Engineering Mononucleosomes for Single-Pair FRET Experiments
In DNA nanotechnology, DNA is used as a structural material, rather than as an information carrier. The structural organization of the DNA itself determines accessibility to its underlying information content in vivo. Nucleosomes form the basic level of DNA compaction in eukaryotic nuclei. Nucleosomes sterically hinder enzymes that must bind the nucleosomal DNA, and hence play an important role in gene regulation. In order to understand how accessibility to nucleosomal DNA is regulated, it is necessary to resolve the molecular mechanisms underlying conformational changes in the nucleosome. Exploiting bottom-up control, we designed and constructed nucleosomes with fluorescent labels at strategically chosen locations to study nucleosome structure and dynamics in molecular detail with single-pair Fluorescence Resonance Energy Transfer (spFRET) microscopy. Using widefield total internal reflection fluorescence (TIRF) microscopy on immobilized molecules, we observed and quantified DNA breathing dynamics on individual nucleosomes. Alternatively, fluorescence microscopy on freely diffusing molecules in a confocal detection volume allows a fast characterization of nucleosome conformational distributions.
Key wordsSingle-molecule fluorescence Nucleosome Reconstitution Fluorescence resonance energy transfer
We thank Andrew Routh (MRC Cambridge) for samples of micrococcal nuclease-digested nucleosome core particles and useful discussion, Alexander Brehm (University of Marburg) for histone octamer preparations, and Jürgen Groll (RWTH Aachen) for providing samples of the NCO-star PEG material and support with the coating procedure.
This work is part of the research programme of the “Stichting voor Fundamenteel Onderzoek der materie (FOM),” which is financially supported by the “Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO).”