Single-Molecule Sensing inside a Stereo- and Regiodefined Hetero-Nanopore

Channel forming proteins often contain recognition pattern or stereo specific selection filter. This ability of the channel confinement inspired us for application towards single molecule sensing. To bypass possible difficulties in purification, misfolding and possible channel gating of biological nanopores, here we show an In situ strategy via single molecule chemical modification of a heptameric protein to build a stereo and regiospecific heteromeric nanopore (hetero-nanopore) with a subunit stoichometric ratio of 3:4. Single-molecule experiments and all-atom molecule dynamic (MD) simulations reveal that the produced hetero-nanopore is indeed featured with an asymmetric stereo- and regio defined organization. In a proof-of-concept single-molecule experiment, the hetero-nanopore exhibited nearly 100% accuracy for label-free discrimination of four peptide stereoisomers with the structural and chiral differences of single amino acid in the mixture. We anticipate to customize multiple hetero-nanopores for single-molecule sensing.