Abstract
DNA unwinding is an important cellular process involved in DNA replication, transcription and repair. In cells, molecular crowding caused by the presence of organelles, proteins, and other molecules affects numerous internal cellular structures. Here, we visualize plasmid DNA unwinding and binding dynamics to an oligonucleotide probe as functions of ionic strength, crowding agent concentration, and crowding agent species using single-molecule CLiC microscopy. We demonstrate increased probe–plasmid interaction over time with increasing concentration of 8 kDa polyethylene glycol (PEG), a crowding agent. We show decreased probe–plasmid interactions as ionic strength is increased without crowding. However, when crowding is introduced via 10% 8 kDa PEG, interactions between plasmids and oligos are enhanced. This is beyond what is expected for normal in vitro conditions, and may be a critically important, but as of yet unknown, factor in DNA’s proper biological function in vivo. Our results show that crowding has a strong effect on the initial concentration of unwound plasmids. In the dilute conditions used in these experiments, crowding does not impact probe–plasmid interactions once the site is unwound.
Highlights
IntroductionEmulating cellular environments with current techniques remains a significant challenge due to the complexity of living cells
Biological sciences have long sought to mimic in vivo conditions in vitro
We have demonstrated a decrease in probe–plasmid binding, that, in turn, indicates a decrease in DNA unwinding at physiological salt conditions
Summary
Emulating cellular environments with current techniques remains a significant challenge due to the complexity of living cells. Cellular DNA, for example, is exposed to a wide variety of solutes of various sizes and physical properties including dissolved salts, proteins, and lipids [1]. 30% of the cell is occupied by solute molecules, though locally this value can rise to as high as 40% [1,2]. Current thinking in biology is that cells are highly compartmentalized, concentrating specific groups of molecules into droplet-like formations, enhancing this crowding effect [3]. This so-called crowding may have an effect on cellular interactions and structures [4,5]
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