Talk abstracts
Talk on Tuesday 09:15-09:30am submitted by Jenny Le
Probing Nucleosome Stability with a DNA Origami Caliper
Jenny V. Le (Biophysics Graduate Program), Yi Luo (Wyss Institute, Harvard), Michael A. Darcy (Department of Biophysics, OSU), Michael G. Poirier (Department of Physics, OSU), Carlos E. Castro (Department of Mechanical Engineering and Aerospace, OSU)
Abstract:
Nucleosomes, consisting of DNA wrapped around a protein core, assemble into higher orders of structure as chromatin. However, we lack methods to probe chromatin structure and structural changes relevant for gene regulation over the 10-100nm mesoscopic length scale. To address this challenge, we designed, constructed, and implemented a nanocaliper via DNA origami, a method that uses DNA as building blocks to assemble complex 3D nanostructures. Our nanocalipers are hinge-like joints that consist of two 70nm rigid arms, each made up of bundled DNA helices, connected by single stranded DNA.
The nanocaliper has two DNA ends of a nucleosome bound to the ends of its arms. The hinge angle reads out the nucleosome end-to-end distance. We demonstrate that nucleosomes integrated with 6, 26, and 51 base pair (bp) linker DNA are partially unwrapped by the nanocaliper by an amount consistent with previously observed structural transitions. Also, the nanocaliper detects transcription activator Gal4-VAP16 binding and significantly increases the probability of Gal4-VP16 occupancy at its target site by partially unwrapping the nucleosome. This suggests that our DNA nanocalipers can read out biologically relevant conformational changes and manipulate nucleosomes to test their function. We also investigate the nanocaliper’s ability to monitor nucleosome wrapping using histone linker H1 and chromatin remodeler Swi6. Preliminary results for H1 show increased wrapping of a 75bp linker nucleosome. Furthermore, no nonspecific binding of Swi6 was evident without a methylated Lysine 9 on histone H3, suggesting a negative control.
This project provides a foundation of future mesoscale studies of nucleosome and chromatin structural dynamics. More broadly, such studies provide insight into molecular events that lead to misregulation of oncogenes and tumorigenesis. Ultimately, these tools could directly monitor or manipulate local chromatin structure and dynamics in single living cells.
References:
[1] Sadowski, I., et al., Gal4-VP16 is an unusually potent transcriptional activator. Nature, 1988. 335(6190): p. 563-4.
[2] Luo, Y., et al., Nucleosomes accelerate transcription factor dissociation. Nucleic Acids Res, 2014. 42(5): p. 3017-27.
[3] Le, J.V. et al. Probing Nucleosome Stability with a DNA Origami Caliper ACS Nano 2016
Keywords: DNA nanotechnology, Nucleosomes, DNA origami