Rapid nanopore discrimination between single polynucleotide molecules

TLDR

Nanopores can rapidly discriminate unlabeled DNA molecules at low copy number, offering a potential low‑cost, high‑throughput method for analyzing DNA polynucleotides. DNA polymers were electrophoretically driven through an α‑hemolysin nanopore in a lipid bilayer. Single‑channel recordings revealed distinct current‑duration patterns for each polymer, allowing discrimination of sequences with similar length and composition, and showed that translocation time scales as ∼T⁻², with a strong correlation between temperature dependence and secondary‑structure propensity.

Abstract

A variety of different DNA polymers were electrophoretically driven through the nanopore of an α-hemolysin channel in a lipid bilayer. Single-channel recording of the translocation duration and current flow during traversal of individual polynucleotides yielded a unique pattern of events for each of the several polymers tested. Statistical data derived from this pattern of events demonstrate that in several cases a nanopore can distinguish between polynucleotides of similar length and composition that differ only in sequence. Studies of temperature effects on the translocation process show that translocation duration scales as ∼ T −2 . A strong correlation exists between the temperature dependence of the event characteristics and the tendency of some polymers to form secondary structure. Because nanopores can rapidly discriminate and characterize unlabeled DNA molecules at low copy number, refinements of the experimental approach demonstrated here could eventually provide a low-cost high-throughput method of analyzing DNA polynucleotides.