1 research outputs found
Exploring Nucleosome Unwrapping Using DNA Origami
We
establish a DNA origami based tool for quantifying conformational
equilibria of biomolecular assemblies as a function of environmental
conditions. As first application, we employed the tool to study the
salt-induced disassembly of nucleosome core particles. To extract
binding constants and energetic penalties, we integrated nucleosomes
in the spectrometer such that unwrapping of the nucleosomal template
DNA, leading from bent to more extended states was directly coupled
to the conformation of the spectrometer. Nucleosome unwrapping was
induced by increasing the ionic strength. The corresponding shifts
in conformation equilibrium of the spectrometer were followed by direct
conformation imaging using negative staining TEM and by FRET read
out after gel electrophoretic separation of conformations. We find
nucleosome dissociation constants in the picomolar range at low ionic
strength (11 mM MgCl<sub>2</sub>), in the nanomolar range at intermediate
ionic strength (11 mM MgCl<sub>2</sub> with 0.5–1 M NaCl) and
in the micromolar range at larger ionic strength (11 mM MgCl<sub>2</sub> with ≥1.5 M NaCl). Integration of up to four nucleosomes
stacked side-by-side, as it might occur within chromatin fibers, did
not appear to affect the salt-induced unwrapping of nucleosomes. Presumably,
such stacking interactions are already effectively screened at the
nucleosome unwrapping conditions. Our spectrometer provides a modular
platform with a direct read out to study conformational equilibria
for targets from small biomolecules up to large macromolecular assemblies