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

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.

Bridging the spatiotemporal scales of macromolecular transport in crowded biomimetic systems

“…The degree of compaction or volume reduction has been reported to depend on the shape, structure, and concentration of crowders. 6,8,15,17,39,[41][42][43][44][45][46] While the compaction we measure likely arises from a similar effect, our results are the first, to our knowledge, to report on depletion-driven compaction in highly entangled, viscoelastic systems or when the crowders are comparable in size or larger than the compacted polymer. The varying degree of compaction in different networks that are at equal molarity further indicates the important role that molecular entanglements and stiffness can play in the excluded volume effect.…”

“…1, Methods). 3,6,32 Specifically, we evaluate the distribution of shapes and sizes of molecular conformations and determine how the measured conformations change over time. The distributions of DNA coil sizes Rcoil = [½(Rmax 2 + Rmin 2 )] 1/2 for each condition clearly show that all cytoskeleton networks induce some degree of DNA compaction compared to buffer conditions (Fig.…”

“…Previous experiments on large DNA crowded by synthetic polymers have also reported molecular compaction arising from the well-known depletion interaction in crowded systems. 6,39,40 Namely, to maximize the volume available to small crowders, and thus maximize their entropy, large macromolecules are forced to condense to reduce the excluded volume or 'depletion zone' that their contours impose. The degree of compaction or volume reduction has been reported to depend on the shape, structure, and concentration of crowders.…”

“…The impact of crowding on macromolecular transport and conformations is a topic of intensive theoretical and experimental research. [1][2][3][4][5][6][7][8][9] The primary inspiration for much of this work is to understand how large biomacromolecules such as nucleic acids and proteins can function in the highly crowded environment of the cell. For example, DNA transport through the crowded cytoplasm is essential for transformation, transcription, looping, gene expression, viral infection and gene therapy.…”

“…The wide spatiotemporal range and variety of measurement deliverables is made possible by integrating two recently developed complementary techniques: selective-plane illumination differential dynamic microscopy (SPIDDM) 31 and single-molecule conformational tracking (SMCT). 3,6,32 SPIDDM couples light-sheet microscopy (LSM), an optical sectioning microscopy technique that allows for minimum exposure to excitation light, [33][34][35] with differential dynamic microscopy (DDM), a type of digital Fourier microscopy that quantifies intensity fluctuations within a time series of images. 36 Importantly, DDM does not require that individual DNA molecules be localized, let alone tracked, in order to determine their transport dynamics.…”