title: Temperature and electrolyte optimization of the α-hemolysin latch sensing zone for detection of base modification in double-stranded DNA.
abstract: The latch region of the wild-type protein pore α-hemolysin (α-HL) constitutes a sensing zone for individual abasic sites (and furan analogs) in double-stranded DNA (dsDNA). The presence of an abasic site or furan within a DNA duplex, electrophoretically captured in the α-HL vestibule and positioned at the latch region, can be detected based on the current blockage prior to duplex unzipping. We investigated variations in blockage current as a function of temperature (12-35°C) and KCl concentration (0.15-1.0 M) to understand the origin of the current signature and to optimize conditions for identifying the base modification. In 1 M KCl solution, substitution of a furan for a cytosine base in the latch region results in an ∼ 8 kJ mol(-1) decrease in the activation energy for ion transport through the protein pore. This corresponds to a readily measured ∼ 2 pA increase in current at room temperature. Optimal resolution for detecting the presence of a furan in the latch region is achieved at lower KCl concentrations, where the noise in the measured blockage current is significantly lower. The noise associated with the blockage current also depends on the stability of the duplex (as measured from the melting temperature), where a greater noise in the measured blockage current is observed for less stable duplexes.
authors.affiliation: Department of Chemistry, University of Utah, Salt Lake City, Utah.
authors.forename: Robert P
authors.initials: RP
authors.lastname: Johnson
authors.affiliation: Department of Chemistry, University of Utah, Salt Lake City, Utah.
authors.forename: Aaron M
authors.initials: AM
authors.lastname: Fleming
authors.affiliation: Department of Chemistry, University of Utah, Salt Lake City, Utah.
authors.forename: Qian
authors.initials: Q
authors.lastname: Jin
authors.affiliation: Department of Chemistry, University of Utah, Salt Lake City, Utah.
authors.forename: Cynthia J
authors.initials: CJ
authors.lastname: Burrows
authors.affiliation: Department of Chemistry, University of Utah, Salt Lake City, Utah. Electronic address: white@chem.utah.edu.
authors.forename: Henry S
authors.initials: HS
authors.lastname: White
mesh_terms: D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry*; D003596:Cytosine / Q000737:chemistry; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D004573:Electrolytes / Q000737:chemistry*; D006460:Hemolysin Proteins / Q000737:chemistry*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011189:Potassium Chloride / Q000737:chemistry*; D013696:Temperature; D044366:Transition Temperature; D018631:ras Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D001427:Bacterial Toxins; D004573:Electrolytes; D006460:Hemolysin Proteins; C021091:staphylococcal alpha-toxin; D011189:Potassium Chloride; D003596:Cytosine; D004247:DNA; D018631:ras Proteins