High-throughput, high-force probing of DNA-protein interactions with magnetic tweezers
详细信息 查看全文
- 作者：Bojk A. Berghuis ; Mariana Kö ; ber ; Theo van Laar ; Nynke H. Dekker ; n.h.dekker@tudelft.nl" class="auth_mail" title="E-mail the corresponding author
- 关键词：Magnetic tweezers ; High-throughput ; DNA construct design ; Tus-Ter ; SV40 large T antigen helicase
- 刊名：Methods
- 出版年：2016
- 出版时间：1 August 2016
- 年：2016
- 卷：105
- 期：Complete
- 页码：90-98
- 全文大小：1502 K
文摘
Recent advances in high-throughput single-molecule magnetic tweezers have paved the way for obtaining information on individual molecules as well as ensemble-averaged behavior in a single assay. Here we describe how to design robust high-throughput magnetic tweezers assays that specifically require application of high forces (>20 pN) for prolonged periods of time (>1000 s). We elaborate on the strengths and limitations of the typical construct types that can be used and provide a step-by-step guide towards a high tether yield assay based on two examples. Firstly, we discuss a DNA hairpin assay where force-induced strand separation triggers a tight interaction between DNA-binding protein Tus and its binding site Ter, where forces up to 90 pN for hundreds of seconds were required to dissociate Tus from Ter. Secondly, we show how the LTag helicase of Simian virus 40 unwinds dsDNA, where a load of 36 pN optimizes the assay readout. The approaches detailed here provide guidelines for the high-throughput, quantitative study of a wide range of DNA-protein interactions.