title: Unzipping of A-Form DNA-RNA, A-Form DNA-PNA, and B-Form DNA-DNA in the α-Hemolysin Nanopore.
abstract: Unzipping of double-stranded nucleic acids by an electric field applied across a wild-type α-hemolysin (αHL) nanopore provides structural information about different duplex forms. In this work, comparative studies on A-form DNA-RNA duplexes and B-form DNA-DNA duplexes with a single-stranded tail identified significant differences in the blockage current and the unzipping duration between the two helical forms. We observed that the B-form duplex blocks the channel 1.9 ± 0.2 pA more and unzips ∼15-fold more slowly than an A-form duplex at 120 mV. We developed a model to describe the dependence of duplex unzipping on structure. We demonstrate that the wider A-form duplex (d = 2.4 nm) is unable to enter the vestibule opening of αHL on the cis side, leading to unzipping outside of the nanopore with higher residual current and faster unzipping times. In contrast, the smaller B-form duplexes (d = 2.0 nm) enter the vestibule of αHL, resulting in decreased current blockages and slower unzipping. We investigated the effects of varying the length of the single-stranded overhang, and studied A-form DNA-PNA duplexes to provide additional support for the proposed model. This study identifies key differences between A- and B-form duplex unzipping that will be important in the design of future probe-based methods for detecting DNA or RNA.
authors.affiliation: Department of Chemistry, University of Utah, Salt Lake City, Utah.
authors.forename: Rukshan T
authors.initials: RT
authors.lastname: Perera
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: Amberlyn M
authors.initials: AM
authors.lastname: Peterson
authors.affiliation: Department of Chemistry, University of Utah, Salt Lake City, Utah.
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Heemstra
authors.affiliation: Department of Chemistry, University of Utah, Salt Lake City, Utah. Electronic address: burrows@chem.utah.edu.
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* / Q000378:metabolism; D020029:Base Pairing; D001483:Base Sequence; D044704:DNA, A-Form / Q000737:chemistry* / Q000378:metabolism; D059371:DNA, B-Form / Q000737:chemistry* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D058608:Nanopores; D011485:Protein Binding; D012313:RNA / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D044704:DNA, A-Form; D059371:DNA, B-Form; D004277:DNA, Single-Stranded; D006460:Hemolysin Proteins; C021091:staphylococcal alpha-toxin; D012313:RNA