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

Abstract

Dynamic force spectroscopy is a valuable technique to explore the energylandscape of molecular interactions. Polymer spacers are typically used to couplethe binding partners to the surfaces. To illustrate the impact of polymer spacerson the measured rupture force and loading rate distributions we used a MonteCarlo simulation, which was adjusted step by step towards realistic experimentalconditions. We found that the introduction of a polymer spacer with adiscrete length had only a marginal effect. However, a distribution ofpolymer spacers with different lengths may induce drastic changes on thedistributions.Three different methods for data analysis were then tested with regard to theirability to reproduce the input values of the Monte Carlo simulations. We foundthat simple linearization of all data points leads to an analysis error up to oneorder of magnitude for the dissociation rate and one-third for the potential width.The best results are achieved by determining the dissociation rate and thepotential width directly with a probability density function for the rupture forcesand the loading rates as a fit function that uses the dissociation rate and thepotential width as fit parameters. By applying this method the analysis errorscould be reduced below 25% for the dissociation rate and only 3% for thepotential width.Applied to a set of experimental data this method proved to be extremely usefuland provided detailed information on the distributions. We are able todiscriminate specific and non-specific contributions of an aptamer–ligandinteraction and correct for the non-specific background. In addition, thisprocedure allowed us to account for the low force instrumentation cut-off andreconstruct the rupture force and force rate distributions.