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Switchable Triggered Interconversion and Reconfiguration of DNA Origami-Dimers and Their Use for Programmed Catalysis Jianbang Wang, Zhixin Zhou, Liang Yue, Shan Wang, and Itamar Willner Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b00793 • Publication Date (Web): 14 Mar 2018 Downloaded from http://pubs.acs.org on March 15, 2018

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Switchable Triggered Interconversion and Reconfiguration of DNA Origami-Dimers and Their Use for Programmed Catalysis Jianbang Wang, Zhixin Zhou, Liang Yue, Shan Wang and Itamar Willner* Institute of Chemistry; The Center for Nanoscience and Nanotechnology The Hebrew University of Jerusalem, Jerusalem 91904, Israel Corresponding Author *E-mail: [email protected]; Tel: +972-2-6585272; Fax: +972-2-6527715.

ABSTRACT: The switchable reconfiguration of a mixture of two dimers of DNA origami tiles AB and CD into a mixture of two DNA origami dimers composed of AD and CB, using collection of fuel and anti-fuel strands, is presented. The reversible reconfiguration of the mixture of dimers AB/CD into AD/CB followed by labeling each of the tile with 0, 1, 2 and 3 4× hairpins labels and by imaging the dimer structures by AFM. Subjecting the reconfigurable dimer mixtures to a collection of Mg2+-dependent DNAzyme subunits and the substrates consisting of the ROX/BHQ2-modified substrate and the FAM/BHQ1-modified substrate leads to the triggered and programmed switchable operation in the presence of appropriate fuel and anti-fuel strands. In the presence of the AB/CD mixture the DNAzyme subunits cleaving the ROX/BHQ2-modified substrate is switched “ON”, leading to the 1 ACS Paragon Plus Environment

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fluorescence of ROX. The reconfiguration of the AB/CD dimers mixture to the AD/CB dimers mixture leads to the assembly of the DNAzyme subunits that switch “ON” the cleavage of the FAM/BHQ1-modified substrate and to the fluorescence of FAM. By the cyclic and reversible reconfiguration of the AB/CD dimers mixture to the AD/CB dimers mixture, in the presence of the appropriate fuel and anti-fuel strands the switchable operation of the two Mg2+-ion dependent DNAzyme leading to fluorescence of ROX or FAM is demonstrated.

KEYWORDS:

DNA

switch;

DNAzyme;

Hairpin

label;

DNA

machine;

Nanotechnology

The programmed construction of 2D or 3D DNA origami-nanostructures represents a major advance in DNA nanotechnology.1,2 Ingenious, 2D shapes and 3D structures were reported by the dictated folding of the long-chain M13 phage DNA with appropriate “staple” units and by the secondary folding of two-dimensional origami tiles into three dimensional networks.3-6 In addition the origami systems provided nanoscale

structures

for

the

programmed

assembly

of

molecular,7,8

macromolecular9,10 or nanoparticle11,12 structures that revealed unique physical13,14 or chemical15 functionalities that originate from the nanoscale ordering of the chemical components on the origami scaffolds. For example, 2D origami scaffolds were used as “playgrounds” for the ordered assembly of enzymes and the operation of bio-catalytic cascades,15 for the triggered operation of DNA machines,16,17 and the ordered 2 ACS Paragon Plus Environment

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assembly of nanoparticles.11,18 Also, 3D origami scaffolds were used as templates for the assembly of chiroplasmonic nanoparticles,19-24 scaffolds for the operation of chiroplasmonic machines,22,25 and signal-triggered nano-capsules for controlled release.26,27 Recent efforts were directed to the conjugation of origami rafts into predesigned patterns,28 the expansion of origami structures by the self-assembly of Jigsaw origami pieces,29 and the replication of dimer origami structures.30 Specifically, our laboratory has introduced the concept of “origami chemistry” where the origami tiles were inter-connected into dimer or trimer origami structures using stimuli-responsive bridging units,31-33 such as pH-bridges, e.g., i-motif or triplex,31 cofactor-dependent DNAzyme,32 aptamer-ligand complexes.33 The selective cleavage of these structures and the dictated reconfiguration of the assemblies were demonstrated. The further development on reconfigurable conjugated origami dimers of enhanced complexities require, however, the introduction of means to statistically analyze the different structures and specifically, to introduce controllable functionalities into the conjugated origami structures. In previous reports, as well as in our studies, biotin labels associated with the origami tiles and biotin-avidin complexes as labels to identify each of these structures were used. Nonetheless, the use of the biotin/avidin labels to identify systems of enhanced complexities is limited, due to the yield of the biotin-avidin complexes that result in incomplete labeled structures upon increasing the number of avidin units on the origami. In the present study, we present the triggered reversible inter-conversion and reconfiguration of two pairs of origami-dimers. In addition to the enhanced complexity introduced by this system to 3 ACS Paragon Plus Environment

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the area of conjugated origami structures, we demonstrate that by the tethering of DNAzyme subunits to the origami-dimer structures, switchable and reversible catalytic are achieved. It should be noted that in order to label the origami tiles we apply nucleic acid hairpins (4× identical hairpins as a label) linked to the origami tiles as labels. This allows us to yield distinguishable origami tiles for AFM imaging of the components. The principle to inter-connect the origami tiles involves the binding of the origami tiles with duplex nucleic acids that include a toehold strand. The toehold mediated strand displacement process in the presence of fuel or anti-fuel strands provides the mechanisms to reconfigure the origami-tiles structures.34,35 In the first step, the assembly of two origami tiles dimers AB and CD and their separation and re-assembly in the presence of appropriate fuel and anti-fuel strands were examined. The origami dimer AB (marked with 0 and 1 4× hairpins labels, (0, 1)), Figure 1(A), was assembled by two sets of crosslinking duplex bridges: 3× AB-U/BA-U and 3× AB-D/BA-D. Each of these sets of bridges included a toehold strand (ab-1 or ab-6, respectively). Subjecting the dimer AB to the fuel strands AB-U’ and AB-D’ results in the toehold-mediated separation of the tiles, where the strands AB-U and AB-D are blocked by the fuel strands AB-U’ and AB-D’, and each of these duplexes includes a toehold strand associated with AB-U’ and AB-D’, respectively. Subjecting the separated dimer AB to the anti-fuel strands AB-U” and AB-D” results in the toehold-mediated strand displacement of the fuel strands by the formation of the energetically-stabilized duplexes AB-U’/AB-U” and AB-D’/AB-D” as “waste” 4 ACS Paragon Plus Environment

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products. The uncaging of the AB-U’ and AB-D’ strands leads to the re-assembly of the duplex AB. By the cyclic treatments of the dimer AB with the fuel strands (AB-U’ and AB-D’) and with the anti-fuel strands (AB-U” and AB-D”) the reversible separation and re-assembly of the dimer AB proceed. Figure 1(B) depicts the AFM images of the origami dimers before (I) and after (II) subjecting to the strands AB-U’ and AB-D’ and after the subsequent treatment of the separated tiles (image II) with the strands AB-U” and AB-D” (III). The AMF images (also see enlarged insets) clearly confirm the reversible transitions from the (0, 1) dimer to the monomer tiles, and back to the (0, 1) dimer. Furthermore, cross-section analyses of the different states of the system, Figure 1(C) confirm nicely the reversible transitions. In state I, the length of the origami tiles is ca. 200 nm with an apparent bump, height ca. 1 nm, that corresponds to the label associated with one of the tiles. The length of the structure, ca. 200 nm is consistent with the combined dimer structure, where each of the tiles is ca. 100 nm long. In state II of the system the cross-section analysis of the two types is demonstrated. One tile, ca. 100 nm long, does not include any label, while the second class of tiles includes one label. The state II, in the presence of the appropriate triggers, regenerate state I, as evident by the cross-section analysis. Figure 1(D) shows the population of the dimers/monomers statistical analysis of many samples (see Table S1). The results demonstrate that the 0, 1 4× hairpins labels provide a useful means to identify the structures. Also, in the primary configuration I the content of the dimers predominates while in state II most of the structures correspond to monomer tiles, with only a trace residue of dimers. The treatment of 5 ACS Paragon Plus Environment

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system II with the anti-fuel strands results in an origami mixture that consists of re-assembled dimers as the major constituent.

Figure 1. (A) Schematic separation and re-assembly of the AB dimer using fuel and anti-fuel strands. The dimer is labeled with 0, 1 4× hairpins labels. The “waste” duplexes generated upon the re-assembly of the dimer are presented. (B) AFM images 6 ACS Paragon Plus Environment

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of: (I) As prepared AB dimers. Inset: enlarged image of the (0, 1) AB dimer. (II) The separated dimer components upon the treatment of the AB dimer with the fuel strands. Inset: enlarged image of the separated units A and B. (III) The re-assembly of the dimer AB after subjecting the monomers to the anti-fuel strands. Inset: enlarged image of the (0, 1) AB dimer. (C) Cross-section analysis of the AFM images corresponding to the transition of dimer AB to the monomers and back to the dimer AB. (D) Statistical analysis of the components constituting states I, II and III (regenerated state I). Similarly, the formation of the CD dimers crosslinked by the 3× CD-U/DC-U duplexes and 3× CD-D/DC-D duplexes were constructed, Figure 2(A). The tiles C were labeled with two 4× hairpins labels whereas the tiles D were labeled with three 4× hairpins labels, thus the overall imaging of the dimers including a combination of (2, 3) 4× hairpins labels. The specific domains associated with the bridging units are displayed in Figure 2(A). Subjecting the dimers to the fuel strands CD-U’ and CD-D’ results in the separation of the dimers to monomer tiles, and the subsequent treatment of the monomers with the anti-fuel strand CD-U” and CD-D” results in the displacement of the CD-U’ and CD-D’ in the form of the “waste” duplexes CD-U”/CD-U’ and CD-D”/CD-D’ and the re-assembly of the initial dimers. Figure 2(B) depicts the AFM images of the CD dimers prior to the addition of the fuel strands (I), the separation of the dimers to monomers after the addition of the fuel strands (II), and upon the subsequent re-assembly of the dimers in the presence of the anti-fuel strands (III). The AFM images (also see enlarged insets) clearly confirm the 7 ACS Paragon Plus Environment

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reversible transitions from the (2, 3) dimer structure to the monomer tiles, labeled with two 4× hairpins and three 4× hairpins, respectively, and back to the (2, 3) dimer structure. These transitions are further supported by cross-section analyses of the respective systems, Figure 2(C). In the (2, 3)-dimer state I, ca. 200 nm long tiles (consistent with the dimer composition) are observed, and four bumps corresponding to the hairpin labels associated with the two tiles are observed (note that the fifth hairpin label, is out of the linear cross-section). In the presence of the appropriate triggers the dimer is separated into two tiles that are ca. 100 nm height bumps of the respective labels, as expected. The subsequent treatment of state II with the respective triggers restores state I with its characteristic cross-section pattern. Figure 2(D) presents the statistical population of the structures in states I, II and III (regenerated state I) upon imaging many surface domains (see Table S2). The results show that the (2, 3) 4× hairpins labels associated with the dimers allow the effective imaging of the different states. In addition, Figures 2(B) and 2(D) confirm the existence of states I and III as rich population of the dimer CD, whereas in state II the monomer tiles C and D predominate.

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Figure 2. (A) Schematic separation and re-assembly of the CD dimer using fuel and anti-fuel strands. The dimer is labeled with (2, 3) 4× hairpins labels. The “waste” duplex generated upon the re-assembly of the dimer is presented. (B) AFM images of: (I) The assembled CD dimer. Inset: enlarged image of the (2, 3) CD dimers. (II) The separated dimer components upon the treatment of the dimer with the fuel strands. 9 ACS Paragon Plus Environment

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Inset: enlarged image of the separated units C and D. (III) The re-assembly of the dimer CD after subjecting the monomers to the anti-fuel strands. Inset: enlarged image of the (2, 3) CD dimers. (C) Cross-section analysis of the AFM images corresponding to the transition of dimer CD to the monomers and back to the dimer CD. (D) Statistical analysis of the components constituting states I, II and III (regenerated state I). In the next step we examined the triggered reversible reconfiguration of a mixture of two dimers composed of the dimeric tiles AB (0, 1 label) and the dimeric tiles CD (2, 3 labels), Figure 3. The schematic reversible reconfiguration of the two dimers is presented in Figure 3(A), top, with the reconfiguration of the mixture of dimer AB, CD into the new mixture AD and CB, and back.

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Figure 3. (A) Cyclic reconfiguration of the dimer mixture AB/CD (labeled 0, 1 and 2, 3, respectively) into the dimer mixture AD/CB (labeled 0, 3 and 2, 1, respectively) using appropriate fuel and anti-fuel strands. (B) AFM images corresponding to the cyclic reconfiguration of the mixtures of dimer AB/CD to AD/CB and back: (I) and 11 ACS Paragon Plus Environment

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(III) mixtures containing AB/CD, (II) and (IV) mixtures containing AD/CB. (Insets show the enlarged structures of AB/CD (0, 1 and 2, 3) and of AD/CB (0, 3 and 2, 1). (C) Statistical analysis of the constituents upon the cyclic reconfiguration of the mixtures between AB/CD and AD/CB. To reach this goal, Figure 3(A) outlines the specific sequences and hybridization features of the different strands associated with the respective dimers and the sequences corresponding to the respective fuel and anti-fuel strands that are used to induce the reversible reconfiguration process. In order to drive the reconfiguration process, the different tiles were modified at one of their edges with four sets of strands, where each set included three identical strands. For example, tile A was modified at its edge with 3× AB-U, 3× AD-U, 3× AB-D and 3× AD-D, while tile B was functionalized at its edge with 3× BA-U, 3× BC-U, 3× BA-D and 3× BC-D. In addition, blocker strands AD-U’ and AD-D’ are hybridized with the strands AD-U and AD-D to prevent any interactions with the crosslinking units of the other dimers. Under these conditions, the dimer AB is crosslinked by the set of duplexes AB-U/BA-U and AB-D/BA-D. Similarly, Figure 3(A), left (bottom) shows the sequences complementarity and the respective blocker units associated with the dimer CD. In this case, the dimers are crosslinked by the 3× duplexes, CD-U/DC-U and 3× CD-D/DC-D. In addition, the strands CB-U and CB-D are blocked by the strands CB-U’ and CB-D’, respectively. Also, Figure 3(A) depicts the set of eight fuel strands and anti-fuel strands and marks the complementation of these domains to the different strands of the crosslinking units associated with dimers AB/CD and AD/CB, 12 ACS Paragon Plus Environment

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respectively. Subjecting the mixture of dimers AB/CD to the set of eight strands AB-U’, AB-D’, AD-U”, AD-D”, CD-U’, CD-D’, CB-U” and CB-D” results in the separation of the respective duplexes, and the reconfiguration of the AB (0, 1)/CD (2, 3) dimer mixture into the AD (0, 3)/CB (2, 1) mixture. The subsequent treatment of the reconfigured, dimer mixture with the strands AB-U”, AB-D”, AD-U’, AD-D’, CD-U”, CD-D”, CB-U’ and CB-D’ results in the separation of the AD/CB dimer mixture, through the formation of the respective duplex “wastes” outlined in the figure, and the reconfiguration of the dime mixture AB/CD. Figures 3(B) and 3(C) depict the AFM images and their statistical analysis corresponding to the reversible reconfiguration of the mixture AB/CD into the mixture AD/CB and back (see Table S3). In state I, the dimer mixture exists in the AB (0, 1) and CD (2, 3) state while no dimers AD/CB are observed. Subjecting the mixture shown in state I to the respective strands yields the dimer mixture AD (0, 3) and CB (2, 1) in state II. The reversible treatment of the mixture AD/CB with the respective strands regenerates state I, and the subsequent interaction of the resulting AB/CD mixture with the respective strands regenerates state II composed of AD/CB. It should be noted the use of the many fuel and anti-fuel strands applied to transform any one of the systems from one state to another many duplexes acting as “waste” are formed, and these “waste” products might interfere with the reversible switching of the system between the states. To avoid such interference each cyclic transition was followed by a centrifugation step (see supporting information). By this way, the excess of fuel/anti-fuel constituents were separated from the origami 13 ACS Paragon Plus Environment

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structures. Using this method we were able to switch the respective transitions of the states in the different systems, for at least seven times, with no noticeable effect on the yield of the transition efficiency (nonetheless after this number of cycles the content of origami structures decreased by ca. 50% due to incomplete recovery of the origami structures). After showing the switchable reconfiguration of the AB/CD dimer mixture into the AD/CB dimer mixture and back, it was important to demonstrate that this dimer exchange process lead to switchable functionality, e.g., switchable catalytic processes. Towards this goal, we made use of the fact that different metal-ion-dependent DNAzymes are known.36-39 In these DNAzymes the loop domain defines the ion specificity of the DNAzymes. The tethered substrate binding “arms” can be, however, diversified to bind different fluorophore/quencher functionalized substrates, and thus the activity of the metal-dependent DNAzymes can be dictated by the engineered “arms” associated with the DNAzymes. To highlight the origami-dimer stimulated switchable functionalities upon the reconfiguration of the dimers AB/CD into AD/BC dimers we utilized the diversity of engineered Mg2+-ion dependent DNAzymes, Figure 4. We note that the CD dimer includes the strands CB-U’ and CB-D’ hybridized with the strands CB-U and CB-D of the origami tile C. These hybridized units include single strands cb-5’ and cb-10’ as tethers. Similarly, the origami-tile D is functionalized with the single strands DA-U (composed of domains ad-2’, ad-3’ and ad-4’) and DA-D (composed of ad-7’, ad-8’ and ad-9’). Subjecting the AB/CD dimer mixture to the mixture of eight Mg2+-ion-dependent subunits (I to VIII) and two kinds 14 ACS Paragon Plus Environment

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of fluorophore/quencher-functionalized substrate (substrate 1 and substrate 2), subunits I, II, III and IV and substrate 1 bind to the complementary tethers of dimer CD. Under these conditions, and in the presence of Mg2+-ions the ROX/BHQ2 functionalized substrate 1 is cleaved, leading to the switched-on fluorescence of ROX. Note that under these conditions, the dimer AB cannot select active DNAzyme units and only inactive subunits may associate subunits VI and VIII to the tile B. The reconfiguration of the dimer mixture AB/CD into the dimer mixture AD/CB in the presence of the appropriate strands, Cf. Figure 3, leads to the functionalization of dimer CB with the tethers that generate new DNAzyme structures. The tile C includes the cd-5’ tethered strand CD-U’ hybridized with the strand CD-U, and the cd-10’ tethered strand of CD-D’ that is hybridized with CD-D. Also, tile B is modified with the tethers BA-U (composed of ab-2’, ab-3’ and ab-4’) and BA-D (composed of domains ab-7’, ab-8’ and ab-9’). Under these conditions the dimer mixture CB selects the Mg2+-dependent DNAzyme subunits V, VI, VII and VIII and the substrate 2 that bind to dimer CB. This results in the cleavage of the FAM/BHQ1-functionalized substrate and the switched-on fluorescence of FAM. Note that the dimer AD accompanying the dimer mixture cannot select any active Mg2+-ion-dependent DNAzyme structure. Thus, by the reversible reconfiguration of the dimer mixtures from states AB/CD to AD/CB the programmed switchable activation of two different DNAzymes proceeds. While the mixture AB/CD leads to the switched-on fluorescence of ROX and switched-off fluorescence of FAM, the origami dimer

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mixture AD/CB yields the switched-on fluorescence of FAM and switched-off fluorescence of ROX, Figure 4(B).

Figure 4. Cyclic and reversible operation of two different Mg2+-ion dependent DNAzymes by the switchable reconfiguration of the AB/CD dimer mixture to the AD/CB dimer mixture and back. (A) The collection of enzyme subunits and their 16 ACS Paragon Plus Environment

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fluorophore-quencher substrates that interact selectively with either the CD dimer in the AB/CD mixture or with the CB dimer in the mixture AD/CB. Note that for simplicity only the dimers carrying the DNAzyme subunits are presented, yet the dimers AB and AD lacking the DNAzyme units are always present in the mixtures. The DNAzyme shown in state A catalyzes the cleavage of the ROX/quencher modified substrate that leads to the switched-on fluorescence of ROX, whereas in state B, the DNAzyme catalyzed cleavage of the FAM/quencher-modified substrates, leading to the switched-on fluorescence of FAM. (C) Cyclic and switchable fluorescence changes upon the reversible reconfiguration of the dimer mixtures AB/CD to the dimer mixture AD/CB and back and the operation of the DNAzymes in state A or state B, respectively. In conclusion, the present study has demonstrated the reversible and switchable structural reconfiguration of two pairs of origami dimers by applying auxiliary “fuel” and “anti-fuel” strands that intervene with nucleic acids that crosslink the dimer structures. The programmed separation of the crosslinking units and their dictated re-formation of exchangeable crosslinking components led to the reconfiguration of the origami-dimers structures. An important result of the study rests on the demonstration that switchable catalytic activities emerge from the structural reconfiguration of the origami tiles. The study paves the way to construct other stimuli-triggered origami-dimer structures that could switch sequestered chemical processes, e.g., enzyme cascades or plasmonic interactions.

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ASSOCIATED CONTENT

Supporting Information. Details on the protocols for the assembly of the origami tiles, the dimer origami tiles and the mixture of two dimers tiles, AB/CD are provided. Also, the experimental conditions to reversibly separate the dimer origami tiles and reassemble the dimers are described. In addition, the experimental condition to reversibly reconfigure the dimers tile mixture AB/CD to AD/CB and back are presented, and the experimental details on the programmed operation of the switchable DNAzymes upon the reversible reconfiguration of the dimers AB/CD to AD/CB and back are detailed. Also, details on the statistical analysis of the tile components and dimer constituents in the different systems are summarized in Table S1, Table S2 and Table S3.In addition, the sequences of the different staple strands, hairpin markers, linker strands, blocker strands and fuel and anti-fuel strands are detailed. Also, the sequences of the DNAzyme subunits strands and their substrates are provided.

Notes The authors declare no competing financial interests. ACKNOWLEDGMENT This research is supported by the Israel Science Foundation and by the Minerva Center for Biohybrid Complex Systems.

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REFERENCES (1) Endo, M.; Sugiyama, H. Single-Molecule Imaging of Dynamic Motions of Biomolecules in DNA Origami Nanostructures Using High-Speed Atomic Force Microscopy. Acc. Chem. Res. 2014, 47, 1645-1653. (2) Hong, F.; Zhang, F.; Liu, Y.; Yan, H. DNA Origami: Scaffolds for Creating Higher Order Structures. Chem. Rev. 2017, 117, 12584-12640. (3) Rothemund, P. W. K. Folding DNA to create nanoscale shapes and patterns. Nature 2006, 440, 297-302. (4) Andersen, E. S.; Dong, M.; Nielsen, M. M.; Jahn, K.; Subramani, R.; Mamdouh, W.; Golas, M. M.; Sander, B.; Stark, H.; Oliveira, C. L. P.; Pedersen, J. S.; Birkedal, V.; Besenbacher, F.; Gothelf, K. V.; Kjems, J. Self-assembly of a nanoscale DNA box with a controllable lid. Nature 2009, 459, 73-76. (5) Ke, Y. G.; Sharma, J.; Liu, M. H.; Jahn, K.; Liu, Y.; Yan, H. Scaffolded DNA Origami of a DNA Tetrahedron Molecular Container. Nano Lett. 2009, 9, 2445-2447. (6) Zhao, Z.; Liu, Y.; Yan, H. Organizing DNA Origami Tiles into Larger Structures Using Preformed Scaffold Frames. Nano Lett. 2011, 11, 2997-3002. (7) Voigt, N. V.; Tørring, T.; Rotaru, A.; Jacobsen, M. F.; Ravnsbæk, J. B.; Subramani, R.; Mamdouh, W.; Kjems, J.; Mokhir, A.; Besenbacher, F.; Gothelf, K. V. Single-molecule chemical reactions on DNA origami. Nat. Nanotechnol. 2010, 5, 200-203.

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