Quantifying Local Molecular Tension Using Intercalated DNA Fluorescence

Abstract

The ability to measure mechanics and forces in biological nanostructures, such as DNA, proteins and cells, is of great importance as a means to analyze biomolecular systems. However, current force detection methods often require specialized instrumentation. Here, we present a novel and versatile method to quantify tension in molecular systems locally and in real time, using intercalated DNA fluorescence. This approach can report forces over a range of at least ∼0.5–65 pN with a resolution of 1–3 pN, using commercially available intercalating dyes and a general-purpose fluorescence microscope. We demonstrate that the method can be easily implemented to report double-stranded (ds)DNA tension in any single-molecule assay that is compatible with fluorescence microscopy. This is particularly useful for multiplexed techniques, where measuring applied force in parallel is technically challenging. Moreover, tension measurements based on local dye binding offer the unique opportunity to determine how an applied force is distributed locally within biomolecular structures. Exploiting this, we apply our method to quantify the position-dependent force profile along the length of flow-stretched DNA and reveal that stretched and entwined DNA molecules—mimicking catenated DNA structures in vivo—display transient DNA–DNA interactions. The method reported here has obvious and broad applications for the study of DNA and DNA–protein interactions. Additionally, we propose that it could be employed to measure forces in any system to which dsDNA can be tethered, for applications including protein unfolding, chromosome mechanics, cell motility, and DNA nanomachines.

Molecular structure, DNA binding mode, photophysical properties and recommendations for use of SYBR Gold

“…To assess to what extent SYBR Gold or SYBR Green I binding lengthens the DNA contour, we first performed DNA stretching experiments in the presence of increasing concentrations of dye on torsionally relaxed (nicked) DNA. We focused on the force regime <5 pN for our force–extension measurements: in this regime the DNA extension is well described by the worm-like chain (WLC) model of entropic stretching elasticity ( 45 ) and we can neglect enthalpic contributions to stretching and the force-dependence of intercalative binding ( 3 , 15 , 46 ). In the absence of dye the response of DNA to force shows the characteristic response of entropic stretching elasticity (Figure 2B , dark blue circles).…”

Skeletal Muscle Atrophy in Simulated Microgravity Might Be Triggered by Immune-Related microRNAs

“…Our in silico analysis showed that let-7 targets vinculin (VC), a protein connecting integrins to actin filaments, and that is recruited to focal adhesions (FAs) in response to force. VCL is suggested to be linked to FA mechanosensitivity and it is essential for FA stabilization under force (King et al, 2018). Migration of satellite cells (SC), necessary for skeletal muscle development and regeneration, is strictly dependant on the formation of mature FA connecting the cell to the extracellular matrix (Goetsch et al, 2014).…”

Single-molecule measurements reveal that PARP1 condenses DNA by loop formation

“…Condensation commenced at the free-end of the molecule, where a compact structure initially formed and then gradually increased in intensity as it moved towards the anchor point ( Figure 1C). It is important to note that the hydrodynamic flow stretches DNA molecules with a line-tension that decreases from a maximum value at the molecule's attachment point to zero at the free end 36,37 . Hence, while our TIRF results clearly demonstrate DNA condensation by PARP1 is initiated at the free end, it is not possible to distinguish whether this is caused by the presence of a double-strand break or the lower tension towards the end.…”