A novel, interferometry-based approach to micropipette aspiration overcomes the spatial resolution limit of current methods, simplifies data retrieval, and enables the study of the dynamic mechanical behavior of biomembranes at the nanoscale.
DNA phase transitions drive life processes and are key to the development of DNA-based biotechnologies. Accordingly, quantifying the physical properties of DNA is an essential endeavor. However, the narrow width (2 nm) of the DNA molecule prohibits direct visualization of its structural dynamics using optical microscopy. To address this challenge, we employ concurrent polarization imaging and DNA manipulation to probe the orientations and rotational dynamics of DNA-intercalated dyes—small fluorescent molecules that bind between adjacent DNA base pairs. The method uses optical tweezers to precisely extend, align and (re)orient a single DNA molecule within the image plane of a fluorescence microscope. Our data shows that at extensions beyond the so-called “overstretching transition” intercalators adopt a dramatically tilted orientation relative to the DNA-axis (approx. 54 degrees), distinct from the perpendicular orientation (approx. 90 degrees) normally observed at lower extensions. Strikingly, by imaging single intercalated dye molecules with polarized illumination, we also demonstrate that intercalators rapidly rotate (i. e. “twirl”) about the DNA-axis, revealing underlying Brownian twisting dynamics of the DNA substrate. Taken together, these results shed new insight on S-DNA: a DNA phase that forms under tension that, at present, is not well understood.
Dense coverage of DNA by proteins is a ubiquitous feature of cellular processes such as DNA organization, replication and repair. We present a single-molecule approach capable of visualizing individual DNA-binding proteins on densely covered DNA and in the presence of high protein concentrations. Our approach combines optical tweezers with multicolor confocal and stimulated emission depletion (STED) fluorescence microscopy. Proteins on DNA are visualized at a resolution of 50 nm, a sixfold resolution improvement over that of confocal microscopy. High temporal resolution (<50 ms) is ensured by fast one-dimensional beam scanning. Individual trajectories of proteins translocating on DNA can thus be distinguished and tracked with high precision. We demonstrate our multimodal approach by visualizing the assembly of dense nucleoprotein filaments with unprecedented spatial resolution in real time. Experimental access to the force-dependent kinetics and motility of DNA-associating proteins at biologically relevant protein densities is essential for linking idealized in vitro experiments with the in vivo situation.
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