Measurement of single molecule reactions can elucidate microscopic mechanisms that are often hidden from ensemble analysis. We describe the acid-base titration of a single DNA duplex confined within the α-hemolysin (α-HL) nanopore for up to 4 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 decreases, while the rate for base flipping from the extra-helical state to the intra-helical state remains unchanged. 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+ into the α-HL nanopore. 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.











Henry White (University of Utah)