Tethered Particle Motion Technique in Crowded Media: Compaction of DNA by Globular Macromolecules

TL;DR

This study explores the use of Tethered Particle Motion (TPM) to analyze DNA compaction by globular macromolecules in crowded fluids, revealing limitations and proposing modifications for better applicability in complex biological environments.

Contribution

The paper demonstrates through simulations that standard TPM faces constraints in crowded media and suggests alternative approaches like Tethered Fluorophore Motion for improved DNA characterization.

Findings

TPM has limitations in crowded environments due to simulation constraints.

Tethered Fluorophore Motion may be more suitable than standard TPM.

Sample preparation techniques need adjustment for crowded media.

Abstract

Tethered Particle Motion (TPM) is a single molecule technique, which consists in tracking the motion of a nano-particle (NP) immersed in a fluid and tethered to a glass surface by a DNA molecule. The present work addresses the question of the applicability of TPM to fluids which contain crowders at volume fractions ranging from that of the nucleoid of living bacteria (around 30%) up to the jamming threshold (around 66%). In particular, we were interested in determining whether TPM can be used to characterize the compaction of DNA by globular crowders. To this end, extensive Brownian Dynamics simulations were performed with a specifically built coarse-grained model. Analysis of the simulations reveals several effects not observed in dilute media, which impose constraints on the TPM set-up. In particular, the Tethered Fluorophore Motion (TFM) technique, which consists in replacing the NP by a much smaller fluorophore, is probably better suited than standard TPM. Moreover, a sample preparation technique which does not involve hydrophilic patches may be required. Finally, the use of a DNA brush may be needed to achieve DNA concentrations close to in vivo ones.