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Tethered Particle Motion: An Easy Technique for Probing DNA Topology and Interactions with Transcription Factors

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Single Molecule Analysis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1665))

Abstract

Tethered Particle Motion (TPM) is a versatile in vitro technique for monitoring the conformations a linear macromolecule, such as DNA, can exhibit. The technique involves monitoring the diffusive motion of a particle anchored to a fixed point via the macromolecule of interest, which acts as a tether. In this chapter, we provide an overview of TPM, review the fundamental principles that determine the accuracy with which effective tether lengths can be used to distinguish different tether conformations, present software tools that assist in capturing and analyzing TPM data, and provide a protocol which uses TPM to characterize lac repressor-induced DNA looping. Critical to any TPM assay is the understanding of the timescale over which the diffusive motion of the particle must be observed to accurately distinguish tether conformations. Approximating the tether as a Hookean spring, we show how to estimate the diffusion timescale and discuss how it relates to the confidence with which tether conformations can be distinguished. Applying those estimates to a lac repressor titration assay, we describe how to perform a TPM experiment. We also provide graphically driven software which can be used to speed up data collection and analysis. Lastly, we detail how TPM data from the titration assay can be used to calculate relevant molecular descriptors such as the J factor for DNA looping and lac repressor–operator dissociation constants. While the included protocol is geared toward studying DNA looping, the technique, fundamental principles, and analytical methods are more general and can be adapted to a wide variety of molecular systems.

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Acknowledgements

We would like to thank Kathleen Matthews for graciously providing us with the lac repressor used in this work. We also acknowledge former Finzi Lab members Chiara Zurla, Carlo Manzo, Suleyman Ucuncuoglu, and Sandip Kumar who forged earlier versions of our TPM protocols. This work was supported by the NIH, Grant: R01 GM084070.

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Authors and Affiliations

  1. Department of Physics, Emory University, 400 Dowman Dr, Atlanta, GA, 30322, USA

    Daniel T. Kovari, Yan Yan, Laura Finzi & David Dunlap

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  1. Daniel T. Kovari
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  2. Yan Yan
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  3. Laura Finzi
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  4. David Dunlap
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Correspondence to David Dunlap .

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  1. Department of Physics & Astronomy, Vrije Universiteit, Amsterdam, The Netherlands

    Erwin J. G. Peterman

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Kovari, D.T., Yan, Y., Finzi, L., Dunlap, D. (2018). Tethered Particle Motion: An Easy Technique for Probing DNA Topology and Interactions with Transcription Factors. In: Peterman, E. (eds) Single Molecule Analysis. Methods in Molecular Biology, vol 1665. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7271-5_17

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  • DOI: https://doi.org/10.1007/978-1-4939-7271-5_17

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7270-8

  • Online ISBN: 978-1-4939-7271-5

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