3D DNA Origami Cuboids as Monodisperse Patchy Nanoparticles for

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3D DNA Origami cuboids as monodisperse patchy nanoparticles for switchable hierarchical self-assembly Thomas Tigges, Thomas Heuser, Rahul Tiwari, and Andreas Walther Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.6b04146 • Publication Date (Web): 01 Nov 2016 Downloaded from http://pubs.acs.org on November 1, 2016

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3D DNA Origami cuboids as monodisperse patchy nanoparticles for switchable hierarchical self-assembly Thomas Tigges2, Thomas Heuser2, Rahul Tiwari2, Andreas Walther1* 1

Institute of Macromolecular Chemistry, Albert-Ludwigs-University of Freiburg, Stefan-Meier-Str. 31, 79104 Freiburg, Germany. 2

DWI – Leibniz-Institute for Interactive Materials, Forckenbeckstraße 50, 52074 Aachen, Germany.

Corresponding author: [email protected] Tel: +4915140749934

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Abstract: The rational design of anisotropic interaction patterns is a key step for programming colloid and nanoparticle self-assembly and emergent functions. Herein, we demonstrate a concept for harnessing the capabilities of 3D DNA origami for extensive supracolloidal self-assembly, and showcase its use for making truly monodisperse, patchy, divalent nanocuboids that can self-assemble into supracolloidal fibrils via programmable DNA hybridization. A change in the number of connector duplexes at the patches reveals that multivalency and cooperativity play crucial roles to enhance superstructure formation. We further show thermal and chemical switching of the superstructures as first steps towards reconfigurable self-assemblies. This concept lays the groundwork for merging monodisperse 3D DNA origami, featuring programmable patchiness and interactions, with nanoparticle self-assembly.

Keyword: DNA-Origami, Hierarchical Self-Assembly, Patchy Particles, Anisotropic Nanoparticles, Colloids


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Progress in colloid and nanoparticle self-assembly relies on the development of preparation pathways able to generate functional and highly defined building blocks with controlled interaction patterns (patchiness) and shape anisometry.1-3 The field witnessed significant advances in the last years by utilizing self-assembly of block copolymers, emulsion-based synthesis routes, microfluidics or topdown processing methods to generate complex patchy colloids on different length scales.4-11 Interaction chemistries for directional self-assembly diversified from solvophobic interactions, via depletion interactions, supramolecular bonds up to DNA recognition.12-19 Unsolved challenges include finding combinations of straightforward preparation pathways towards truly monodisperse particles with controlled patch size and distributions, as well as highly defined, at best, orthogonal interaction chemistries. An emerging method for the fabrication of highly complex and uniform nanoparticles is the direct built-up by 3D DNA origami.20,21 Therein, the base pair coding principle is utilized to program the folding of a large circular strand of DNA by addition of staples strands with matching sequence. Folding via slow temperature reduction yields the designed DNA nanoobjects in high yield and purity. This field has so far mostly focused on creating individual objects for drug-delivery, biosensing or complex optical materials.22-25 Few studies reported more extended 1D structures by utilizing staples to bridge several circular strands into tight, non-responsive structures using intimate connections that do not allow disassembly into individual objects.20,26Dietz and co-workers created isolated superstructures of 3D DNA origami by cation-induced shape-recognition, and Kostiainen and co-worker created dimers to tune enzymatic reactions.28,29 In the more established 2D origami field, switchable structures were recently reported, showing pH-responsive assembly of origami tiles.27 Here, we go beyond isolated objects and demonstrate that 3D DNA origami cuboids can be turned into versatile patchy nanoobjects able to direct and control self-assembly into fibrillar superstructures. We control the hierarchical self-assembly by utilizing complementary connector strands on the opposing sides (patches) of a divalent 3D DNA origami that have a melting point far below that of the DNA origami body itself. This provides the key to separate DNA cuboid formation (intraparticular) from supraparticular self-assembly into fibrillar structures (interparticular).


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Figure 1: Divalent 3D DNA origami cuboids. a) Folding of the circular M13mp18 scaffold ss-DNA in presence of a tenfold excess of 220 ss-DNA staples and poly-T passivated side strands at the patches (cyan) in 20 mM MgCl2. b,c) TEM and gel electrophoreses (GEP) of single stranded M13mp18 DNA in comparison to folded cuboids.

It affords distinct patchy nanoparticles. We describe in detail the effect of connector multivalency, uniquely tunable by bottom up 3D DNA origami, and reveal criteria for switching such self-assemblies using temperature and (dis-) connector strands as triggers. As model patchy nanoobject we design a 3D DNA origami cuboid with a divalent interaction pattern (cyan), based on a 7249 base pair M13mp18 single stranded scaffold DNA using the open source software CaDNAno (Supporting Information for details).30 The end caps (i.e. the patches; cyan) can be modified with different strands to prevent or induce switchable interactions. For a basic characterization of the cuboid, we block all end caps first with strands containing nine free thymine bases to inhibit undesired aggregation (poly-T). Folding via temperature decrease then yields welldefined, isolated 3D DNA origami cuboids (Figure 1). For folding, all staple and poly-T strands are added in a 10-fold excess to the scaffold strand. Further experimental details can be found in the Supporting Information. Transmission electron microscopy (TEM) and the narrow band in gel electrophoreses (GEP) confirm the successful folding of the scaffold DNA into cuboidal origami ACS Paragon Plus Environment

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objects with the dimensions of 24 nm x 36 nm x 18 nm. The dimensions of the DNA cuboid are defined by the number of bases inside each double helix (95), the number of adjacent double helices per layer (12), and the number of stacked layers (6). To gain an understanding on the amount of connector strands needed to control higher level selfassembly, we first replaced one of the poly-T strands on each side of the particles by the connector pair able to induce selective hybridization between the two sides (red/green sequence) prior to the folding procedure (Figure 2a-b). The DNA cuboids again do not show any tendency for self-assembly into fibrils during the initial folding (Figure 2c). This can be explained by the fact that the complementary strands are in a 10-fold excess, meaning the sticky ends are saturated by other free connector strands. Moreover, the system at room temperature is almost non-dynamic with respect to exchange of the duplex at the connector strands, because the melting temperature, Tm, of the connector pairs is at 32 °C under dilute conditions.

Figure 2: Self-assembly of divalent 3D DNA origami cuboids with a single connector pair. a) Scheme of the hierarchical self-assembly of cuboids into fibrils starting from ss-DNA. Folding of scaffold DNA with staple strands and a single pair of connector strands yields monomeric DNA cuboids. Annealing or removal of excess staple strands yields DNA cuboid oligomers. b) Overview of different strand types. The fading grey indicates longer sequences to bind the connector strands to the scaffold. c-e) Unstained and negatively stained (insets) TEM micrographs directly after folding, annealing and removal of excess staples. ACS Paragon Plus Environment

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However, upon annealing in the range of the Tm (35 °C, 15 h), short cuboid oligomers can be observed, even still in presence of the excess staple strands. The self-assembly is allowed by the higher dynamics of the duplex of the complementary pairs around the Tm. Oligomerization occurs despite the presence of an excess of staples, and is favored for entropic reasons (release of ss-DNA from the patches, Figure 2d). A slightly increased oligomerization can be observed after purification of the DNA cuboids to remove excess staples (Figure 2e). In this case, oligomers are found shortly after the purification step, indicating that removal of free connector strands enhances the dimerization of bound connectors and thus the formation of cuboid oligomers. To get further insights on the effect of tailored multivalency, we next replaced eight of the poly-T passivated side strands on each side of the cuboid with complementary connectors (Figure 3a). After folding, substantially longer and more oligomers can be observed (compare Figure 2c for one connector). The mean number average degree of polymerization ܺ୬ is 4 with oligomers going up to a length of 14 (Figure 3b). This behavior was initially unexpected, because every connector strand still has a 9-fold excess of its partner in solution. However, the strong ability for self-assembly into fibrils despite this large excess of staple strands is caused by the multivalency and cooperativity of the process. This can be understood as follows: When a single cuboid-cuboid-bond forms, the remaining, unpaired connectors at the patches are brought into immediate vicinity, facilitating the hybridization between neighboring connector pairs like a molecular zipper, and thus the formation of fibrils is favored (Figure 3c). The released ss-DNA connector strands hybridize in solution, and contribute to the entropy of the system and largely shift the equilibrium to the side of the fibrils. The length of the observed fibrils significantly increases with an additional annealing for 15 h at 35 °C (Figure 3d). Long supracolloidal polymers form with a Xn of 11, and length up to ca. 56 units (Figure 3d). A large portion of the fibrils displays micrometer length, which may even make it possible to visualize the processes by fluorescence microscopy in future. A further change of the connector pairs to 3 and comparative analysis of the influence of 1, 3, and 8 connector pairs on the degree of polymerization under the standardized conditions (15 h at 35 °C; 20 nM scaffold) confirms the tendency for longer fibrils with increasing number of connector pairs, as Xn and the length steadily increases (Figure SI1).


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Figure 3: Self-assembly of divalent 3D DNA origami cuboids with eight connector pairs. a) Schematic representation of the self-assembly process. Folding directly yields DNA origami oligomers. Additional annealing at 35 °C for 15 h then yields highly elongated fibrils. b) Histogram and c,d) corresponding negatively stained (bottom) and unstained (top) TEM micrographs showing (c) oligomeric and (d) polymeric DNA origami fibrils before and after annealing.

The next step in harnessing 3D DNA origami structure for programmable nanoparticle self-assemblies is to understand under which conditions they can be switched. While switchability is well-known for molecular DNA systems or DNA-coated colloids, principles for colloidal 3D DNA origami have yet to be established.31,32 To this end we first focus on thermal switching. This can be enabled by having purposefully designed the melting point of the connector pairs substantially lower (Tm ≈ 32 °C) compared to the duplexes inside the DNA origami (Tm ≈ 48 – 73 °C). Indeed, heating of the selfassembled fibrils to 40 °C leads to disassembly into monomers and few short oligomers while the DNA cuboids themselves retain their shape. Subsequent cooling and annealing at 35 °C then reassembles the origami fibrils due to reconnection of the connector pairs at the patches (Figure 4a). While we here established a transition around body temperature, the thermal disassembly can be manipulated up to the ACS Paragon Plus Environment

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Tm of the duplexes stabilizing the cuboid body itself. The Tm can be varied by changing the hybridization length, the sequence or encoding base pair mismatches. Hence, different DNA duplex connectors can give rise to orthogonal switching of self-sorting self-assemblies containing connectors with different Tm in future. In a complementary approach, we raised the question how additional strands can be used as a signal to control the self-assembly state. Above, we derived that the DNA cuboids based on 8 connector pairs self-assemble into polymeric fibrils even in the presence of an excess of staple strands. Hence, adding both connector strands simultaneously will not lead to breakage of the fibrils. We hypothesized that adding only one strand of the connector pair would allow to impart changes, because the partnering strand for forming a duplex would only be available from the patch of a cuboid-cuboid contact. To realize this scenario, we added connector type 1 in a tenfold excess to the assembled fibrils. Indeed, it acts as a ‘disconnector’ by replacing cuboid-cuboid connector pairs and hybridizing the connector type 2 strands. Consequently, the fibrils are broken and the equilibrium shifts to the monomeric 3D DNA origami cuboids (Figure 4b, center). Subsequent addition of the second, complementary strand (connector type 2) provides new partners in solution and again shifts the equilibrium to the polymeric origami fibrils, as the free strands again dimerize, and allow for multivalent and cooperative hybridization between the origamis (Figure 4b, right). A further refinement of this switching process may be in reach using the concept of fuel strands with increasing binding affinity for each step.


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Figure 4: Thermal and strand-induced (chemical) switching of the self-assembled state of the DNA cuboid fibrils with 8 connector pairs. a) Thermally induced switching: From left to right: Selfassembled fibrils after annealing at 35 °C for 15 h. Colloidal fibrils disassemble into monomers and short oligomers via heating to 40 °C. Cooling and annealing at 35 °C then again yields fibrils. b) Strand-induced switching: From left to right: Self-assembled fibrils disconnect when a single type of connector strand is added. Addition of the second complementary connector strand then leads to dimerization of the free strands and simultaneous formation of origami fibrils.


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In conclusion, we have established first design principles of using 3D DNA origami objects for switchable nanoparticle self-assemblies. This strategy will foster the merger of these two relatively independent fields and help to provide monodisperse and tunable building blocks for fundamental studies and applications in self-assembling colloidal systems. Looking into the future, the method presented here provides a starting point to diversify the structures of the 3D DNA origami objects (shape and valency), their hybridization with inorganic nanoparticles in co-assembly concepts, and calls for the implementation of more refined switching patterns, possibly taking inspiration of switchable non-DNA supramolecular chemistry.

ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Andreas Walther, DWI – Leibniz-Institute for Interactive Materials, Forckenbeckstraße 50, 52074 Aachen, Germany. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT We thank the DFG for financial support in the WA3084/4-1 grant. Parts of this work were performed at the Center for Chemical Polymer Technology, supported by the EU and the state of North RhineWestphalia (Grant No. EFRE 30 00 883 02).


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