Skip to Main Content
 

Global Search Box

 
 
 

ETD Abstract Container

Abstract Header

Tunable Nanocalipers to Probe Structure and Dynamics in Chromatin

Le, Jenny Vi, Le
http://rave.ohiolink.edu/etdc/view?acc_num=osu1543163132011865

Abstract Details

2018, Doctor of Philosophy, Ohio State University, Biophysics.
Scaffolded DNA origami uses DNA as building blocks to create 3D nanostructures. Dynamic 3D DNA origami nanostructures have strong potential in biophysical experiments and applications. This includes measuring biomolecular forces, particularly on the nanoscale. Current biophysical methods lack the ability and/or specificity to measure on such a scale, in addition to being challenging and expensive. Dynamic DNA origami can address such a problem by providing a malleable material to accommodate shape and function on the 10-100nm length scale. The major goal of this work is to leverage DNA origami to develop tools to study biophysical properties of the biomolecular complexes. 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 structure and dynamics at the 10-100nm length scale (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 accessibility and processing. We designed, constructed, and implemented a nanocaliper via DNA origami. Our nanocalipers are hinge-like joints that consist of two 70nm rigid arms, each made up of bundled DNA helices, connected along one edge by single stranded DNA. For proof-of-concept, we first studied a single nucleosome by binding the two nucleosomal DNA ends to the ends of nanocaliper arms. Here, the caliper angle reports the nucleosome end-to-end distance. Moreover, the caliper angle is sensitive to nucleosome stability as a function of NaCl concentration. 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 unwrap the nucleosome. This suggests that our DNA nanocalipers can report biologically relevant conformational changes and manipulate nucleosomes to test and engineer 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 biomolecule and then apply a tensile/compressive force. This research provides a foundation of future mesoscale studies of nucleosome arrays and chromatin structural dynamics. Broader impacts include these tools directly monitoring or manipulating local chromatin structure and dynamics in single living cells.
Carlos Castro (Advisor)
Michael Poirier (Committee Member)
Marcos Sotomayor (Committee Member)
Jonathan Song (Committee Member)
242 p.
Biochemistry; Biomechanics; Biophysics; Mechanical Engineering
DNA origami; nucleosomes; structural DNA nanotechnology; tunable nanocaliper; 3D origami; nucleosome arrays; nucleic acids

Recommended Citations

Citations

  • Le, Le, J. V. (2018). Tunable Nanocalipers to Probe Structure and Dynamics in Chromatin [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1543163132011865

    APA Style (7th edition)

  • Le, Le, Jenny. Tunable Nanocalipers to Probe Structure and Dynamics in Chromatin. 2018. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1543163132011865.

    MLA Style (8th edition)

  • Le, Le, Jenny. "Tunable Nanocalipers to Probe Structure and Dynamics in Chromatin." Doctoral dissertation, Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1543163132011865

    Chicago Manual of Style (17th edition)

osu1543163132011865
66
© 2018, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.