Single-molecule visualization of the effects of ionic strength and crowding on structure-mediated interactions in supercoiled DNA molecules

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.

Differential Impact of Salinity Stress on Seeds Minerals, Storage Proteins, Fatty Acids, and Squalene Composition of New Quinoa Genotype, Grown in Hyper-Arid Desert Environments

“…Under saline conditions, osmolyte destabilization of DNA has important cellular effects. Salts that accumulate during salt stress can also overstabilize the double helix, which can adversely inhibit DNA function in replication and transcription ( Scott et al, 2019 ). Proline can also be a substitute energy source if necessary; proline degrades and releases energy as a reducing agent ( Polavarapu et al, 2014 ).…”

Non-equilibrium structural dynamics of supercoiled DNA plasmids exhibits asymmetrical relaxation

“…Convex lens-induced confinement ( 3 , 5 , 27 ) was used in all microscopy experiments to confine molecules in 3 µm wide, 500 nm deep pits. Pits were etched into D263 cover glass using UV lithography as described in prior work ( 4 ).…”

“…The most easily unwound region occurs upstream from the origin of replication, and the second region is associated with the ampicillin resistance gene. The region upstream from the origin of replication is our focus of interest, both in our previous work ( 4 , 5 ) and in this work.…”

“…In previous work, we used this technique to study supercoiling-induced unwinding in plasmid DNA ( 4 , 5 ). We introduced a fluorescently labeled oligonucleotide complementary to a sequence-specific unwinding site that would only bind to that site if the site was in the single-stranded DNA conformation.…”

Biophysical Reviews ‘Meet the Editors Series’ — a profile of Sabrina Leslie

“…2 ). Over the next ten years, we developed CLiC imaging into a platform and used it for new single-molecule studies of nucleic acids and their interactions (Henkin et al 2016 ) and how these interactions are mediated by DNA structure (Scott et al 2018 , 2019 ); proteins (Elting et al 2013 ); protein droplets and polymer models (Shayegan et al 2019 ); DNA polymers and the effects of confinement (Leith et al 2016 ) as well as applications to genomics (Berard et al 2014 ); as well as cells (Jia et al 2016 ) and their response to therapeutics (Thiombane et al 2019 ). After promotion to Associate Professor with tenure in 2017, I used my sabbatical to co-found a start-up company ScopeSys — which I envision as a vehicle for practical translation of our work and an output of my research which simultaneously enables new career opportunities for Canadian biophysicists.…”