Construction of nanopores using β-hairpin peptides synthesized by a cell-free system.

Abstract

Biological nanopore is a sensor of single-molecule detection, and its reading resolution should be improved in terms of the congeniality between the target molecules and nanopore. Recently, de novo design, which designs the intended structure by artificially designed sequences, has been also applied to the design of nanopores to complement their structural variety. We previously reported a de novo designed nanopore with a peptide, SVG28, which assembles to form a stable β-barrel structure in a bilayer lipid membrane. A comprehensive search of SVG28 sequences like molecular evolution engineering may enable further optimization such as target-specificity and target capturing frequency. Such a comprehensive screening method requires cell-free synthesis which is a method to synthesize peptides/proteins parallelly. In this study, we tried to express the strongly hydrophobic SVG28 using the PURE system. Since SVG28 has too hydrophobic membrane-embedding sequences to synthesize with the cell-free system, internal hydrophilic mutations were introduced. Hydrophilic mutations incorporated into SVG28 enabled the cell-free synthesis, and the appropriate expression was confirmed using MALDI-TOF/MS and CD spectrum. Pore-opening states from the nanopore measurement showed retained pore-forming ability, and the number of monomers of hydrophilic mutants converged to be 5- and 6-mer with a diameter of approx. 1.1 and 1.5 nm, compared to the SVG28 5∼7-mer structure of 1.1∼1.7 nm. Destabilization of peptides due to hydrophilic mutations may have reduced the construction of large pore structures. To test the detection capability of the hydrophilic variant as the nanopores, we attempted single-molecule detection of oligopeptide RRRRRRRG (R7G). The blocking current signals suggesting the R7G translocation were observed with larger pores which were not the main configuration and were rarely observed, suggesting a pore structure passing molecules and low passage probability due to the reduced pore diameter.