title: Energetics of base flipping at a DNA mismatch site confined at the latch constriction of α-hemolysin.
abstract: 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 α-hemolysin (αHL) 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°. 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 αHL are used to derive thermodynamic (ΔH, ΔS) and kinetic (EA) 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 αHL 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 αHL nanopore is a useful tool for measuring conformational changes in dsDNA at the single molecule level.
authors.affiliation: Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT 84112-0850, USA. burrows@chem.utah.edu white@chem.utah.edu.
authors.forename: Robert P
authors.initials: RP
authors.lastname: Johnson
authors.affiliation: Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT 84112-0850, USA. burrows@chem.utah.edu white@chem.utah.edu.
authors.forename: Rukshan T
authors.initials: RT
authors.lastname: Perera
authors.affiliation: Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT 84112-0850, USA. burrows@chem.utah.edu white@chem.utah.edu.
authors.forename: Aaron M
authors.initials: AM
authors.lastname: Fleming
authors.affiliation: Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT 84112-0850, USA. burrows@chem.utah.edu white@chem.utah.edu.
authors.forename: Cynthia J
authors.initials: CJ
authors.lastname: Burrows
authors.affiliation: Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT 84112-0850, USA. burrows@chem.utah.edu white@chem.utah.edu.
authors.forename: Henry S
authors.initials: HS
authors.lastname: White
mesh_terms: D003596:Cytosine / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D006460:Hemolysin Proteins / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D013816:Thermodynamics
chemical_list: D006460:Hemolysin Proteins; D003596:Cytosine; D004247:DNA