title: Combining single-molecule manipulation and imaging for the study of protein-DNA interactions.
abstract: The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.
authors.affiliation: LENS - European Laboratory for Non-linear Spectroscopy, University of Florence; Chemistry Research Laboratory, University of Oxford.
authors.forename: Carina
authors.initials: C
authors.lastname: Monico
authors.affiliation: LENS - European Laboratory for Non-linear Spectroscopy, University of Florence.
authors.forename: Gionata
authors.initials: G
authors.lastname: Belcastro
authors.affiliation: LENS - European Laboratory for Non-linear Spectroscopy, University of Florence; Department of Biology, University of Florence.
authors.forename: Francesco
authors.initials: F
authors.lastname: Vanzi
authors.affiliation: LENS - European Laboratory for Non-linear Spectroscopy, University of Florence; Department of Physics and Astronomy, University of Florence; National Institute of Optics-National Research Council, Italy; International Center of Computational Neurophotonics.
authors.forename: Francesco S
authors.initials: FS
authors.lastname: Pavone
authors.affiliation: LENS - European Laboratory for Non-linear Spectroscopy, University of Florence; Department of Physics and Astronomy, University of Florence; capitan@lens.unifi.it.
authors.forename: Marco
authors.initials: M
authors.lastname: Capitanio
mesh_terms: D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D061925:Immobilized Nucleic Acids / Q000737:chemistry / Q000378:metabolism; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D056968:Lac Repressors / Q000737:chemistry / Q000378:metabolism; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods; D061848:Optical Imaging / Q000295:instrumentation / Q000379:methods; D052898:Optical Tweezers
chemical_list: D004268:DNA-Binding Proteins; D061925:Immobilized Nucleic Acids; D055767:Immobilized Proteins; D056968:Lac Repressors; D004247:DNA