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Dynamic single-molecule force spectroscopy: bond rupture analysis with variable spacer length

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.