Probing Nucleosome Stability with a DNA Origami Nanocaliper

Abstract

The organization of eukaryotic DNA into nucleosomes and chromatin undergoes dynamic structure changes to regulate genome processing, including transcription and DNA repair. Critical chromatin rearrangements occur over a wide range of distances including the mesoscopic length scale of tens of nanometers. However, there is a lack of methodologies that probe changes over this mesoscopic length scale within chromatin. We have designed, constructed, and implemented a DNA-based nanocaliper that probes this mesoscopic length scale. We developed an approach of integrating nucleosomes into our nanocaliper at two attachment points with over 50% efficiency. Here, we focused on attaching the two DNA ends of the nucleosome to the ends of the two nanocaliper arms, so the hinge angle is a readout of the nucleosome end-to-end distance. We demonstrate that nucleosomes integrated with 6 bp, 26 bp and 51 bp linker DNA are partially unwrapped by the nanocaliper by an amount consistent with previously observed structural transitions. In contrast, the nucleosomes integrated with the longer 75 bp linker DNA remains fully wrapped. We found that the nanocaliper angle is a sensitive measure of nucleosome disassembly and can read out transcription factor (TF) binding to its target site within the nucleosome. Interestingly, the nanocaliper not only detects TF binding, but it significantly increases the probability of TF occupancy at its site by partially unwrapping the nucleosome. These studies demonstrate the feasibility of using DNA nanotechnology to both detect and manipulate nucleosome structure, which provides a foundation of future mesoscale studies of nucleosome and chromatin structural dynamics.

A DNA-based synthetic apoptosome

“…DNA origami-based nanostructures, in particular, are well-suited as synthetic platforms as their unique addressability allows for precise assembly of multiple non-identical proteins with nanometer accuracy [18][19][20] . The DNA origami technique has found broad applicability as an experimental tool for spatial organization of native multi-protein systems, such as amyloid fibrils 21 , membrane fusion proteins 22 , nucleosomes 23,24 , and intrinsically disordered proteins 25 . Additionally, these platforms have been used to engineer localized genetic circuits 26 , to study confinement-induced enzyme activity 27 , and to investigate scaffolded metabolic cascades [28][29][30][31] .…”

Integrating computer-aided engineering and computer-aided design for DNA assemblies

“…The precise control over geometry of DNA assemblies 16-19 make them highly attractive for applications such as drug delivery 20 , templating a variety of materials or molecules 21-25 , nanoscale measurement tools 26,27 , and molecular robotics 28-33 . However, DNA-based design approaches have largely overlooked material properties, which limits the structural, mechanical, and functional complexity.…”

“…The recent emergence of high fidelity coarse-grained molecular dynamics (MD) simulation tools for DNA nanostructures 10-15 provide an opportunity to realize CAE for DNA-based design to enable systems with new levels of structural complexity that can also be tailored for functional properties such as reconfiguration, mechanical properties, or stimulus response. Here we present an ICME approach for DNA assemblies that relies on a custom CAD tool with several features that enhance the scope of geometric design and facilitate tight integration with coarse-grained MD models [10][11][12] to enable CAE for complex DNA assemblies.The precise control over geometry of DNA assemblies 16-19 make them highly attractive for applications such as drug delivery 20 , templating a variety of materials or molecules 21-25 , nanoscale measurement tools 26,27 , and molecular robotics 28-33 . However, DNA-based design approaches have largely overlooked material properties, which limits the structural, mechanical, and functional complexity.…”

MrDNA: A multi-resolution model for predicting the structure and dynamics of nanoscale DNA objects

“…The mrdna framework can be used to predict the average structure and the conformational fluctuations of such objects. For example, the Dietz and Castro groups have designed nanoscale calipers using DNA origami that allow measurement of intermolecular forces between proteins attached to each arm of the caliper 55,56 . TEM can be used to determine the distribution of angles of the caliper with and without attached proteins, allowing inference of intermolecular forces.…”