Poster abstracts
Poster number 35 submitted by Jenny Le
Tunable DNA Nanocalipers to Probe Structure and Dynamics of Chromatin
Jenny V. Le (Biophysics Interdisciplinary Program), Dengke Zhao (Biophysics Interdisciplinary Program), Michael A. Darcy (Physics), Cassady Croy (Columbus State Community College), Ralf Bundschuh, Michael G. Poirier (Physics), Carlos E. Castro (Mechanical and Aerospace Engineering)
Abstract:
Nucleosomes , consisting of genomic DNA wrapped around a protein core, assemble into higher orders of chromatin structure to compact DNA. Tools to probe site-specific chromatin at the 10-100nm lengthscale (relevant for gene regulation) and to apply tensile or compressive forces at targeted sites could greatly improve insight into how chromatin structural dynamics regulate DNA processing. We designed, constructed, and implemented a nanocaliper via DNA origami, a method using 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.
For proof-of-concept, we bind the two nucleosomal DNA ends to the ends of nanocaliper arms. Here, the caliper angle reports the nucleosome end-to-end distance. We demonstrated the nanocaliper can detect nucleosome conformational changes via transcription activator Gal4-VP16 binding. The caliper also significantly increases the probability of Gal4-VP16 occupancy by applying a tension to partially unwraps the nucleosome. This suggests that our DNA nanocalipers can report biologically relevant conformational changes and manipulate nucleosomes to test their function.
We developed a model that accurately describes our nucleosome-nanocaliper assemblies as concomitantly (simultaneous but independent) unwrapping, further validated by hexasome-nanocaliper measurements. This model demonstrates nucleosome unwrapping is sensitive to the caliper’s applied force, motivating a design that applies tunable tensile/compressive forces. We created a new nanocaliper with tunable stiffness and equilibrium angle, for example, to incorporate a sample and then apply a tensile/compressive force.
This project provides a foundation of future mesoscale studies of nucleosome arrays and chromatin structural dynamics. 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