We study the translocation dynamics of a single protein molecule attached to a double-stranded DNA that is threaded through a solid-state nanopore by optical tweezers and an electric field (nanopore force spectroscopy). We find distinct asymmetric and retarded force signals that depend on the protein charge, the DNA elasticity and its counterionic screening in the buffer. A theoretical model where an isolated charge on an elastic, polyelectrolyte strand is experiencing an anharmonic nanopore potential was developed. Its results compare very well with the measured force curves and explain the experimental findings that the force depends linearly on the applied electric field and exhibits a small hysteresis during back and forth translocation cycles. Moreover, the translocation dynamics reflects the stochastic nature of the thermally activated hopping between two adjacent states in the nanopore that can be adequately described by Kramers rate theory.

Dynamic force spectroscopy is a well-established tool to study molecular recognition in a wide range of binding affinities on the single-molecule level. The theoretical interpretation of these data is still very challenging and the models describe the experimental data only partly. In this paper we reconsider the basic assumptions of the models on the basis of an experimental data set and propose an approach of analyzing and quantitatively evaluating dynamic force spectroscopy data on single ligand-receptor complexes. We present our procedure to process and analyze the force-distance curves, to detect the rupture events in an automated manner, and to calculate quantitative parameters for a biophysical characterization of the investigated interaction.

In dynamic force spectroscopy, a (bio-)molecular complex is subjected to a steadily increasing force until the chemical bond breaks. Repeating the same experiment many times results in a broad distribution of rupture forces, whose quantitative interpretation represents a formidable theoretical challenge. In this study we address the situation that more than a single molecular bond is involved in one experimental run, giving rise to multiple rupture events that are even more difficult to analyze and thus are usually eliminated as far as possible from the further evaluation of the experimental data. We develop and numerically solve a detailed model of a complete dynamic force spectroscopy experiment including a possible clustering of molecules on the substrate surface, the formation of bonds, their dissociation under load, and the postprocessing of the force extension curves. We show that the data, remaining after elimination of obvious multiple rupture events, may still contain a considerable number of hidden multiple bonds, which are experimentally indistinguishable from true single bonds, but which have considerable effects on the resulting rupture force statistics and its consistent theoretical interpretation.

The forced rupture of single chemical bonds under external load is addressed. A general framework is put forward to optimally utilize the experimentally observed rupture force data for estimating the parameters of a theoretical model. As an application, we explore to what extent a distinction between several recently proposed models is feasible on the basis of realistic experimental data sets.

Electron-tunneling data suggest that a noncovalently-bonded complex of three molecules, two recognition molecules that present hydrogen-bond donor and acceptor sites via a carboxamide group, and a DNA base, remains bound for seconds. This is surprising, given that imino-proton exchange rates show that basepairs in a DNA double helix open on millisecond timescales. The long lifetime of the three-molecule complex was confirmed using force spectroscopy, but measurements on DNA basepairs are required to establish a comparison with the proton-exchange data. Here, we report on a dynamic force spectroscopy study of complexes between the bases adenine and thymine (A-T, two-hydrogen bonds) and 2-aminoadenine and thymine (2AA-T, three-hydrogen bonds). Bases were tethered to an AFM probe and mica substrate via long, covalently linked polymer tethers. Data for bond-survival probability versus force and the rupture-force distributions were well fitted by the Bell model. The resulting lifetime of the complexes at zero pulling force was ~2 s for two-hydrogen bonds (A-T) and ~4 s for three-hydrogen bonds (2AA-T). Thus, DNA basepairs in an AFM pulling experiment remain bonded for long times, even without the stabilizing influence of base-stacking in a double helix. This result suggests that the pathways for opening, and perhaps the open states themselves, are very different in the AFM and proton-exchange measurements.