?-Hemolysin Nanopore Is Sensitive to Guanine-to-Inosine Substitutions in Double-Stranded DNA at the Single-Molecule Level.

ABSTRACT: Biological nanopores provide a unique single-molecule sensing platform to detect target molecules based on their specific electrical signatures. The ?-hemolysin (?-HL) protein produced by Staphylococcus aureus is able to assemble into an octamer nanopore with a ?2.3 nm diameter ?-barrel. Herein, we demonstrate the first application of ?-HL nanopore for DNA structural analysis. To optimize conditions for ion-channel recording, the properties of the ?-HL pore (e.g., conductance, voltage-dependent gating, and ion-selectivity) were characterized at different pH, temperature, and electrolyte concentrations. The optimal condition for DNA analysis using ?-HL corresponds to 3 M KCl, pH 5, and T = 20 °C. The ?-HL protein nanopore is able to translocate dsDNA at about ?20 bp/ms, and the unique current-signature of captured dsDNA can directly distinguish guanine-to-inosine substitutions at the single-molecule level with ?99% accuracy. The slow dsDNA threading and translocation processes indicate this wild-type ?-HL channel has potential to detect other base modifications in dsDNA.

SUBMITTER: Tan CS 

PROVIDER: S-EPMC6242276 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature