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 C2 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 C2 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.