High-throughput, high-force probing of DNA-protein interactions with magnetic tweezers

High-throughput, high-force probing of DNA-protein interactions with magnetic tweezers

Berghuis, B.A. (TU Delft BN/Nynke Dekker Lab; Kavli institute of nanoscience Delft)
Köber, M. (TU Delft BN/Nynke Dekker Lab; Kavli institute of nanoscience Delft)
van Laar, T. (TU Delft BN/Nynke Dekker Lab; Kavli institute of nanoscience Delft)
Dekker, N.H. (TU Delft BN/Nynke Dekker Lab; Kavli institute of nanoscience Delft)

2016-08-01

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.

DNA construct design
High-throughput
Magnetic tweezers
SV40 large T antigen helicase
Tus-Ter

http://resolver.tudelft.nl/uuid:ebfe1d22-88f0-4445-bd55-5e23d2af9d7a

https://doi.org/10.1016/j.ymeth.2016.03.025

1046-2023

Methods, 105, 90-98

Institutional Repository

journal article

© 2016 B.A. Berghuis, M. Köber, T. van Laar, N.H. Dekker