title: Tethered particle analysis of supercoiled circular DNA using peptide nucleic acid handles.
abstract: This protocol describes how to monitor individual naturally supercoiled circular DNA plasmids bound via peptide nucleic acid (PNA) handles between a bead and a surface. The protocol was developed for single-molecule investigation of the dynamics of supercoiled DNA, and it allows the investigation of both the dynamics of the molecule itself and of its interactions with a regulatory protein. Two bis-PNA clamps designed to bind with extremely high affinity to predetermined homopurine sequence sites in supercoiled DNA are prepared: one conjugated with digoxigenin for attachment to an anti-digoxigenin-coated glass cover slide, and one conjugated with biotin for attachment to a submicron-sized streptavidin-coated polystyrene bead. Plasmids are constructed, purified and incubated with the PNA handles. The dynamics of the construct is analyzed by tracking the tethered bead using video microscopy: less supercoiling results in more movement, and more supercoiling results in less movement. In contrast to other single-molecule methodologies, the current methodology allows for studying DNA in its naturally supercoiled state with constant linking number and constant writhe. The protocol has potential for use in studying the influence of supercoils on the dynamics of DNA and its associated proteins, e.g., topoisomerase. The procedure takes ~4 weeks.
authors.affiliation: The Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Kamilla
authors.initials: K
authors.lastname: Norregaard
authors.affiliation: 1] The Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark. [2].
authors.forename: Magnus
authors.initials: M
authors.lastname: Andersson
authors.affiliation: Department of Cellular and Molecular Medicine, Faculty of Health and Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Peter Eigil
authors.initials: PE
authors.lastname: Nielsen
authors.affiliation: The Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Stanley
authors.initials: S
authors.lastname: Brown
authors.affiliation: The Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Lene B
authors.initials: LB
authors.lastname: Oddershede
mesh_terms: D001710:Biotin / Q000378:metabolism; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004278:DNA, Superhelical / Q000737:chemistry*; D004076:Digoxigenin / Q000378:metabolism; D018715:Microscopy, Video; D008863:Microspheres; D020135:Peptide Nucleic Acids / Q000737:chemistry; D011137:Polystyrenes; D011485:Protein Binding
chemical_list: D004278:DNA, Superhelical; D020135:Peptide Nucleic Acids; D011137:Polystyrenes; D001710:Biotin; D004076:Digoxigenin