Osmolyte Effects on the Conformational Dynamics of a DNA Hairpin at Ambient and Extreme Environmental Conditions

Abstract

The structural dynamics of a DNA hairpin (Hp) are studied in the absence and presence of the two natural osmolytes trimethylamine-N-oxide (TMAO) and urea at ambient and extreme environmental conditions, including high pressures and high temperatures, by using single-molecule Förster resonance energy transfer and fluorescence correlation spectroscopy. The effect of pressure on the conformational dynamics of the DNA Hp is investigated on a single-molecule level, providing novel mechanistic insights into its conformational conversions. Different from canonical DNA duplex structures of similar melting points, the DNA Hp is found to be rather pressure sensitive. The combined temperature and pressure dependent data allow dissection of the folding free energy into its enthalpic, entropic, and volumetric contributions. The folded conformation is effectively stabilized by the compatible osmolyte TMAO not only at high temperatures, but also at high pressures and in the presence of the destabilizing co-solute urea.

Crowders and Cosolvents—Major Contributors to the Cellular Milieu and Efficient Means to Counteract Environmental Stresses

“…Further, using high pressure FTIR, our laboratory could show that the structure of phenylalanine transfer RNA is also hardly perturbed by pressure . Conversely, non‐canonical DNA structures, such as G‐quadruplex DNA and a DNA hairpin with an oversized poly‐dA loop, are more sensitive to pressure …”

“…hydrophilic vs. hydrophobic surface, charge distribution, SASA, packing density). In case of RNAs, the m ‐value further correlates with the length of the duplex, the degree to which RNA structures fold, and the ionic conditions . Especially, ambivalent effects of TMAO on secondary and tertiary interactions of nucleic acids have been reported.…”

A generalized purification step for viral particles using mannitol flocculation

“…An unusual observation during this work is that DNA appears to also aggregate in the presence of mannitol because it is removed less than what should have been the negative control of water (see Figure F and F). The more compact shape of DNA and RNA was favored in high TMAO concentrations. It is also possible that the neutral mannitol could cause DNA to lose its charge, similar to the study of silica particles being neutralized in the presence of the neutral, protecting osmolyte, glycerol .…”

“…There appeared to be flocculation of the DNA along with the virus in the mannitol system. TMAO has been shown to enhance compact conformations of DNA and RNA, but little is known on how mannitol affects nucleic acids. The mechanism of osmolyte flocculation of nucleic acids should be explored and may be a future method to purify plasmid and other DNA or RNA therapeutic products.…”

The Deep Sea Osmolyte TrimethylamineN‐Oxide and Macromolecular Crowders Rescue the Antiparallel Conformation of the Human Telomeric G‐Quadruplex from Urea and Pressure Stress

“…The single‐molecule fluorescence resonance energy transfer (smFRET) technique as used herein allows to distinguish different folded conformations . Recently, we were also able to setup a HHP smFRET stage (Figure S1 in the Supporting Information) allowing us to reach pressures up to ∼2 kbar (200 MPa), covering the whole relevant pressure range on Earth . We used a DNA construct comprised of a duplex stem and a single stranded region containing the human telomeric repeats (Figure ), which was also previously used in ambient pressure smFRET studies …”