Crowding Induces Entropically-Driven Changes to DNA Dynamics That Depend on Crowder Structure and Ionic Conditions

Area of Science:

• Biophysics
• Molecular Biology
• Polymer Science

Background:

• Macromolecular crowding is crucial for biological processes like gene expression and viral infection.
• The effect of crowding on nucleic acid dynamics is not fully understood.
• Investigating DNA dynamics under crowding conditions is essential for understanding cellular processes.

Purpose of the Study:

• To investigate how macromolecular crowding affects the transport and conformational dynamics of large DNA molecules.
• To determine the influence of crowder structure, concentration, and ionic conditions on DNA diffusion and configuration.
• To elucidate the mechanisms behind crowding-induced changes in DNA behavior.

Main Methods:

• Single-molecule fluorescence microscopy was employed to track DNA molecules.
• Custom particle-tracking algorithms were developed to analyze diffusion and conformational changes.
• Experiments were conducted using various in vitro solutions with different crowding polymers and ionic strengths.

Main Results:

• Branched, compact crowders (PEG, Ficoll) induced DNA compaction.
• Linear, flexible crowders (dextran) caused DNA elongation.
• DNA diffusion reduction was independent of crowder structure, despite altered DNA configurations.
• DNA diffusion and size showed non-monotonic salt dependence under crowding, unlike in dilute solutions.

Conclusions:

• Crowder architecture dictates DNA conformation, influencing biological processes.
• DNA mobility is consistently reduced by crowding, irrespective of crowder type.
• Ionic conditions introduce complex, non-monotonic effects on DNA dynamics in crowded environments.
• These findings provide critical insights into DNA behavior within the crowded cellular milieu.