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.

The formation of insulator-supported functional molecular structures requires a firm anchoring of the molecular building blocks to the underlying surface. With a suitable anchoring mechanism, the functionality of single molecules can be maintained and molecular reaction routes for advanced fabrication can be realized to ultimately produce a functional unit. Here, we demonstrate the anchoring of a functionalized ferrocene molecule 1,1′-ferrocenedicarboxylic acid (FDCA) to the CaF2(111) surface. Due to the large band gap and high purity of CaF2 crystals, as well as the presence of particularly large, defect-free terraces, CaF2(111) is a prototypical insulator surface most suitable for the fabrication of molecular devices. Noncontact atomic force (NC-AFM) and scanning tunneling microscopy (STM) experiments performed on CaF2 bulk and CaF2/CaF1/Si­(111) thin film samples reveal the formation of ultrasmall molecular FDCA islands composed of only a few molecules. This molecular assembly is stable even at room temperature and at temperatures as low as 5 K. A comparison of the experimental data with results of density functional theory (DFT) calculations indicates that the exceptional stability is based on a robust quadruped binding motif. This quadruped anchoring bears strong potential for creating tailored molecular structures on CaF2(111) surfaces that are stable at room temperature.

A double sample holder supporting both a metal sample and an insulator crystal for high-resolution scanning probe microscopy experiments is described. The metal sample serves as a substrate for tip preparation and tip functionalization to efficiently and reliably enable high-resolution studies of the adjacent insulator surface. Imaging of Ag(111)/mica, Au(111)/mica, CaF2(111), and calcite(104) surfaces is demonstrated at 5 K, including images on calcite(104) produced with a CO terminated tip, which was prepared on the adjacent metal sample.

A distinct dumbbell shape is observed as the dominant contrast feature in the experimental data when imaging 1,1’-ferrocene dicarboxylic acid (FDCA) molecules on bulk and thin film CaF2(111) surfaces with non-contact atomic force microscopy (NC-AFM). We use NC-AFM image calculations with the probe particle model to interpret this distinct shape by repulsive interactions between the NC-AFM tip and the top hydrogen atoms of the cyclopentadienyl (Cp) rings. Simulated NC-AFM images show an excellent agreement with experimental constant-height NC-AFM data of FDCA molecules at several tip–sample distances. By measuring this distinct dumbbell shape together with the molecular orientation, a strategy is proposed to determine the conformation of the ferrocene moiety, herein on CaF2(111) surfaces, by using the protruding hydrogen atoms as markers.

In typical force spectroscopy experiments, a small biomolecule is attached to a soft polymer linker that is pulled with a relatively large bead or cantilever. At constant force, the total extension stochastically changes between two (or more) values, indicating that the biomolecule undergoes transitions between two (or several) conformational states. In this work, we consider the influence of the dynamics of the linker and mesoscopic pulling device on the forcedependent rate of the conformational transition extracted from the timedependence of the total extension, and the distribution of rupture forces in force-clamp and force-ramp experiments, respectively. For the different pulling experiments, we derive analytic expressions for the observables that account for the mechanical response and dynamics of the pulling device and linker. Possible artifacts arise when the characteristic times of the pulling device and linker become comparable to, or slower than, the lifetimes of the metastable conformational states, and when the highly anharmonic regime of stretched linkers is probed at high forces. We also revisit the problem of relating force-clamp and force-ramp experiments, and identify a linker and loading-rate dependent correction to the rates extracted from the latter. The theory provides a framework for both the design and the quantitative analysis of force spectroscopy experiments by highlighting, and correcting for, factors that complicate their interpretation.