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

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.

References in corpus (17)

• Jamming transitions in amorphous packings of frictionless spheres occur over a continuous range of volume fractions
• Excluded-Volume Effects in Tethered-Particle Experiments: Bead Size Matters
• Onset of DNA Aggregation in Presence of Monovalent and Multivalent Counterions
• Depletion forces near curved surfaces
• Physical descriptions of the bacterial nucleoid at large scales, and their biological implications
• Compaction of bacterial genomic DNA: Clarifying the concepts
• Stepwise bending of DNA by a single TATA-box Binding Protein
• Probing DNA conformational changes with high temporal resolution by Tethered Particle Motion
• Segregative phase separation scenario of the formation of the bacterial nucleoid
• Semi-flexible chain condensation by neutral depleting agents: role of correlations between depletants
• Multiple Lac-mediated loops revealed by Bayesian statistics and tethered particle motion
• In vivo compaction dynamics of bacterial DNA: A fingerprint of DNA/RNA demixing ?
• DNA sequence-dependent mechanics and protein-assisted bending in repressor-mediated loop formation
• Preferential localization of the bacterial nucleoid
• Bacterial nucleoid: Interplay of DNA demixing and supercoiling
• Organization of the bacterial nucleoid by DNA-bridging proteins and globular crowders
• Coarse-Grained model of the demixing of DNA and non-binding globular macromolecules