title: High-throughput, high-force probing of DNA-protein interactions with magnetic tweezers.
abstract: 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 (>20pN) for prolonged periods of time (>1000s). 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 90pN 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 36pN optimizes the assay readout. The approaches detailed here provide guidelines for the high-throughput, quantitative study of a wide range of DNA-protein interactions.
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
authors.forename: Bojk A
authors.initials: BA
authors.lastname: Berghuis
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
authors.forename: Mariana
authors.initials: M
authors.lastname: Köber
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
authors.forename: Theo
authors.initials: T
authors.lastname: van Laar
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands. Electronic address: n.h.dekker@tudelft.nl.
authors.forename: Nynke H
authors.initials: NH
authors.lastname: Dekker
mesh_terms: D004247:DNA / Q000737:chemistry; D004265:DNA Helicases / Q000737:chemistry* / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D052898:Optical Tweezers; D013539:Simian virus 40 / Q000201:enzymology; D000072760:Single Molecule Imaging / Q000379:methods*
chemical_list: D004268:DNA-Binding Proteins; D004247:DNA; D004265:DNA Helicases
keywords: DNA construct design; High-throughput; Magnetic tweezers; SV40 large T antigen helicase; Tus-Ter