title: Directly interrogating single quantum dot labelled UvrA2 molecules on DNA tightropes using an optically trapped nanoprobe.
abstract: In this study we describe a new methodology to physically probe individual complexes formed between proteins and DNA. By combining nanoscale, high speed physical force measurement with sensitive fluorescence imaging we investigate the complex formed between the prokaryotic DNA repair protein UvrA2 and DNA. This approach uses a triangular, optically-trapped "nanoprobe" with a nanometer scale tip protruding from one vertex. By scanning this tip along a single DNA strand suspended between surface-bound micron-scale beads, quantum-dot tagged UvrA2 molecules bound to these '"DNA tightropes" can be mechanically interrogated. Encounters with UvrA2 led to deflections of the whole nanoprobe structure, which were converted to resistive force. A force histogram from all 144 detected interactions generated a bimodal distribution centered on 2.6 and 8.1 pN, possibly reflecting the asymmetry of UvrA2's binding to DNA. These observations successfully demonstrate the use of a highly controllable purpose-designed and built synthetic nanoprobe combined with fluorescence imaging to study protein-DNA interactions at the single molecule level.
authors.affiliation: School of Biological Sciences, University of Essex, Essex, CO4 3SQ, UK.
authors.forename: Michelle
authors.initials: M
authors.lastname: Simons
authors.affiliation: Central Laser Facility, Research Complex at Harwell, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Didcot, OX11 0FA, UK.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Pollard
authors.affiliation: School of Biological Sciences, University of Essex, Essex, CO4 3SQ, UK.
authors.forename: Craig D
authors.initials: CD
authors.lastname: Hughes
authors.affiliation: Central Laser Facility, Research Complex at Harwell, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Didcot, OX11 0FA, UK.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Ward
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh Cancer Institute, Hillman Cancer Research Pavilion, 5117 Centre Ave, Pittsburgh, PA, USA 15213.
authors.forename: Bennett
authors.initials: B
authors.lastname: Van Houten
authors.affiliation: Central Laser Facility, Research Complex at Harwell, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Didcot, OX11 0FA, UK.
authors.forename: Mike
authors.initials: M
authors.lastname: Towrie
authors.affiliation: Central Laser Facility, Research Complex at Harwell, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Didcot, OX11 0FA, UK.
authors.forename: Stan W
authors.initials: SW
authors.lastname: Botchway
authors.affiliation: Central Laser Facility, Research Complex at Harwell, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Didcot, OX11 0FA, UK.
authors.forename: Anthony W
authors.initials: AW
authors.lastname: Parker
authors.affiliation: School of Biosciences, University of Kent, Canterbury, Kent, CT2 7NH, UK.
authors.forename: Neil M
authors.initials: NM
authors.lastname: Kad
mesh_terms: D004247:DNA / Q000378:metabolism*; D045643:DNA Repair Enzymes / Q000378:metabolism*; D053758:Nanoparticles / Q000737:chemistry*; D052898:Optical Tweezers; D045663:Quantum Dots / Q000378:metabolism*; D012996:Solutions; D013194:Staining and Labeling
chemical_list: D012996:Solutions; D004247:DNA; D045643:DNA Repair Enzymes