Single-molecule force spectroscopy using optical tweezers continues to provide detailed insights into the behavior of nanoscale systems. Obtaining precise measurements of their mechanical properties is highly dependent on accurate instrument calibration. Therefore, instrumental drift or inaccurate calibration may prevent reaching an accuracy at the theoretical limit and may lead to incorrect conclusions. Commonly encountered sources of error include inaccuracies in the detector sensitivity and trap stiffness and neglecting the non-harmonicity of an optical trap at higher forces. Here, we first quantify the impact of these artifacts on force-extension data and find that a small deviation of the calibration parameters can already have a significant downstream effect. We then develop a method to identify and remove said artifacts based on differences in the theoretical and measured noise of bead fluctuations. By applying our procedure to both simulated and experimental data, we can show how effects due to miscalibration and trap non-linearities can be successfully removed. Most importantly, this correction can be performed post-measurement and could be adapted for data acquired using any force spectroscopy technique.

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The genome is organized by diverse packaging mechanisms like nucleosome formation, loop extrusion and phase separation, which all compact DNA in a dynamic manner. Phase separation additionally drives protein recruitment to condensed DNA sites and thus regulates gene transcription. The cohesin complex is a key player in chromosomal organization that extrudes loops to connect distant regions of the genome and ensures sister chromatid cohesion after S-phase. For stable loading onto the DNA and for activation, cohesin requires the loading complex Scc2/4. As the precise loading mechanism remains unclear, we investigated whether phase separation might be the initializer of the cohesin recruitment process. We found that, in absence of cohesin, budding yeast Scc2/4 forms phase separated co-condensates with DNA, which comprise liquid-like properties shown by droplet shape, fusion ability and reversibility. We reveal in DNA curtain and optical tweezer experiments that these condensates are built by DNA bridging and bending through Scc2/4. Importantly, Scc2/4-mediated condensates recruit cohesin efficiently and increase the stability of the cohesin complex. We conclude that phase separation properties of Scc2/4 enhance cohesin loading by molecular crowding, which might then provide a starting point for the recruitment of additional factors and proteins.