Biomolecular Detection with Engineered Robust Nanopores

Nanopores have become indispensable, powerful tools for bimolecular detections and molecular manipulations at single-molecule level, and they have been realized as an alternative to current analytical devices in biomedical molecular diagnosis. With a limited number of protein nanopores available, designing robust, functionally-intact protein scaffolds under various detection conditions is a daunting challenge in developing protein nanopore-based technologies. We used ferric hydroxamate uptake component A (FhuA), a bacterial β-barrel membrane protein as a template for protein engineering to produce an exceptionally-functional nanopore for sampling biomolecular events in harsh environments.FhuA has a luminal cross-section of ∼3.1×4.4 nm and is plugged by a globular N-terminal cork domain (C). Single-channel electrical recordings with extensive protein redesign of FhuA resulted in identifying four long extracellular loops (4L) that partially block the lumen upon the removal of the cork. The newly engineered protein, FhuA ΔC/Δ4L, was the result of a removal of almost 33% of the total number of amino acids of the wild-type FhuA (WT-FhuA) protein. The crown achievement in this work was combining direct genetic engineering with a refolding approach to produce this unusually-stable protein nanopore. Critical to its future nanotechnological applications, FhuA ΔC/Δ4L was functional under structure-altering conditions, including low ion concentration and highly acidic aqueous phase.To tailor the FhuAΔC/Δ4L protein to the use in nanopore-based detection devices, we show that the FhuA-based nanopores function as stochastic biosensing elements. For example, we monitored the proteolytic activity of an enzyme at highly acidic pH and we were able to determine the kinetics of protein-DNA aptamer interactions at physiological salt concentration. These two assays have not been demonstrated with the existing nanopores.This work is funded in part by grants from the US National Science Foundation (DMR-1006332, L.M.) and the National Institutes of Health (R01 GM088403, L.M.).