Aptamer-functionalized interface nanopores enable amino acid-specific single-molecule proteomics

Abstract

Abstract Single-molecule proteomics based on nanopore technology has made significant advances in recent years. However, to achieve nanopore sensing with single amino acid resolution, several bottlenecks must be tackled: controlling nanopore sizes with nanoscale precision and slowing molecular translocations events. Herein, we address these challenges by coupling amino acid-specific DNA aptamers to interface nanopores with dynamically tunable pores. A phenylalanine aptamer was used as a proof-of-concept; aptamer recognition of phenylalanine moieties led to retention of specific peptides, slowing translocation speeds. We decoupled specific binding between aptamers and peptides with phenylalanine moieties from nonspecific interactions (e.g., electrostatics) using optical waveguide lightmode spectroscopy. Aptamer-modified interface nanopores differentiated peptides containing phenylalanine vs. control peptides with structurally similar amino acids (i.e., tyrosine and tryptophan). When the duration of aptamer-target interactions inside the nanopore were prolonged by lowering the applied voltage, discrete ionic current levels with repetitive motifs were observed. Such reoccurring signatures in the measured signal suggest that the proposed method has the possibility to resolve amino acid-specific aptamer recognition, a step towards single-molecule proteomics.