Abstract
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, 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.
Highlights
Biological cells are highly crowded by macromolecules of varying sizes and structures
To determine the role that crowder structure plays in crowding-induced changes to DNA conformation, we look to the measured major and minor axis lengths Rmax and Rmin
We have investigated the role of crowder structure, size, and concentration, as well as ionic conditions, on the diffusion and conformational dynamics of large DNA molecules
Summary
Biological cells are highly crowded by macromolecules of varying sizes and structures. This complex crowded environment has been shown to directly impact key DNA processes and functions including replication, transcription, transformation, gene expression, and chromosomal compaction [1,2,3,4,5,6]. The exact effect that crowding has on DNA mobility and conformation remains poorly understood. The wide range of differing results presented in the literature likely stems from the myriad of sizes and types of crowders, as well as the varying ionic conditions, used in in vitro experiments— both of which directly impact the effect of crowding on DNA
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