Construction of a Polyhedral DNA 12-Arm Junction for Self

Wireframe structures, on the other hand, are less susceptible to salt ..... free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b0...
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Construction of a Polyhedral DNA 12-Arm Junction for Self-Assembly of Wireframe DNA Lattices Ilenia Manuguerra,†,‡,§ Guido Grossi,†,‡,∥ Rasmus P. Thomsen,†,‡,∥ Jeppe Lyngsø,‡ Jan S. Pedersen,‡ Jørgen Kjems,†,‡,∥ Ebbe S. Andersen,†,‡,∥ and Kurt V. Gothelf*,†,‡,§ †

Center for DNA Nanotechnology, ‡Interdisciplinary Nanoscience Center, §Department of Chemistry, and ∥Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark S Supporting Information *

ABSTRACT: A variety of different tiles for the construction of DNA lattices have been developed since the structural DNA nanotechnology field was born. The majority of these are designed for the realization of close-packed structures, where DNA helices are arranged in parallel and tiles are connected through sticky ends. Assembly of such structures requires the use of cation-rich buffers to minimize repulsion between parallel helices, which poses limits to the application of DNA nanostructures. Wireframe structures, on the other hand, are less susceptible to salt concentration, but the assembly of wireframe lattices is limited by the availability of tiles and motifs. Herein, we report the construction of a polyhedral 12-arm junction for the self-assembly of wireframe DNA lattices. Our approach differs from traditional assembly of DNA tiles through hybridization of sticky ends. Instead, the assembly approach presented here uses small polyhedral shapes as connecting points and branch points of wires in a lattice structure. Using this design principle and characterization techniques, such as transmission electron microscopy, single-particle reconstruction, patterning of gold nanoparticles, dynamic light scattering, UV melting analyses, and small-angle X-ray scattering among others, we demonstrated formation of finite 12-way junction structures, as well as 1D and 2D short assemblies, demonstrating an alternative way of designing polyhedral structures and lattices. KEYWORDS: DNA nanotechnology, DNA junction, DNA lattice, DNA tiles, hierarchical assembly, TEM, DLS

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multiarm junctions has never been accomplished due to the flexibility of such junctions, yielding undefined orientation of the arms radiating from the branch point. Furthermore, tilebased DNA assemblies rely on weak reversible interactions through short sticky ends, making the lattice sensitive to variation in temperature and salt concentration. This issue has been recently overcome by introducing external agents, such as chemical cross-linkers, during the post-assembly stabilization process, making the formation of stable lattices a two-step process.16−19 Here, in the attempt to form stable lattices in a one-step process, the use of sticky ends was avoided by designing junctions that can act both as branch points and as connecting points among different tiles (Figure 1).

ince its inception, the major goal of structural DNA nanotechnology has been to use oligonucleotides as structural building material to create shapes and patterns with nanometer-scale precision. Many DNA-based building blocks have been designed and tested for their ability to selfassemble in a periodic manner1−5 forming one-, two-, and three-dimensional (1D, 2D, and 3D, respectively) lattices potentially useful in the formation of protein arrays6,7 and crystallography.8 Tiles are often designed to form close-packed 2D and 3D structures, where bundles of helices are arranged in close proximity.1−6 Therefore, relatively high salt concentrations are required to stabilize the folding of these structures, limiting the applicability of such DNA architectures to in vitro systems.9,10 Oppositely, wireframe structures show stability in physiological conditions and are cost-effective, due to the minimal amount of DNA used.11,12 The development of alternative DNA motifs, branched junctions, in particular, is essential for the assembly of complex wireframe structures and lattices. The production of star-like DNA junctions bearing 4, 5, 6, 8, and 12 arms has been reported,13−15 but the construction of lattices using complex © 2017 American Chemical Society

RESULTS AND DISCUSSION Design Principles. Here, we report the construction of a DNA 12-way junction with a predefined tertiary structure. The Received: May 20, 2017 Accepted: August 14, 2017 Published: August 14, 2017 9041

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Figure 1. Schematic representation of the possible hierarchical folding processes for a wireframe DNA lattice. (a) Traditional hierarchical folding, where junctions are formed first (monomer) and then lattice assembles through short sticky ends on the arms. (b) Inverse hierarchical folding employed in this study, where wires are self-assembled first (monomer) and then branched junctions are formed in the lattice.

Figure 2. Geometry of the 12-way junction. (a) Light blue lines represent the cuboctahedron; black lines indicate double-stranded DNA. Each triangular face of the polyhedron includes one three-way junction. (b) Rendered image of the 12-way junction (on the black skeleton from (a)) with angular views after 45 and 90° rotation from the original.

junction is designed to have 12 arms symmetrically arranged around the center in three dimensions. The DNA components self-assemble along a thermal annealing ramp into a wireframe cuboctahedron, forming the core of the junction, which has arms protruding from each of its 12 vertices (Figure 2 and Supporting Figure S1). Such design does not allow free movement of the junctions nor the arms. Hence, it represents a more suitable junction for developing complex DNA architectures compared to previously reported 12-way junctions.13 The design principles hereby described allow assembly of the 12-way junction both as monodispersed cuboctahedron structure (0D) and as monomeric unit for 1D, 2D, and 3D structures (Figure 3). The 0D structure is built using 24 DNA strands that pair forming 12 “half double-stranded tiles” (HDSTs). Each HDST is composed by one double-stranded moiety (8 bp) that constitutes the arms of the junction and two single-stranded moieties (16 nt each) that anneal to complementary sequences on two other HDSTs (annealing for 8 bp with each additional HDST, Supporting Figure S2a), resulting in a wireframe cuboctahedron unit exposing 12 protruding arms. In order to produce 1D and 2D lattices, HDSTs were employed in combination with “double-stranded tiles” (DSTs). A typical DST is constituted by two DNA strands that pair

along the central region of the tile (16 bp) exposing four unpaired ends (16 nt each). Similarly to the HDSTs, each 16 nt single-stranded end hybridizes with two other DSTs (annealing for 8 bp with each additional DST), forming a three-way junction (Supporting Figure S2a). Due to the different lengths of the complementary regions, the lattice forms following a hierarchical self-assembly process where the DSTs form first and subsequently assemble into either 1D or 2D lattices. According to the proposed design, lattice formation does not happen through sticky end interactions among preformed junctions, as in previously reported works,20−24 but along the formation of small polyhedral nanostructures. We therefore define this process “inverse hierarchical assembly” for its ability to assemble wires first and junctions later of a wider lattice structure (Figure 1). Characterization of the 0D 12-Way Junction. The assembly of the 0D structure was first analyzed by an electrophoretic mobility shift assay (EMSA; Supporting Figure S2). Native gel analysis shows slower electrophoretic mobility with increasing number of components present in the annealed mixture, indicating that the HDSTs self-assemble in one defined structure. Negative stain transmission electron microscopy (nsTEM) was used to verify the shape of 0D. In spite of the small size of the wireframe nanostructure, raw nsTEM images support 9042

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Figure 3. Graphic representation of the assembly scheme of 0D, 1D, 2D, and 3D lattices. Different pools of oligonucleotides form half doublestranded tiles (HDSTs) and double-stranded tiles (DSTs). The 12-way junction (0D) is composed by 12 HDSTs, whereas the 3D lattice (3D) is composed by 6 DSTs. The 1D and 2D lattices (1D and 2D, respectively) are formed by different combinations of HDSTs and DSTs. The assembly process follows a cooling ramp where tiles are formed first and lattices later.

of the population that measured ∼250 nm (Figure 5h). The analysis showed that most of the structures were close to the size of the 0D, probably due to low efficiency in polymerization along one dimension. TEM micrographs (Supporting Figure S7) also confirm that the sample contains short oligomers but is mainly composed by monomers, accounting for the short size of the 1D lattice (∼18 nm) found by DLS. The formation of a small population that measures ∼250 nm may be due to larger 1D assemblies not present in the TEM study with AuNPs, due to the large cargo attached to the structure that makes it more unstable. DLS analysis of the 2D lattice showed particles of 40 ± 0.5 nm in size, with a fraction of highly polymerized structures that measured ∼400 nm (Figure 5l, negative control in Supporting Figure S6). These data indicate formation of large 2D assemblies, as confirmed by TEM analyses, where more extended even though less ordered structures are visible (Supporting Figure S8). The fact that more extended assemblies are formed when DTSs are increased from one to two indicates that the monomers polymerize in a specific manner using the DTSs, as intended from the rational design. Three-Dimensional Lattice. The design of the 12-way junction allows production of extended 3D lattices with octahedral symmetry by using 12 DSTs and no HDSTs (Figure 3). Due to the complex nature of the resulting wireframe DNA lattice, the assembly product could not be characterized by either EMSA, nsTEM (Supporting Figure S9), or DLS. However, self-assembly of the 3D structure led to

correct formation of a hollowed structure (Figure 4). Further 2D and 3D single-particle reconstruction yields strong support of correctly assembled cuboctahedral 12-way junctions. Size and features of the computed projections of the 0D are in good agreement with the model shown in Figure 4 (Supporting Figures S3 and S4). As expected, the short protruding arms of the 0D are not visible due to the limited resolution of the nsTEM technique. The size distribution profile of 0D in solution was determined by dynamic light scattering (DLS). The hydrodynamic diameter of the 0D was calculated to be 15 ± 0.6 nm (Figure 5d), a value that corresponds to the theoretical diameter of the cuboctahedral structure (see Supporting Figure S5 for calculations) and is in agreement with the TEM analysis (Figure 4). Characterization of 1D and 2D Polyhedral Lattices. A biotin-modified HDST was employed to scaffold streptavidin− gold nanoparticle conjugates (STV-AuNPs) on the structure, for enabling characterization of self-assembled 1D and 2D lattices without the use of stain. Patterning of STV-AuNPs was obtained for both 1D and 2D lattices. Up to six consecutive AuNPs were visualized on 1D (Figure 5g), and fully occupied arrays of 3 × 4 AuNPs were identified on 2D (Figure 5k). Furthermore, the size distribution profile of 1D and 2D lattices in solution was measured by DLS. The average hydrodynamic diameter of the 1D lattice was determined to be 18 ± 1.3 nm for the majority of the structures in the sample, but with ∼5% 9043

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Figure 4. Structural characterization of the 12-way junction by nsTEM. Left panel: Typical nsTEM raw image that shows the formation of hollowed wireframe structures. Scale bar: 50 nm. Right panel: Model, class average, and 3D reconstruction of the particles from different points of view. Protruding arms are not visible due to low electron density of the sample. Scale bar: 10 nm.

design principles because when one tile is missing the structure loses stability.

samples with turbid appearance (Figure 5o), indicating that complex structures with size larger than the wavelength of visible light are formed. Since noncrystalline order in the 3D assembly was observed by small-angle X-ray scattering (SAXS) (Supporting Figure S11), we speculate that the 3D structure most likely resembles a DNA hydrogel, where branched structuresmade of double-stranded DNAare in a semiordered state.25 We speculate that the lack of crystalline order may be due to an inherent twist of the structure: the DSTs form 16 bp between two junction monomers, but this distance does not correspond to exactly 1.5 turns (i.e., 540°); after 16 bp, the strands have rotated ∼549° (1 bp = 34.3°). This means that two consecutive junctions are slightly tilted relative to each other, and since such effect is not counter-balanced, it propagates along the lattice, extending the local distortion to a global defect. To further investigate the formation of such structure, melting curves of the 3D lattice and variants were measured. The 3D lattice showed a melting temperature of 50 °C (Supporting Figure S10b, upper panel), but when the same lattice is assembled in the absence of one DST (3D−DST), a lower melting temperature is measured, indicating reduced stability of the structure (Supporting Figure S10b, lower panel). Furthermore, the comparison of the melting curves for the 3D lattice, the 3D−DST and a single-stranded DNA that shows minimal secondary structure (ssDNA), indicating that the 3D lattice consists of a double-stranded structure at the beginning of the heating ramp, whereas the 3D−DST and the ssDNA samples consist of ∼40 and ∼80% of single strands, respectively (Figure 5n). After the denaturing ramp, the samples were cooled and heated again. The resulting curves show that the 3D and the 3D−DST structures do not fold back into the lattice structure (Supporting Figure S10a). These results show that a 12 h cooling ramp is necessary to form a large double-stranded structure, but it also suggests that the structure follows the

CONCLUSIONS We have introduced an approach to assemble DNA tiles in a hierarchical fashion. Instead of assembling the junctions of the lattice first, and then in a second step assemble them by short sticky end extensions, we have applied an “inverse” approach where the wires (DSTs) are formed first, and upon interaction of these, the junctions form. To verify this, we have rationally designed a polyhedral 12way junction, which was shown to form starting from 12 half double-stranded tiles. The structure was characterized by EMSA, DLS, nsTEM, and single-particle reconstruction. The design principle underlying this structurally defined junction was further exploited to obtain 1D and 2D lattices used to scaffold AuNPs in ordered patterns. Furthermore, it was attempted to form a 3D lattice with octahedral symmetry, upon polymerization in three dimensions. We obtained formation of a complex double-stranded structure that resembles a DNA hydrogel, but no crystalline order was observed by SAXS. We believe that this “inverse” hierarchical assembly strategy has potential as an alternative approach to form wireframe 3D lattice structures and that future design with optimized geometry may provide crystalline order. MATERIALS AND METHODS Reagents. All reagents were purchased from Sigma-Aldrich Co. unless otherwise stated. All DNA strands were acquired from Integrated DNA Technologies, Inc. Folding Protocol. The samples were assembled in TAE 1× buffer with 11 mM MgCl2 at 12 μM concentration, except for the following: samples for DLS that were folded at 1 μM final concentration in 100 μL final volume, samples for the melting curves that were assembled at 25 μM and then diluted to 500 μL with buffer prior the analysis, and 9044

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Figure 5. Nanostructure and lattice characterization overview. (a−c) Rendered model (a), class average (b), and 3D reconstruction (c) of 0D from nsTEM imaging. (e−g) Rendered model of 1D (e), rendered model (f), and TEM micrograph (g) of STV-AuNPs patterned on 1D. (i− k) Rendered model of 2D (i), rendered model (j), and TEM micrograph (k) of STV-AuNPs patterned on 2D. (d,h,l) DLS analysis of the 0D, 1D, and 2D. (m,n) Rendered model (m) and melting curve analysis (n) of 3D. (o) Images of 3D sample (left) and control sample containing a single-stranded DNA that does not show secondary structures (right) after self-assembly reaction. Scale bars: 10 nm. samples for SAXS that were folded at 1 mg/mL (7.6 μM) in 50 μL final volume. All the structures were assembled using a thermal gradient from 95 to 23 °C in 12 h. DLS Protocol. Samples for DLS were folded at 1 μM final concentration, diluted to 300 μL final volume with buffer, and analyzed using Nanosizer Nano from Malvern. Data were processed using the appropriate software distributed by Malvern. Sample Preparation for nsTEM. Folded samples (5 μL) were deposited on glow-discharged 200 mesh carbon-coated copper TEM grids (Ted Pella Inc.) for 30 s and stained with 1% uranyl formate solution for 1 min. A TEM FEI Tecnai G2 Spirit (Bio)twin was used at 120 kV acceleration voltage, and images were recorded and analyzed with EM-Menu4 software (TVIPS). Single-Particle Reconstruction Protocol. Imaging was done using a FEI Tecnai G2 Spirit electron microscope operated at 120 kV. Automated image acquisition was performed using a bottom-mounted TVIPS CMOS 4k camera (TEM-cam-F416) and setup by Leginon software. A total of 513 images was acquired at a magnification of ×67k, a pixel size of 1.57 Å, and with defocus in the range of 0.5 to 1.5 μm. A raw micrograph is shown in Figure 4.

Particles were picked automatically by dogpicker which resulted in 217509 particles picked which were used for RELION 1.4 singleparticle processing. Particle cleaning by 2D class average clustering resulted in 87683 well-defined particles. These were used in the 3D classification step of three classes where C1 symmetry was imposed using a 7.5° angular interval throughout 25 iterations. The first class (32781 particles) was chosen for further refinement by RELION 3D auto refinement (C1 symmetry). In all 3D processing steps, a simple sphere was used as the initial reference. Processing steps are outlined in Supporting Figure S3, and the angular coverage of the processing is shown in Supporting Figure S4. Sample Preparation for STV-AuNP Patterning and Visualization with TEM. The sample was folded using Dark Blue2_Biotin instead of Dark Blue2. Dark Blue2_Biotin is modified with biotin through a terminal C6 spacer linker. STV-AuNPs (NANOCS GNA5, 0.1% Au) were added to the folded sample in a 1:1 biotin/STV-AuNP ratio, followed by incubation at room temperature for 2 h. The lattice was purified from the unbound STV-AuNP by gel filtration (Illustra MicroSpin S-200 HR columns): the column was washed two times with buffer before adding the sample and centrifuged for 1 min to elute the sample. Purified samples were deposited on glow-discharged 200 mesh carbon-coated copper TEM grids (Ted Pella Inc.) for 30 s. No 9045

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ACS Nano stain was used. A TEM FEI Tecnai G2 Spirit (Bio)twin was used at 120 kV acceleration voltage, and images were recorded and analyzed with EM-Menu4 software (TVIPS). Melting Curve Analysis. The folded sample was diluted to 500 μL with buffer and analyzed in a Thermo Scientific Evolution 260 Bio at 260 nm, 2 nm bandwidth, with heating and cooling ramps of 1 °C/ min. A cycle of heating (melting I), cooling (annealing), and heating (melting II) was performed. Resulting values were normalized between 0 and 1 and fitted to a dose−response curve using the software Originpro. For melting temperature calculations, the values resulting from first denaturing curve were derived (first derivative) and plotted, where the highest value corresponds to the melting temperature of the structure. Small-Angle X-ray Scattering. The SAXS data were recorded at the instrument at Aarhus University. It is a NanoSTAR from Bruker AXS26 with a liquid metal jet source from Excillum AB, Sweden, and with home-built scatterless slits.27 A home-built flow-through quartz capillary sample cell was used for the measurements. The sample is changed by an autosampler based on Gilson components. It also cleans and dries the capillary between measurements. The data are displayed as a function of the modulus of the scattering vector

edges M. De Stefano for help in drawing the rendered image of the cuboctahedron unit.

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q = 4π sin(θ)/λ where 2θ is the scattering angle and λ is the X-ray wavelength. The data were modeled using a structural arrangement in agreement with the design of the structures, except that a polymer-like contribution had to be added to describe the scattering from imperfections, random structures, and non-assembled strings. The DNA helices were modeled by simply placing spheres along the helices of the structure.28

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b03538. Nanostructure design schematics, supporting text, additional data, figures and DNA sequences (PDF) Video showing a comparison of the 12-way junction monomer model with the 3D image generated by singleparticle reconstruction (AVI)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Jeppe Lyngsø: 0000-0002-6301-1300 Kurt V. Gothelf: 0000-0003-2399-3757 Author Contributions

I.M. designed the DNA sequences, performed EMSA, DLS, STV-AuNP patterning and TEM studies. G.G. helped with the design of the nanostructures and performed the nsTEM experiments. R.P.T. performed the single-particle reconstruction. J.L. and J.S.P performed the SAXS experiments and the data analysis. K.V.G conceived and supervised the project. J.K. and ESA co-supervised the project. I.M. and K.V.G. wrote the manuscript with assistance from all other authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS The work was financially supported by the European School of DNA Nanotechnology (EScoDNA), a Marie Curie ITN under FP7 (Grant Number 317110) and by the Danish National Research Foundation (Grant Number DNRF81). I.M. acknowl9046

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