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Exploring nucleosome unwrapping using DNA origami Jonas Jörg Funke, Philip Ketterer, Corinna Lieleg, Philipp Korber, and Hendrik Dietz, Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.6b04169 • Publication Date (Web): 09 Nov 2016 Downloaded from http://pubs.acs.org on November 10, 2016
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Exploring nucleosome unwrapping using DNA origami
Jonas J. Funke1, Philip Ketterer1, Corinna Lieleg4, Philipp Korber4, and Hendrik Dietz1,2,3 *
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Physik Department and 2Institute for Advanced Study, Technische Universität München, Am Coulombwall 4a, Garching bei München, Germany; 3Center for Integrated Protein Science, Munich; 4Biomedical Center, Molecular Biology, LMU Munich, Martinsried near Munich, Germany; * corresponding author’s e-mail address:
[email protected] 1
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ABSTRACT We establish a DNA origami based tool for quantifying conformational equilibria of biomolecular assemblies as a function of environmental conditions. As first application, we employed the tool to study the salt-induced disassembly of nucleosome core particles. To extract binding constants and energetic penalties we integrated nucleosomes in the spectrometer such that unwrapping of the nucleosomal template DNA from bent to more extended states was directly coupled to the conformation of the spectrometer. Nucleosome unwrapping was induced by increasing the ionic strength. The corresponding shifts in conformation equilibrium of the spectrometer were followed by direct conformation imaging using negative staining TEM and by FRET read out after gel electrophoretic separation of conformations. We find nucleosome dissociation constants in the picomolar range at low ionic strength (11 mM MgCl2), in the nanomolar range at intermediate ionic strength (11 mM MgCl2 with 0.5-1 M NaCl) and in the micromolar range at larger ionic strength (11 mM MgCl2 with ≥1.5 M NaCl). Integration of up to four nucleosomes stacked side-by-side, as it might occur within chromatin fibers, did not appear to affect the saltinduced unwrapping of nucleosomes. Presumably, such stacking interactions are already effectively screened at the nucleosome unwrapping conditions. Our spectrometer provides a modular platform with a direct read out to study conformational equilibria for targets from small biomolecules up to large macromolecular assemblies.
Keywords: DNA origami, nucleosome, FRET, TEM, force spectroscopy
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Programmable self-assembly with DNA origami allows creating custom-shaped nanoscale objects (1-6). Through this capacity, DNA origami enables constructing custom instruments to perform precision measurements of molecular interactions and structure, with enhanced control over positioning, orientating and manipulating the molecules under study. Researchers have just begun to exploit this capacity, for example, to study the collective behavior of molecular motors (7), to augment stochastic sensing with nanopores (8-10), to enhance NMR-based structure measurements (11, 12), to study vesicle fusion (13), and to perform measurements of weak intermolecular forces such as basepair stacking (14) or the interactions between nucleosomes (15). Here, we establish a tool for quantifying conformational equilibria that leverages the control over the placement of molecules on the nanoscale. The system enables coupling and amplifying structural changes of the molecule under study for detection via transmission electron microscopy (TEM) imaging, fluorescence measurements, and gel electrophoresis.
Our system builds on a previously described object consisting of two rigid DNA origami beams connected by a hinge (16, 17). The object was previously used for controlling the distance between two molecules with subnanometer accuracy (16). The hinge acts as a torsional spring that biases the configuration of the device toward a subset of preferred opening angles (15). Through this property, the object may be employed to exert forces on molecular binding partners, and it becomes thus a type of force spectrometer (15). One may use the device, for example, to quantify the forces between two molecular surfaces by analyzing the change in the distribution of opening angles that are adopted by the device with and without the forces between the molecular surfaces under study. Using this concept, we previously determined the distancedependent energy landscape for the weak stacking interactions between two nucleosome core 3
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particles (henceforth just “nucleosomes”). Also, Castro and coworkers recently employed a related device to explore the linker-length dependent partial unwrapping of nucleosomes (18).
Our device should also allow studying conformational equilibria of biomolecular assemblies as a function of environmental conditions. Inter- and intramolecular conformational equilibria may, of course, be studied by various existing methods, in particular using FRET spectroscopy (1922). However, the DNA origami spectrometer system offers a number of features that promise added value in such measurements: First, the binding partners under study may be displayed in close vicinity, in controlled orientations, and in controlled stoichiometry, which provides opportunities for interrogating multivalent interactions, next-neighbor interactions, and the effect of relative orientations. Second, a force bias may be exerted in a particular way to prepare otherwise rarely populated states. Third, the signals reported by the device (opening angles or FRET efficiencies) are distance-calibrated with near-atomic accuracy (16). Fourth, the dynamic range for molecular distance changes may be adjusted and expanded much beyond the typical range of FRET by placing the dye pair at different positions on the device (16). Fifth, simple gel electrophoresis may be used in a standardized fashion as read out for the conformational equilibrium. Potential difficulties arise from the need to conjugate the molecular targets to the spectrometer, but for conventional FRET studies also the fluorescent dyes would need to be conjugated to the molecule(s) under study.
The goal of the present study is to use the spectrometer for quantifying conformational equilibria and their dependence on environmental conditions which requires establishing the necessary experimental methods and data analysis. To this end we use imaging by TEM to track 4
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the conformational equilibrium of a molecular complex with direct structural feedback on the single particle level. On the ensemble-level and complementary to TEM, we monitor the equilibrium using FRET. As a first application, we study the salt-induced disassembly (“unwrapping”) of nucleosomes. The nucleosome is a disc-shaped DNA-protein complex with a diameter of ~10.5 nm and a height of ~4.5 nm (23-25) in which a 147 base pairs (bp) long DNA double-strand is wrapped 1.65-times around an octamer of histone proteins. Two copies of each histone protein (H2A, H2B, H3 and H4) form the histone octamer. In a nucleosome, the positively charged proteins interact with the negatively charged DNA. Therefore, the stability of this DNA-protein complex strongly depends on the ionic strength of the solution (26, 27). In vitro, the nucleosome can be reconstituted from a mixture of template DNA and histone octamers by salt gradient dialysis (28). The assembly proceeds through three major steps (23, 29, 30): the (H3-H4)2 tetramer associates to the DNA template at ~1 M NaCl forming the intermediate tetrasome; the H2A-H2B dimers associate to the tetrasome at ~0.8 M NaCl forming a partly wrapped nucleosome that relaxes at 0.6 M NaCl to a completely wrapped state. The saltinduced disassembly follows the pathway in reverse order (29).
We integrated the nucleosomes in the spectrometer so that a 41 bp-long segment of the template DNA connects both beams of the spectrometer (Fig. 1, Fig. S1). In the wrapped versus unwrapped state of the nucleosome, the template segment will be bent versus extended, respectively, which in turn will cause the spectrometer to adopt a smaller versus larger opening angle (Fig. 2A). The opening angle thus becomes an indicator for the conformation of the nucleosome, specifically, for its degree of unwrapping. In addition, we configured the spectrometer so that it may host up to four nucleosomes in an arrangement which may be likened 5
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to a stack of tires (Fig. 1A). This arrangement allows determining penalties for integration and enables also testing whether nucleosome stacking interactions affect unwrapping transitions. For the coupling, we used nucleosomes based on the Widom 601 sequence (31) that bear DNA single-strands as previously described (15) (Fig. S2). The nucleosomes were reconstituted by salt gradient dialysis with a branched DNA template such that two DNA single strands protruded radially and opposite from each other from the nucleosome. We hybridized those strands to complementary single strands that are displayed at selected positions on the two beams of the spectrometer (Fig. 1 A, B and Fig. S1, S3).
RESULTS AND DISCUSSION We prepared spectrometer variants featuring one, two, or four nucleosomes (Fig. 1C) and imaged these samples using negative-staining transmission electron microscopy (TEM). The micrographs showed clearly disc-shaped features at the designed positions and in the designed orientations (Fig. 1C, D). Moreover, the transmission contrast of the disc features also depended clearly on the number of nucleosomes hosted by the spectrometer (Fig. 1D). For example, the disc-features in the samples designed to include one versus four nucleosomes had the lowest versus highest transmission contrast (Fig. 1D). Thus, TEM imaging confirmed the successful incorporation of nucleosomes into the spectrometer according to design.
To reveal the salt-induced disassembly of nucleosomes (Fig. 2A) by TEM, we imaged spectrometer particles containing one nucleosome (same sample as in Fig. 1C) in the presence of four different concentrations of sodium chloride (see Fig S4 and S5 for representative micrographs). As the nucleosomes could be clearly discerned in individual particle micrographs, 6
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we could sort the image data according to whether or not the particles featured a nucleosome. We measured the opening angles of individual spectrometer particles and determined the frequency at which particular angles were populated in the particle subsets with and without nucleosome. The frequency of particles featuring nucleosomes clearly decreased with increasing salt concentration (Fig. 2B, C). In the absence of sodium chloride, 52% of the particles had integrated nucleosomes, and the distribution of opening angles for those particles peaked at 50° opening angle. The particles lacking a nucleosome sampled a broader spectrum of opening angles that was shifted toward greater opening angles (Fig. 2C). The nucleosome-free distribution was consistent with previous measurements (15) and reflects the conformational freedom of the spectrometer without an element connecting the two beams. In the presence of 500 mM sodium chloride, the fraction of particles with nucleosomes remained approximately constant at 58% but the angle distribution for particles with nucleosomes became narrower compared to the one obtained in the absence of sodium chloride (Fig. 2C). Small opening angles below 40° were more rarely populated in the presence of 500 mM NaCl than in the absence of NaCl. Close inspection of the particles having small opening angles
