title: Characterization of Interstrand DNA-DNA Cross-Links Using the α-Hemolysin Protein Nanopore.
abstract: Nanopore-based sensors have been studied extensively as potential tools for DNA sequencing, characterization of epigenetic modifications such as 5-methylcytosine, and detection of microRNA biomarkers. In the studies described here, the α-hemolysin protein nanopore embedded in a lipid bilayer was used for the detection and characterization of interstrand cross-links in duplex DNA. Interstrand cross-links are important lesions in medicinal chemistry and toxicology because they prevent the strand separation that is required for read-out of genetic information from DNA in cells. In addition, interstrand cross-links are used for the stabilization of duplex DNA in structural biology and materials science. Cross-linked DNA fragments produced unmistakable current signatures in the nanopore experiment. Some cross-linked substrates gave irreversible current blocks of >10 min, while others produced long current blocks (10-100 s) before the double-stranded DNA cross-link translocated through the α-hemolysin channel in a voltage-driven manner. The duration of the current block for the different cross-linked substrates examined here may be dictated by the stability of the duplex region left in the vestibule of the nanopore following partial unzipping of the cross-linked DNA. Construction of calibration curves measuring the frequency of cross-link blocking events (1/τon) as a function of cross-link concentration enabled quantitative determination of the amounts of cross-linked DNA present in samples. The unique current signatures generated by cross-linked DNA in the α-HL nanopore may enable the detection and characterization of DNA cross-links that are important in toxicology, medicine, and materials science.
authors.affiliation: Department of Bioengineering and Dalton Cardiovascular Research Center, University of Missouri , Columbia, Missouri 65211, United States.
authors.forename: Xinyue
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Chemistry, University of Missouri , 125 Chemistry Building, Columbia, Missouri 65211, United States.
authors.forename: Nathan E
authors.initials: NE
authors.lastname: Price
authors.affiliation: Department of Bioengineering and Dalton Cardiovascular Research Center, University of Missouri , Columbia, Missouri 65211, United States.
authors.forename: Xi
authors.initials: X
authors.lastname: Fang
authors.affiliation: Department of Chemistry, University of Missouri , 125 Chemistry Building, Columbia, Missouri 65211, United States.
authors.forename: Zhiyu
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Department of Bioengineering and Dalton Cardiovascular Research Center, University of Missouri , Columbia, Missouri 65211, United States.
authors.forename: Li-Qun
authors.initials: LQ
authors.lastname: Gu
authors.affiliation: Department of Chemistry, University of Missouri , 125 Chemistry Building, Columbia, Missouri 65211, United States.|Department of Biochemistry, University of Missouri , Columbia, Missouri 65211, United States.
authors.forename: Kent S
authors.initials: KS
authors.lastname: Gates
mesh_terms: D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004249:DNA Damage; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000378:metabolism*; D058608:Nanopores; D009690:Nucleic Acid Conformation
chemical_list: D003432:Cross-Linking Reagents; D006460:Hemolysin Proteins; D004247:DNA
keywords: abasic site; damaged DNA; interstrand cross-link; nanopore; α-hemolysin