  Macromolecular crowding plays a principal role in a wide range of biological
processes including gene expression, chromosomal compaction, and viral
infection. However, the impact that crowding has on the dynamics of nucleic
acids remains a topic of debate. To address this problem, we use
single-molecule fluorescence microscopy and custom particle-tracking algorithms
to investigate the impact of varying macromolecular crowding conditions on the
transport and conformational dynamics of large DNA molecules. Specifically, we
measure the mean-squared center-of-mass displacements, as well as the
conformational size, shape, and fluctuations, of individual 115 kbp DNA
molecules diffusing through various in vitro solutions of crowding polymers. We
determine the role of crowder structure and concentration, as well as ionic
conditions, on the diffusion and configurational dynamics of DNA. We find that
branched, compact crowders (10 kDa PEG, 420 kDa Ficoll) drive DNA to compact,
whereas linear, flexible crowders (10 kDa, 500 kDa dextran) cause DNA to
elongate. Interestingly, the extent to which DNA mobility is reduced by
increasing crowder concentrations appears largely insensitive to crowder
structure (branched vs linear), despite the highly different configurations DNA
assumes in each case. We also characterize the role of ionic conditions on
crowding-induced DNA dynamics. We show that both DNA diffusion and
conformational size exhibit an emergent non-monotonic dependence on salt
concentration that is not seen in the absence of crowders.
