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

Abstract

Tethered Particle Motion (TPM) is a versatile in vitro technique for monitoring the conformations a linear macro-molecule, such as DNA, can exhibit. The technique involves monitoring the diffusive motion of a tracker particle anchored to a fixed point via the macro-molecule 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 understanding the timescale over which the diffusive motion of the tracker 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 differentiated. Applying those estimates to a lac repressor titration assay, we describe how to perform a TPM experiment. Additionally, 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 DNA looping J factor 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.

Polydopamine Encapsulation of Fluorescent Nanodiamonds for Biomedical Applications

“…The TPM method, in which the Brownian motion of a DNA-tethered bead is monitored by tracking the bead, can report biologically relevant conformational changes of individual DNA molecules. [25] Therefore, TPM has been frequently used to study the physical and mechanical properties of biopolymers such as nucleic acids and a variety of DNA-protein interactions including DNA looping and transcription. [26] Biotinylated FND@PDA was incubated with 90 nM excess of SA.…”

Protein-mediated looping of DNA under tension requires supercoiling

“…Microchambers were prepared similarly to what has been previously described ( 25 , 39 ). In brief, a microchamber with ∼30 μl volume was created between two glass coverslips (Fisherbrand, Thermo Fisher Scientific, Waltham, MA, USA) separated by a parafilm gasket with a narrow inlet and outlet to reduce evaporation of the reaction buffer, and a wide, central observation area ( 40 ). DNA tethers were attached through a digoxigenin at one end to the chamber bottom passivated with anti-digoxigenin (Roche Life Science, Indianapolis, IN, USA) and at the other end to either a 320-nm-diameter streptavidin-coated polystyrene bead (Spherotech, Lake Forest, IL, USA) for TPM experiments or a 1.0-μm–diameter streptavidin-coated paramagnetic bead (Dynabead MyOne Streptavidin T1, Invitrogen, Life Technologies, Grand Island, NY, USA) for magnetic tweezing.…”

Protein-mediated loops in supercoiled DNA create large topological domains

“…Chambers were prepared as previously reported ( 16 , 20 , 21 ). In brief, micro-chambers with an approximate volume of ∼30 μl were prepared by laser-cutting a gasket from parafilm and mildly heating to seal it between two coverslips (Fisherbrand, Thermo Fisher Scientific, Waltham, MA, USA).…”