High-Speed Time-Lapse Atomic Force Microscopy to Capture Nucleosome Dynamics
Transcrição
Nucleosomes — eukaryotic DNA repeating units — consist of DNA segments wrapped around histone protein cores. DNA unwrapping around the histone core is crucial for nucleosome dynamics.
To capture nucleosome dynamics, begin with a smooth, functionalized mica substrate mounted to an atomic force microscope, AFM, scanner stage. Overlay the substrate with a nucleosome-containing buffer.
The negatively charged nucleosome DNA interacts with positive charges on the functionalized mica, immobilizing them on the surface. Wash the immobilized mica substrate with a buffer to prevent the nucleosomes overcrowding.
Transfer the nucleosome-containing scanner stage to an imaging chamber containing a pre-mounted cantilever, with a tip at the edge and facing up toward the nucleosomes.
Fill the chamber with an imaging buffer. Align the laser beam onto the cantilever back to ensure accurate deflection measurements.
The AFM tip oscillates close to the fully wrapped nucleosomes and scans over the surface to measure the nucleosomes' height at regular intervals.
Spontaneous DNA unwrapping increases nucleosome height, alternating the tip's oscillations and leading to cantilever deflection. This cantilever deflection shifts the reflected laser beam position, which is detected by a position-sensitive detector and produces topographic nucleosome images.
In AFM images, the histone core appears as a glowing blob with unwrapped DNA of increasing heights over time, indicative of nucleosome dynamics.
