Calibration of Tethered Particle Motion Experiments

TL;DR

This paper provides a detailed calibration of Tethered Particle Motion (TPM) experiments, analyzing how DNA length, particle size, and experimental parameters influence the measurement, thereby enabling more accurate interpretation of TPM data.

Contribution

It offers a systematic calibration of TPM measurements against DNA length and particle size, improving quantitative analysis of protein-DNA interactions.

Findings

TPM motion depends systematically on DNA length and particle size.

Experimental parameters like acquisition and exposure time significantly affect measured motion.

Comparison with theory clarifies DNA conformational models.

Abstract

The Tethered Particle Motion (TPM) method has been used to observe and characterize a variety of protein-DNA interactions including DNA looping and transcription. TPM experiments exploit the Brownian motion of a DNA-tethered bead to probe biologically relevant conformational changes of the tether. In these experiments, a change in the extent of the bead's random motion is used as a reporter of the underlying macromolecular dynamics and is often deemed sufficient for TPM analysis. However, a complete understanding of how the motion depends on the physical properties of the tethered particle complex would permit more quantitative and accurate evaluation of TPM data. For instance, such understanding can help extract details about a looped complex geometry (or multiple coexisting geometries) from TPM data. To better characterize the measurement capabilities of TPM experiments involving DNA tethers, we have carried out a detailed calibration of TPM magnitude as a function of DNA length and particle size. We also explore how experimental parameters such as acquisition time and exposure time affect the apparent motion of the tethered particle. We vary the DNA length from 200bp to 2.6kbp and consider particle diameters of 200, 490 and 970nm. We also present a systematic comparison between measured particle excursions and theoretical expectations, which helps clarify both the experiments and models of DNA conformation.