Enhanced Tethered-Particle Motion Analysis Reveals Viscous Effects

Abstract

Tethered-particle motion experiments do not require expensive or technically complex hardware, and increasing numbers of researchers are adopting this methodology to investigate the topological effects of agents that act on the tethering polymer or the characteristics of the polymer itself. These investigations depend on accurate measurement and interpretation of changes in the effective length of the tethering polymer (often DNA). However, the bead size, tether length, and buffer affect the confined diffusion of the bead in this experimental system. To evaluate the effects of these factors, improved measurements to calibrate the two-dimensional range of motion (excursion) versus DNA length were carried out. Microspheres of 160 or 240 nm in radius were tethered by DNA molecules ranging from 225 to 3477 basepairs in length in aqueous buffers containing 100 mM potassium glutamate and 8 mM MgCl2 or 10 mM Tris-HCl and 200 mM KCl, with or without 0.5% Tween added to the buffer, and the motion was recorded. Different buffers altered the excursion of beads on identical DNA tethers. Buffer with only 10 mM NaCl and >5 mM magnesium greatly reduced excursion. Glycerol added to increase viscosity slowed confined diffusion of the tethered beads but did not change excursion. The confined-diffusion coefficients for all tethered beads were smaller than those expected for freely diffusing beads and decreased for shorter tethers. Tethered-particle motion is a sensitive framework for diffusion experiments in which small beads on long leashes most closely resemble freely diffusing, untethered beads.

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

“…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). Four seconds is adequate in low viscosity buffers, and such moving averages reveal the coordinates of the attachment point of the tether to the glass.…”

“…A. Schneider, 2012) with forward and reverse primers: 5′-ttaattccgcacagatgcgtaaggagaa-3′ and digoxigenin-labeled 5′-ccaaagactttgatttctcgta-3′ (Integrated DNA Technologies, Coralville, IA, USA), respectively. The DNA tethers for gyrase experiments (336 bp) were produced using pDL611 plasmid as a template with biotin- and digoxigenin-labeled primers and used in calibration experiments as described (Kumar et al, 2014). …”

“…The time series of xy coordinates were analyzed using methods similar to those described previously (Kumar, et al, 2014) to select beads and determine the magnitude of the confined Brownian motion exhibited. First, the mechanical drift was eliminated by subtracting the apparent motion of stuck beads.…”

Quantitation of interactions between two DNA loops demonstrates loop domain insulation in E. coli cells

“…We used the TPM technique to detect and measure DNA looping in vitro (24,(52)(53)(54). For loop interference, we used a DNA tether with the asymmetrical looping arrangement that we used in vivo (Fig.…”

“…S1B) TPM. TPM experiments were performed as described previously (53). The DNA fragment was prepared using PCR with biotin-and digoxigenin-labeled primers and was attached to an anti-digoxigenin-coated coverslip and streptavidin-coated polystyrene beads (160 nm radius; Spherotech Inc.).…”