In Situ Nucleosome Assembly for Single-Molecule Correlative Force and Fluorescence Microscopy

Single-molecule techniques are powerful tools to study the mechanics, coordination, and composition of chromatin systems, and therefore, researchers are always looking for better methods to generate nucleosome substrates. Here, we describe a protocol to form nucleosomes across DNA in situ in a single-molecule correlative force and fluorescence microscope. Typically, nucleosome substrates are made by assembling nucleosomes on DNA containing strong positioning sequences via salt dialysis.

Although this has advantages, it generates artificially stable nucleosomes and is heavy-handed with reagents. Our protocol prepares nucleosome substrates for single-molecule correlated force and fluorescence microscopy without specific DNA sequences and with much less reagents, all within minutes. This protocol enables nucleosome assembly on native DNA sequences, easy adjustment of nucleosome density, as well as less preparation time and use of reagents.

Additionally, the formation of nucleosomal DNA tethers in situ enable simpler experimental workflow and the convenience of built-in single molecule visualization and manipulation. When uniform and specific nucleosome positioning is not an essential part of the experiment, our findings can help scientists study chromatin and its mining proteins at the single molecule level more efficiently. With the time and resources saved, more experiments can be done to further investigate additional variables and conditions.

Applicable research areas that we're currently thinking about include chromatin mechanics that are regulated by histone variants and other post-translational modifications. We're also thinking about the biophysical engagements and kinetics of other chromatin-binding proteins. And finally, we're also thinking about the nucleosome-driven higher order assembly processes, such as biomolecular condensation.

To begin, add 500 microliters of protein-free image buffer to channel three of the flow cell. Mix two nanomolar of Saccharomyces cerevisiae Nap1 and two to five nanomolar of LD655 labeled H4 histone octamer with 500 microliters of 1XHR buffer. Add this mixture to channel four of the flow cell.

Flow all the channels at 1.0 bar for 30 seconds to flush them with samples. Set the flow to 0.2 bar, and move the optical traps to channel 1 to catch a suitable pair of streptavidin-coated beads. Next, move the optical traps to channel 3.

Turn off the flow, and conduct a force calibration for the bead pair. Turn on the red confocal laser, and calibrate the imaging area. After setting the flow to 0.2 bar, move the optical traps to channel 2 to catch biotinylated DNA.

Then move the optical traps to channel 3, and stop the flow. Increase the distance between the optical traps to stretch the DNA, and monitor the associated forced distance curve to confirm the presence of a single DNA tether. Adjust the distance between the optical traps so that the DNA tension reads approximately one piconewton.

Turn on the line scanning to start collecting a kymograph with the red laser on. Then move the optical traps to channel 4 with the flow off to form nucleosomes along the DNA tether. For unwrapping nucleosomes, move the optical traps to channel 3.

Setting the force reading to zero and speed to 0.1 micrometers per second, move the optical trap 1 away from the optical trap 2. Mix 10 nanomolar of Cy3-labeled linker histone H1.4 to 500 microliters of image buffer, and add to channel 5. After forming a DNA tether containing nucleosomes, turn on the green laser in addition to the red laser, and move optical traps to channel 5 for real-time imaging of H1 binding to chromatin.

Nucleosome formation along the DNA tether was visualized as stationary red fluorescent foci on A 2D scan and kymograph. Photobleaching analysis showed one-step or two-step photobleaching events, indicating the presence of mononucleosomes. In the absence of Nap1, histone octamer bound DNA, but remained mostly unwrapped, indicated by the constant tension.

Using Cy3-labeled H2a, similar nucleosome assembly results were obtained as with LD655-labeled H4.Nucleosome unwrapping increased tether distance over time, visible on the kymograph, and generated forced distance curves with characteristic rips that denote the unwrapping of individual nucleosomes. Linker histone H1 binding to chromatin was indicated by green fluorescent foci with dual-color stationary trajectories representing the binding of H1 to nucleosomes and green jagged trajectories representing diffusing H1 trajectories.