Energetics of base flipping at a DNA mismatch site confined at the latch constriction of alpha-hemolysin

摘要

Unique, two-state modulating current signatures are observed when a cytosine-cytosine mismatch pair is confined at the 2.4 nm latch constriction of the alpha-hemolysin (alpha HL) nanopore. We have previously speculated that the modulation is due to base flipping at the mismatch site. Base flipping is a biologically significant mechanism in which a single base is rotated out of the DNA helical stack by 180 degrees. It is the mechanism by which enzymes are able to access bases for repair operations without disturbing the global structure of the helix. Here, temperature dependent ion channel recordings of individual double-stranded DNA duplexes inside alpha HL are used to derive thermodynamic (Delta H, Delta S) and kinetic (E-A) parameters for base flipping of a cytosine at an unstable cytosine-cytosine mismatch site. The measured activation energy for flipping a cytosine located at the latch of alpha HL out of the helix (18 +/- 1 kcal mol(-1)) is comparable to that previously reported for base flipping at mismatch sites from NMR measurements and potential mean force calculations. We propose that the aHL nanopore is a useful tool for measuring conformational changes in dsDNA at the single molecule level.