De novo design of β-barrel nanopore with Gly-kink towards single-molecule detection.

Abstract

Nanopore sensing is a rapid and label-free method for single-molecule detection of DNA, peptides, and protein. However, it remains challenging to adjust the size and geometry of nanopores for each target molecule using biological nanopores. Therefore, constructing size and geometry controllable nanopore is highly demanded. Recently, de novo design has enabled the creation of specific structures and functions of proteins. We have previously designed a de novo β-hairpin peptide named SV28, which is assembled to form a multidisperse-sized β-barrel nanopore in a bilayer lipid membrane (BLM). A monodisperse-size nanopore is required for the highly sensitive detection of a specific target. Toward constructing a monodisperse-sized nanopore, we focused on Gly-kink. In the computational protein design, introducing the Gly-kink in a β-barrel structure makes the packing of the β-strands relaxed and the β-barrel structure to be more rigid. Then, we redesigned SVG28 with the introduction of Gly-kink into SV28. To evaluate the pore-forming ability of SVG28, we conducted channel current measurement with BLM in microdevices and signal classification analysis. As result, the SVG28 nanopore converged relatively monodisperse size (SVG28: φ = 1.7 nm, SV28: φ = 1.7-6.3 nm). Moreover, we carried out molecular dynamics simulations of peptide nanopores, indicating the stabilization of the β-barrel structure by introducing the Gly-kink. For future nanopore amino acid sequencing, we applied SVG28 nanopore to polypeptide detection. The SVG28 nanopore could detect and discriminate molecular weight difference of poly-l-lysine (PLL: φ = 1.1-1.4 nm). Furthermore, the SVG28 nanopore could identify the single amino acid in the oligo-arginine with several different types of single amino acids (R7X: φ = 1.0-1.5 nm). The above achievements are due to size and geometry matching between the SVG28 nanopore and the targets. Now, we are redesigning SVG28 to construct nanopores that have specific large pore sizes for protein detection.