Calibration of Tethered Particle Motion Experiments

Abstract

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 magnitudeas 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 200 bp to 2.6 kbp and consider particle diameters of 200, 490 and 970 nm. We also present a systematic comparison between measured particle excursions and theoretical expectations, which helps clarify both the experiments and models of DNA conformation. 1. Introduction. Single molecule studies are enriching our understanding of biological processes by providing a unique window on the microtrajectories of individual molecules rather than their ensemble-averaged behavior. Many of these studies are devoted to exploring the intricacies of protein-DNA interactions that are central to gene regulation, DNA replication and DNA repair. The resolution of nanometer-scale distances involved in such interactions poses a significant challenge. The emergence of the tethered particle motion (TPM) method offers a practical and relatively simple solution. In this method, a biopolymer is tethered between a stationary substrate and a micrometer-scale sphere (a "bead"), which is large enough to be imaged with conventional optical microscopy (Fig. 1). The constrained Brownian motion of the bead serves as a reporter of the underlying macromolecular dynamics, either by observing its blurred image in a long exposure [5], or by tracking its actual trajectory in time (e.g. as done in [11] and the present work). Changes in the extent of the motion (which we will call "excursion") reflect conformational transformations of the tethered molecule. Such changes may be caused by processive walking of RNA polymerase [12,23], DNA looping [5,17,24,25,22,19 Although TPM is simple in principle, there are a variety of technical challenges that must be addressed for successful implementation. For example, sample preparation can be compromised by multiply-tethered beads, non-specific adsorption, transient sticking events and dissociation of the tether joints [11,19,17,3,9]. In additio...

Bead size effects on protein‐mediated DNA looping in tethered‐particle motion experiments

“…At this frame rate the influence of time averaging by the detector is negligible. 19 The particles are tracked with custom MATLAB software that implements a Gaussian mesh algorithm to locate the center of each nanoparticle. 20 With this algorithm we are able to determine the beads center with an error of less than 5 nm.…”

First-principles calculation of DNA looping in tethered particle experiments

“…Recent work on looping via TPM includes [16–18, 26, 45–47]. TPM experiments do require significant analysis to determine looping state from bead motion, but techniques such as dead-time correction [16] and hidden Markov modeling [17, 18] now exist to handle this.…”

Multiplexed, Tethered Particle Microscopy for Studies of DNA-Enzyme Dynamics

“…The frame rate with 1-ms exposure value was sufficient to observe the beads without blurring due to the fast bead motion (Han et al, 2009; Wong & Halvorsen, 2006). In our tests, video was streamed at 125 MB/s through a Cat 6e network cable between the camera and an Intel Pro 1000 series network adaptor interfaced to a NI-IMAQ driver to manage the data bus.…”

“…〈 x 〉 t and 〈 y 〉 t are moving averages of x and y across the time interval, t, including that frame. This time interval can be determined experimentally and must be of sufficient length to follow the bead as it diffuses throughout the available hemisphere (Han, et al, 2009). It also depends on the diffusion medium, as shown previously (Kumar, et al, 2014).…”