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

Abstract

The structural dynamics of aDNA 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,i ncluding high pressures and high temperatures,byusing single-molecule Fçrster resonance energy transfer and fluorescence correlation spectroscopy. The effect of pressure on the conformational dynamics of the DNAHpisinvestigated on asingle-molecule level, providing novel mechanistic insights into its conformational conversions.D ifferent from canonical DNAd uplex structures of similar melting points,the DNAHpisfound to be rather pressure sensitive.T he combined temperature and pressure dependent data allow dissection of the folding free energy into its enthalpic,e ntropic,a nd volumetric contributions.T he folded conformation is effectively stabilized by the compatible osmolyte TMAOnot only at high temperatures,but also at high pressures and in the presence of the destabilizing co-solute urea.Even though DNAh airpins (Hps) play ap rominent role in many biological processes, [1] as biosensors and in DNA nanotechnology, [2] results on their conformational and free energy landscape remain still controversial. [3][4][5][6][7][8] Here,w e report on the effect of the important natural osmolytes TMAOa nd urea on the conformational dynamics of aw ellknown DNAH p [4] at both ambient and extreme environmental (i.e., high pressure and temperature) conditions by using diffusion-based single-molecule Fçrster resonance energy transfer (smFRET) and fluorescence correlation spectroscopy (FCS). What is still unknown is the combined effect of temperature,p ressure and osmolytes on the conformational dynamics of the nucleic acid on the singlemolecule level. We combined the pressure perturbation approach-to our knowledge for the first time-with smFRET in order to obtain additional mechanistic and volumetric information on the Hp.S uch studies are also of high biological relevance as in the deep sea and sub-seafloor crust, high pressures up to the 1kbar (100 MPa) range are encountered, and living organisms have to cope with such harsh conditions. [9] According to Le Châteliersp rinciple, pressurization shifts chemical equilibria towards conformations occupying smaller partial volumes,t hereby facilitating the experimental characterization of important high-energy conformers,w hich are sparsely populated at ambient condition. [10] First, smFRET measurements (for details see the Supporting Information, SI) were performed on the DNAHpin buffer solution (20 mm Tris-HCl, 50 mm NaCl, pH 8.0) in the absence and presence of TMAOand urea at ambient pressure (1 bar) and temperature (25 8 8C). To this end, donor and acceptor fluorophore labeled oligonucleotide strands were annealed to generate ad ually labeled DNAH p ( Figure 1a). Thedonor-acceptor intensity time trace and the correspond-[*] Dr.Figure 1. a) DNA hairpin including the position of the fluorophores T-Atto 550 (donor) and T-Atto 647 N( acceptor) serving as FRET pair. b) Plots of the...

Lessons from pressure denaturation of proteins

“…Most recent studies using FRET to monitor biomolecular conformational transitions involves single molecule (sm)FRET. To our knowledge, there are only two published studies to date of biomolecular folding based on smFRET under HP, one HP smFRET study on DNA hairpin formation [41] and the other on protein folding, both using a capillary system similar to that previously described for use in HP fluorescence correlation measurements [42]. Progress can still be made in rendering the HP capillary system more reliable and easier to use.…”

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 2F and 5F). The more compact shape of DNA 43 and RNA 44 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 43 and RNA, 44 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.…”

Urea-aromatic interactions in biology

“…These strong urea nucleobase interactions are quantified by various experimental and theoretical approaches (Gao et al 2017;Patra et al 2017;Miner and García 2017). Guinn et al showed relatively favorable interactions of urea compared with water with different regions of nucleic acids structures, like, heterocyclic aromatic ring, methyl, carbonyl and phosphate O, amino N, and sugar (C and O) (Guinn et al 2013).…”

“…Urea has long been used as an analytical tool to characterize the ubiquitous thermodynamic forces stabilizing biomolecular structures. Recent FRET studies indicated an increase in enthalpy and entropy of the RNA/DNA hairpins in the presence of high concentration of urea (Holmstrom and Nesbitt 2014;Holmstrom et al 2015;Patra et al 2017). A rugged folding free energy surface of DNA was observed which involves a number of quasi-open intermediate conformations (Sarkar et al 2009).…”