Enhanced tethered-particle motion analysis reveals viscous effects

Area of Science:

• Biophysics
• Polymer Physics
• Molecular Biology

Background:

• Tethered-particle motion (TPM) is a cost-effective technique for studying polymer dynamics.
• Accurate measurement of polymer effective length changes is vital for TPM applications.
• Factors like bead size, tether length, and buffer composition influence bead diffusion in TPM.

Purpose of the Study:

• To investigate how bead size, DNA tether length, and buffer conditions affect confined diffusion in TPM experiments.
• To calibrate the relationship between bead excursion and DNA length under varying experimental parameters.
• To determine optimal conditions for TPM to accurately reflect polymer behavior.

Main Methods:

• Tethered-particle motion experiments were conducted using DNA tethers of varying lengths (225–3477 bp).
• Microspheres (160 or 240 nm radius) were tethered in different aqueous buffers (varying salt, MgCl2, and Tween concentrations).
• Bead motion and confined diffusion were recorded and analyzed to measure excursion and diffusion coefficients.

Main Results:

• Different buffer compositions significantly altered bead excursion on identical DNA tethers.
• Buffers with high magnesium concentrations (>5 mM) and low salt (10 mM NaCl) markedly reduced excursion.
• Increased viscosity via glycerol slowed diffusion but did not affect excursion; shorter tethers exhibited smaller diffusion coefficients.

Conclusions:

• Buffer composition is a critical variable affecting bead excursion in TPM, necessitating careful control.
• TPM is a sensitive method for diffusion studies, with smaller beads on longer tethers better approximating free diffusion.
• Understanding these parameters enhances the reliability of TPM for investigating polymer physics and molecular interactions.