文章基本信息

标题：Directly interrogating single quantum dot labelled UvrA2 molecules on DNA tightropes using an optically trapped nanoprobe
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作者：Michelle Simons ; Mark R. Pollard ; Craig D. Hughes 等
期刊名称：Scientific Reports
电子版ISSN：2045-2322
出版年度：2015
卷号：5
期号：1
DOI：10.1038/srep18486
语种：English
出版社：Springer Nature
摘要：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.