Subpiconewton Dynamic Force Spectroscopy Using Magnetic Tweezers

Abstract

We introduce a simple method for dynamic force spectroscopy with magnetic tweezers. This method allows application of subpiconewton force and twist control by calibration of the applied force from the height of the magnets. Initial dynamic force spectroscopy experiments on DNA molecules revealed a large hysteresis that is caused by viscous drag on the magnetic bead and will conceal weak interactions. When smaller beads are used, this hysteresis is sufficiently reduced to reveal intramolecular interactions at subpiconewton forces. Compared with typical quasistatic force spectroscopy, a significant reduction of measurement time is achieved, allowing the real-time study of transient structures and reaction intermediates. As a proof of principle, nucleosome-nucleosome interactions on a subsaturated chromatin fiber were analyzed.

Force spectroscopy of chromatin fibers: Extracting energetics and structural information from Monte Carlo simulations

“…From this part approximations for the nucleosome-nucleosome interaction energy have been derived in experimental force-extension curves. 27,29 (iii) The increasing distance between nucleosomes then resulted in an apparent plateau, as nucleosome-nucleosome interactions were still significant ( Figure 1, regime 3). 28,33 The nucleosome interaction energy term and the electrostatic repulsion energy from the DNA decreased upon further extension (Supporting Information Figure S4).…”

“…Previously, the interaction energy between nucleosomes has been calculated by integrating a certain area of experimentally determined force-extension curves, which corresponds to regime 2 or 3 in Figure 1. 27,28 From dissecting the relative contributions of all energetic contributions in our MC simulations, it becomes apparent that all energy terms are significant in these two regimes (Supporting Information Figure S4). The nucleosome-nucleosome interaction energy is responsible for the presence or absence of the initial increase and/or the subsequent plateau of the force extension curve.…”

“…To investigate this experimentally, force spectroscopy studies were conducted. [27][28][29] In these experiments, a nucleosome chain is bound at one end to a solid support and is then extended by pulling at the other end using optical tweezers or an atomic force microscopy setup. The increase in distance of the two ends of the fiber at a given force in the regime from $0.1 pN to $40 pN is then computed as a force-distance curve.…”

“…28 More recently, reconstituted nucleosome arrays have been investigated in which the protein composition is better defined. 27,[29][30][31][32] The values for the nucleosome-nucleosome energy derived from these experiments vary from 3.4 to 17 k B T. Several approaches have been reported to dissect the energetic terms that govern the force-distance curve for a given type of fiber. For the description of the nucleosome chain geometry according to a two-angle model, an analytical approach was used.…”

“…46,47 In contrast, for the chain of 25 nucleosomes analyzed in Figure 5A no unwrapping of nucleosomes below pulling forces of 6-7 pN has been reported. 27,29 This has been deduced from the lack of a hysteresis in the plateau starting at $200 nm extension. In the simulations conducted here for this system with E max 5 18 k B T no significant unwrapping was present in the trajectories up to forces of 4.5 pN.…”