Scientists in Japan say they have designed the first bottom-up peptides that can form artificial nanopores to identify and enable single molecule-sorting of genetic material in a lipid membrane. They believe their achievement could be applied to facilitating the understanding of the relationship between protein structure and function.
Biological nanopores are generally channels made by pore-forming proteins, that can detect specific molecules, but such natural channels are difficult to identify, limiting proposed applications in low-cost, speedy DNA sequencing, small molecule detection and more, according to the research team, which published its study (“De novo design of a nanopore for single-molecule detection that incorporates a β-hairpin peptide”) in Nature Nanotechnology.
“Nanopore sensing is a powerful tool for label-free, single-molecule detection,” said corresponding author Ryuji Kawano, PhD, professor in Tokyo University of Agriculture and Technology (TUAT) in Japan. “This is the first time that DNA and polypeptides were sensed using a de novo–designed nanopore.”
“The amino-acid sequence of a protein encodes information on its three-dimensional structure and specific functionality. De novo design has emerged as a method to manipulate the primary structure for the development of artificial proteins and peptides with desired functionality,” write the investigators.
“This paper describes the de novo design of a pore-forming peptide, named SV28, that has a β-hairpin structure and assembles to form a stable nanopore in a bilayer lipid membrane. This large synthetic nanopore is an entirely artificial device for practical applications. The peptide forms multidispersely sized nanopore structures ranging from 1.7 to 6.3 nm in diameter and can detect DNAs.
“To form a monodispersely sized nanopore, we redesigned the SV28 by introducing a glycine-kink mutation. The resulting redesigned peptide forms a monodisperse pore with a diameter of 1.7 nm leading to detection of a single polypeptide chain. Such de novo design of a β-hairpin peptide has the potential to create artificial nanopores, which can be size adjusted to a target molecule.”
Built from scratch
The de novo-designed nanopores are built “from scratch,” noted Kawano, and have the potential to mimic natural proteins and their ability to detect specific proteins. Crucially, Kawano said, they can also be engineered to act as artificial molecular machines capable of detecting a much wider range of molecules—which may help elucidate the connection between structure and function in target proteins.
“The folded structure of proteins is determined by their linear polypeptide sequence and gives rise to specific protein functionality,” Kawano said, noting that all proteins have a unique structure and size. “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.”
To develop large synthetic nanopores that can better detect and identify molecules for practical applications, Kawano and the team designed a peptide dubbed SV28. With two arms of amino acids bent at a sharp angle, and specific charges at the terminus, the orientation of the hairpin-shaped peptide can be precisely controlled by applying a voltage.
The peptide can assemble to form nanopore structures ranging in size from 1.7 to 6.3 nanometers, suitable for detecting molecules of DNA.
The researchers also modified SV28 by adding a mutation that causes the peptide structure to bend and twist in specific ways. The resulting peptide formed evenly dispersed pores of 1.7 nanometers each, capable of detecting a single polypeptide chain—or one half of a protein.
For the next steps, the team plans to design various peptides and proteins to construct different types of nanopores to aid in peptide sequencing and operate as molecular robots, for example.
