Asymmetric Nanopore Sensing Enables Single-Molecule Identification of Nucleobases in Minimalist Peptide Nucleic Acids

Nanopore sensing offers ultrasensitive detection of biopolymers, yet their diversity and heterogeneity have hindered single-molecule discrimination. A key challenge in this respect lies in controlling the analyte capture under a defined driving force. Here, we implicated electrically neutral molecules represented by 6-mers of peptide nucleic acids and reveal that their current blockade signatures upon capture inside an α-hemolysin (α-HL) nanopore, facilitated by appended polyarginine tags, allow discrimination of corresponding nucleotide bases. We unveil a physical mechanism governing side-dependent capture, conformational dynamics, and translocation through α-HL. Surprisingly, entropy contributions─not enthalpy─seem to dominate the translocation barrier, with the polyarginine tag length playing a decisive role. More broadly, we demonstrate that a charged peptide tail transforms neutral biomolecules into nanopore-readable probes, encoding discriminatory signatures within their blockade patterns. This strategy opens new avenues for high-precision nanopore-based single-molecule analysis of otherwise undetectable targets.