Crowding-Induced Hybridization of Single DNA Hairpins

Abstract

It is clear that a crowded environment influences the structure, dynamics, and interactions of biological molecules, but the complexity of this phenomenon demands the development of new experimental and theoretical approaches. Here we use two complementary single-molecule FRET techniques to show that the kinetics of DNA base pairing and unpairing, which are fundamental to both the biological role of DNA and its technological applications, are strongly modulated by a crowded environment. We directly observed single DNA hairpins, which are excellent model systems for studying hybridization, either freely diffusing in solution or immobilized on a surface under crowding conditions. The hairpins followed two-state folding dynamics with a closing rate increasing by 4-fold and the opening rate decreasing 2-fold, for only modest concentrations of crowder [10% (w/w) polyethylene glycol (PEG)]. These experiments serve both to unambiguously highlight the impact of a crowded environment on a fundamental biological process, DNA base pairing, and to illustrate the benefits of single-molecule approaches to probing the structure and dynamics of complex biomolecular systems.

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

“…Owing to experimental challenges, results on the combined effect of temperature, pressure and cosolvents on the structure and dynamics of proteins and nucleic acids on a single‐molecule level are still scarce. As an example, we show results on a DNA hairpin (Hp) (Figure a) . Using a special microcapillary‐based high‐pressure cell for single‐molecule fluorescence studies, both temperature‐ and pressure‐dependent single‐molecule Förster resonance energy transfer (smFRET) measurements were carried out in the absence and presence of cosolvents and crowders to retrieve enthalpic, entropic and volumetric information associated with the conformational folding transition of the DNA Hp (Figure ).…”

“…As an example, we show resultso na DNA hairpin (Hp) (Figure 8a). [120,232] Using as pecial microcapillary-based high-pressure cell for single-moleculef luorescence studies, both temperature-and pressure-dependents inglemolecule Fçrsterr esonance energy transfer (smFRET) measurementsw erec arried out in the absence and presence of cosolvents and crowders to retrieve enthalpic, entropic and volumetrici nformationa ssociated with the conformational folding transition of the DNA Hp (Figure 8). TMAO andu rea were used as osmolytes and Ficoll 70 was used as am acromolecular crowding agent.…”

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

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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. [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). [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).

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Metallation‐Induced Heterogeneous Dynamics of DNA Revealed by Single‐Molecule FRET

“…To investigate the interaction of organo-osmium anticancer complex Os1ÀCl with DNA, we employed aD NA hairpin consisting of two single-stranded oligonucleotides (H1 andH 2/H3; see Supporting Information, Methods and Materials), with a 20 nucleotide double-stranded handle, a3 2nucleotide polyA loop, and as tem region of 6base pairs (5 GC and 1A T; Figure 1). [17] Hairpins are excellent probesf or DNA hybridisa-tion. [18] Theh airpin used here was labelledw ith fluorophores acting as ad onor-acceptor couple,w ith either Cy3 (H1-Cy3) or Cy3B (H1-Cy3B) as the donor,a nd Cy5 (H2) as acceptor ( Figure 1).…”

“…This is expected to generate al ow FRET signal when the hairpin stem is open (unpaired) and ah igh signal when the stem is closed (paired). [17] Furthermore, the system carriesa biotin moiety,w hich allowed the hairpin to be immobilised on aP EG-functionalised coverslip for single molecule experiments. Os1ÀCl is knownt ob ind specifically to guaninea nd cytosine bases, [15] so the hairpin provides suitable target sites, particularly in the GC-rich stem.…”