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

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