Ren, Hang Cheyne, Cameron G. Fleming, Aaron M. Burrows, Cynthia J. White, Henry S. Single-Molecule Titration in a Protein Nanoreactor Reveals the Protonation/Deprotonation Mechanism of a C:C Mismatch in DNA Measurement of single-molecule reactions can elucidate microscopic mechanisms that are often hidden from ensemble analysis. Herein, we report the acid–base titration of a single DNA duplex confined within the wild-type α-hemolysin (α-HL) nanopore for up to 3 h, while monitoring the ionic current through the nanopore. Modulation between two states in the current–time trace for duplexes containing the C:C mismatch in proximity to the latch constriction of α-HL is attributed to the base flipping of the C:C mismatch. As the pH is lowered, the rate for the C:C mismatch to flip from the intra-helical state to the extra-helical state (<i>k</i><sub>intra-extra</sub>) decreases, while the rate for base flipping from the extra-helical state to the intra-helical state (<i>k</i><sub>extra‑intra</sub>) remains unchanged. Both <i>k</i><sub>intra-extra</sub> and <i>k</i><sub>extra‑intra</sub> are on the order of 1 × 10<sup>–2</sup> s<sup>–1</sup> to 1 × 10<sup>–1</sup> s<sup>–1</sup> and remain stable over the time scale of the measurement (several hours). Analysis of the pH-dependent kinetics of base flipping using a hidden Markov kinetic model demonstrates that protonation/deprotonation occurs while the base pair is in the intra-helical state. We also demonstrate that the rate of protonation is limited by transport of H<sup>+</sup> into the α-HL nanopore. Single-molecule kinetic isotope experiments exhibit a large kinetic isotope effect (KIE) for <i>k</i><sub>intra-extra</sub> (<i>k</i><sub>H</sub>/<i>k</i><sub>D</sub> ≈ 5) but a limited KIE for <i>k</i><sub>extra‑intra</sub> (<i>k</i><sub>H</sub>/<i>k</i><sub>D</sub> ≈ 1.3), supporting our model. Our experiments correspond to the longest single-molecule measurements performed using a nanopore, and demonstrate its application in interrogating mechanisms of single-molecule reactions in confined geometries. α- HL nanopore;k intra-extra;KIE;extra-helical state;DNA;mismatch;intra-helical state;isotope experiments exhibit;single-molecule reactions 2018-03-21
    https://acs.figshare.com/articles/journal_contribution/Single-Molecule_Titration_in_a_Protein_Nanoreactor_Reveals_the_Protonation_Deprotonation_Mechanism_of_a_C_C_Mismatch_in_DNA/6083069
10.1021/jacs.8b00593.s001