Calibration of Tethered Particle Motion Experiments

The Tethered Particle Motion (TPM) method has been used to observe and characterize a variety of protein-DNA interactions including DNA loping 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 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

Single-molecule detection of DNA hybridization

We demonstrate the detection of nanometer-scale conformational changes of single DNA oligomers through a micromechanical technique. The quantity monitored is the displacement of a micrometer-size bead tethered to a surface by the probe molecule undergoing the conformational change. This technique allows probing of conformational changes within distances beyond the range of fluorescence resonance energy transfer. We apply the method to detect single hybridization events of label-free target oligomers. Hybridization of the target is detected through the conformational change of the probe

Undulations of a Straight Polymer Chain Embedded in a Membrane

We consider a sinusoidal undulation of a straight, linear polymer embedded in a flat membrane exposed to a lateral tension. We determine the shape of the membrane and compute the elastic energy associated with the undulation of the polymer. Analysing this energy at different wavelengths of the undulation, we find the criteria of stability of the straight shape of the polymer, and show them to be controlled by the difference of moduli of Gaussian curvature, $\Delta \bar{\kappa}$, of the polymerized and unpolymerized membrane. We also discuss formal analogies and differences between the behaviour of a polymer embedded into a membrane and a charged bilayer membrane immersed in a solution of electrolyte