Ionic Current Blockades from DNA and RNA Molecules in the α-Hemolysin Nanopore

We characterize the substate structure of current blockades produced when single-stranded polynucleotide molecules were electrophoretically driven into the α-hemolysin protein pore. We frequently observe substates where the ionic current is reduced by ∼50%. Most of these substates can be associated with a molecular configuration where a polymer occupies only the vestibule region of the pore, though a few appear related to a polymer occupying only the transmembrane β-barrel region of the pore. The duration of the vestibule configuration depends on polymer composition and on which end of the polymer, 3′ or 5′, subsequently threads into the narrowest constriction and initiates translocation. Below ∼140 mV a polymer is more likely to escape from the vestibule against the applied voltage gradient, while at higher voltages a polymer is more likely to follow the voltage gradient by threading through the narrowest constriction and translocating through the pore. Increasing the applied voltage also increases the duration of the vestibule configuration. A semiquantitative model of these trends suggests that escape has stronger voltage dependence than threading, and that threading is sensitive to polymer orientation while escape is not. These results emphasize the utility of α-hemolysin as a model system to study biologically relevant physical and chemical processes at the single-molecule level.