Talk abstracts
Talk on Wednesday 03:30-03:45pm submitted by Jenny Le
Characterization of nucleosome structure using DNA origami
J.V. Le (Interdisciplinary Biophysics Graduate Program), Yi Luo (Interdisciplinary Biophysics Graduate Program), Christopher Lucas (Department of Mechanical of Engineering and Aerospace), Michael Poirier (Department of Physics), Carlos Castro (Department of Mechanical of Engineering and Aerospace)
Abstract:
While biophysical tools such as single molecule fluorescence and force spectroscopy have been used to study the structural dynamics of individual nucleosomes or large chromatin assemblies, it is challenging to probe conformational dynamics of gene regulation in the critical 10-100nm range. This work aims to develop DNA origami tools to probe the mesoscale structure and dynamics of nucleosomes and chromatin. DNA origami itself is a recently established nanotechnology that enables the self-assembly of precisely designed nanostructures. Here, we implement a DNA origami hinge structure with the length scale of ~100nm as a nano-caliper to study the structure and dynamics of a single nucleosome.
The device was calibrated by integrating DNA linkers to constrain the hinge arms, showing that it is possible to use the hinge angular distribution as a readout for the size of the sample attached between the arms. We further integrated single nucleosomes with varying but symmetric lengths of DNA linkers to verify our ability to detect structural changes in nucleosomes. Our immediate goal is to measure the structural changes in single nucleosomes and nucleosome arrays in response to protein binding. Initial experiments using the hinge-nucleosome construct with GAL4-VP16, a hybrid transcription factor capable of highly-efficient transcription activation when bound near the promoter region of the gene , shows that the angular distribution broadens as a result of increasing TF titrations. Our measurements revealed a dissociation constant in the range of 1-10 nM, which agrees with bulk measurements. Furthermore, preliminary results with H1, which is known to cause compaction of nucleosomes and chromatin, demonstrated a physical shift in nucleosome position rather than the angular distribution.
These results demonstrate the potential of a DNA origami device probing chromatin structure and function in vitro, in particular, the ability to measure structural changes in the range of 10-100nm.
References:
Marras, A. E., L. Zhou, H. J. Su and C. E. Castro. "Programmable Motion of DNA Origami Mechanisms." Proc Natl Acad Sci U S A, (2015).
Poirier, M. G., E. Oh, H. S. Tims and J. Widom. "Dynamics and Function of Compact Nucleosome Arrays." Nat Struct Mol Biol 16, no. 9 (2009): 938-44.
Sadowski, I., J. Ma, S. Triezenberg and M. Ptashne. "Gal4-Vp16 Is an Unusually Potent Transcriptional Activator." Nature 335, no. 6190 (1988): 563-4.
Keywords: DNA origami, nucleosomes, structural dynamics