation of the hairpin-shaped peptide can be precisely controlled. SV28 can also be assembled to create nanopore structures of several different sizes, ranging between 1.7 to 6.3 nanometres in diameter, which are suitable for detecting DNA molecules. The researchers subsequently mutated SV28 to bend and twist it in specific ways, the result of which formed evenly dispersed pores that are 1.7 nanometres in diameter each and can detect a single polypeptide chain.

By addressing some current hurdles faced in nanopore technology, the study has expounded the applications of nanopore sensing of DNA secondary structures and polypeptides. In future, the team intends to design many other peptides and proteins to construct different types of nanopores to advance peptide sequencing and the creation of molecular machines. [APBN]

Source: Shimizu et al. (2021). De novo design of a nanopore for single-molecule detection that incorporates a β-hairpin peptide. Nature Nanotechnology.