Dynamic force spectroscopy is a valuable technique to explore the energy landscape of molecular interactions. Polymer spacers are typically used to couple the binding partners to the surfaces. To illustrate the impact of polymer spacers on the measured rupture force and loading rate distributions we used a Monte Carlo simulation, which was adjusted step by step towards realistic experimental conditions. We found that the introduction of a polymer spacer with a discrete length had only a marginal effect. However, a distribution of polymer spacers with different lengths may induce drastic changes on the distributions. Three different methods for data analysis were then tested with regard to their ability to reproduce the input values of the Monte Carlo simulations. We found that simple linearization of all data points leads to an analysis error up to one order of magnitude for the dissociation rate and one-third for the potential width. The best results are achieved by determining the dissociation rate and the potential width directly with a probability density function for the rupture forces and the loading rates as a fit function that uses the dissociation rate and the potential width as fit parameters. By applying this method the analysis errors could be reduced below 25% for the dissociation rate and only 3% for the potential width. Applied to a set of experimental data this method proved to be extremely useful and provided detailed information on the distributions. We are able to discriminate specific and non-specific contributions of an aptamer–ligand interaction and correct for the non-specific background. In addition, this procedure allowed us to account for the low force instrumentation cut-off and reconstruct the rupture force and force rate distributions.
In this paper, we measure the single chain elasticity of an oligomer single-stranded DNA (ssDNA) in both aqueous and nonaqueous, apolar liquid environments by AFM-based single molecule force spectroscopy. We find a marked deviation between the force-extension relations recorded for the two conditions. This difference is attributed to the additional energy required to break the H-bond-directed water bridges around the ssDNA chain in aqueous solutions, which are nonexistent in organic solvents. The results obtained in 8 M guanidine-HCl solution provide more evidence that water bridges around ssDNA originate the observed deviation. On the basis of the results obtained by an ab initio quantum mechanics calculation, a parameter-free freely rotating chain model is proposed. We find that this model is in perfect agreement with the experimental force-extension curve obtained in organic solvents, which further corroborates our assumption. On the basis of the experimental results, it is suggested that the weak H-bonding between ssDNA and water molecules may be a precondition for stable double-stranded DNA to exist in water.
DNA displays a richness of biologically relevant supramolecular structures, which depend on both sequence and ambient conditions. The effect of dragging double-stranded DNA (dsDNA) from water into poor solvent on the double-stranded structure is still unclear because of condensation. Here, we employed single molecule techniques based on atomic force microscopy and molecular dynamics (MD) simulations to investigate the change in structure and mechanics of DNA during the ambient change. We found that the two strands are split apart when the dsDNA is pulled at one strand from water into a poor solvent. The findings were corroborated by MD simulations where dsDNA was dragged from water into poor solvent, revealing details of the strand separation at the water/poor solvent interface. Because the structure of DNA is of high polarity, all poor solvents show a relatively low polarity. We speculate that the principle of spontaneous unwinding/splitting of dsDNA by providing a low-polarity (in other word, hydrophobic) micro-environment is exploited as one of the catalysis mechanisms of helicases.
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