Single -molecule DNA dynamics revealed by nanopore analysis

Using a protein nanopore detector, end-fraying of a captured DNA molecule is detected as high-frequency, near-full current deflections from a low-frequency conductance state. These spike transients relate to the stability of duplex DNA terminal base pairs. The alpha-hemolysin nanopore was used to examine end-fraying in model DNA substrates for HIV integrase, an enzyme that processes end-fraying target sequences more efficiently than stabilized target ends.;A typical 9 base pair hairpin captured in the alpha-hemolysin nanopore displays characteristic low frequency toggling between three discreet conductance states. An alternating G•C/C•G sequence favors interconversion from B DNA to Z DNA under conditions of high ionic strength or high negative torsion. Hairpins with an alternating G•C/C•G stem exhibit additional, embedded conductance levels that can be prolonged by increasing the degree of substitution with m5dC---a known stabilizer of Z DNA.;A comparison of the 4 possible homotrimer (3dN) overhangs on a standard 9 base pain hairpin reveals that 3dT and 3dG overhangs can be distinguished from each other and from 3dA and 3dC overhangs by their spike amplitudes. Due to their similar spike amplitudes, 3dA and 3dC overhangs are indistinguishable.