Effects of Molecular Crowders on Single-Molecule Nucleic Acid Folding: Temperature-Dependent Studies Reveal True Crowding vs Enthalpic Interactions.

Biomolecular folding in cells can be strongly influenced by spatial overlap/excluded volume interactions (i.e., "crowding") with intracellular solutes. As a result, traditional in vitro experiments with dilute buffers may not accurately recapitulate biomolecule folding behavior in vivo. In order to account for such ubiquitous excluded volume effects, biologically inert polyethylene glycol (PEG) and polysaccharides (dextran and Ficoll) are often used as in vitro crowding agents to mimic in vivo crowding conditions, with a common observation that high concentrations of these polymers stabilize the more compact biomolecule conformation. However, such an analysis can be distorted by differences in polymer interactions with the folded vs unfolded conformers, requiring temperature-dependent analysis of the thermodynamics to reliably assess competing enthalpic vs entropic contributions and thus the explicit role of excluded volume. In this work, temperature-controlled single-molecule fluorescence resonance energy transfer (smFRET) is used to characterize the thermodynamic interaction between nucleic acids and common polymer crowders PEG, dextran, and Ficoll. The results reveal that PEG promotes secondary and tertiary nucleic acid folding by simultaneously increasing the folding rate while decreasing the unfolding rate, with temperature-dependent studies confirming that the source of PEG stabilization is predominantly entropic and consistent with a true excluded volume crowding mechanism. By way of contrast, neither dextran nor Ficoll induces any significant concentration-dependent change in nucleic acid folding stability at room temperature, but instead, stabilization effects gradually appear with a temperature increase. Such a thermal response indicates that both folding enthalpies and entropies are impacted by dextran and Ficoll. A detailed thermodynamic analysis of the kinetics suggests that, instead of true entropic molecular crowding, dextran and Ficoll associate preferentially with the unfolded vs folded nucleic acid conformer as a result of larger solvent accessible surface area, thereby skewing the free energy landscapes through both significant entropic/enthalpic contributions that compete and fortuitously cancel near room temperature.