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Directly interrogating single quantum dot labelled UvrA2 molecules on DNA tightropes using an optically trapped nanoprobe

Simons, Michelle and Pollard, Mark R and Hughes, Craig D and Ward, Andrew D and Van Houten, Bennett and Towrie, Mike and Botchway, Stan W and Parker, Anthony W and Kad, Neil M (2015) 'Directly interrogating single quantum dot labelled UvrA2 molecules on DNA tightropes using an optically trapped nanoprobe.' Scientific Reports, 5 (1). 18486-. ISSN 2045-2322

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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.

Item Type: Article
Uncontrolled Keywords: DNA Repair Enzymes; DNA; Solutions; Staining and Labeling; Quantum Dots; Nanoparticles; Optical Tweezers
Subjects: Q Science > QH Natural history > QH426 Genetics
SWORD Depositor: Elements
Depositing User: Elements
Date Deposited: 15 Jan 2016 16:39
Last Modified: 18 Aug 2022 11:16

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