Nanopores offer highly sensitive, low-cost, and single-molecule sensing capabilities, and the societal impact of this approach is best captured by the advent of nanopore-based DNA detection and sequencing technologies, which extract genomic information without amplification. To address a critical difficulty plaguing such undertakings involving especially protein-based nanopores isolated in lipid bilayers, namely, the formation of a stable, long-lasting single nanopore, we pioneer herein an approach for generating functional nanostructures enabling small single-stranded DNA (ssDNA) detection. We designed a dynamic hybrid construct by appending extramembrane peptide nucleic acid (PNA) segments to the C-terminus of modified ion channel-forming alamethicin monomers. We found that the resulting chimeric molecules successfully coassemble in a voltage-dependent manner in planar lipid membranes generating diameter-variable oligomers. The subsequent interaction at the flexible extramembrane segment of such formed dynamic nanopores with aqueously added complementary ssDNA fragments leads to overall conformational alterations affecting the peptide assembly state kinetics and mediated ionic current. Such recognition events were found specific to the primary structure of target ssDNA and uninhibited the presence of serum. Our platform demonstrates the feasibility of designing an entirely new class of versatile chimeric biosensors, for which, dependent upon the nature of the attached receptor moiety and underlying recognition chemistry, the applicability area may extend to other analytes.
Keywords: alamethicin; biosensor; hybridization; peptide nucleic acid; ssDNA.