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Self-Assembly

DNA Pillars Constructed from an i-Motif Stem and Duplex Branches

Yang Yang, Chao Zhou, Tao Zhang, Enjun Cheng, Zhongqiang Yang,* and Dongsheng Liu*

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As a ‘bottom-up’ nanotechnology, DNA self-assembly has continuously excited the whole world owing to its remark-able molecular recognition, precise addressability, and high fidelity.[1] It has shown that, by using a tile-by-tile method, DNA units containing sticky ends can be used as building blocks to create diverse structures, ranging from ribbons[2] to grids[3] and tubes[4] and, more recently, this method has been extended to build 3D structures, e.g., cubes, tetrahedrons, octahedrons, and icosahedrons.[5] Alternatively, DNA origami is now a well-known strategy to construct desired two- or three-dimensional shapes by folding a single-stranded DNA template into a predesigned structure, aided by multiple ‘staple’ strands.[6] Due to the specificity of Watson–Crick interactions between complementary base pairs, each staple binds to a specific DNA region, thus forming a well-defined nanostructure. It has also been reported that, unlike the above methods, metal ions and covalently modified chemical ligands were used to facilitate the assembly of highly ordered DNA nanostructures.[7] All the DNA self-assembly strategies out-lined above are based on classic Watson–Crick base paring. DNA wires utilizing G-quadruplexes and i-tetraplexes have also been established,[8] however, currently the manipulation and functionalization of the structures assembled using this method have not been sufficiently explored. Inspired by those pioneer studies, we sought to establish a methodology to build up DNA nanostructures; in particular, we employed tetra-plex (i-motif) backbones and periodical duplex branches to form DNA pillars, and further engineered them to direct the assembly of Au nanoparticles (AuNPs).

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DOI: 10.1002/smll.201102061

C. Zhou, Dr. Z. Yang, Prof. D. Liu Key Laboratory of Organic Optoelectronics & Molecular Engineering of the Ministry of EducationDepartment of ChemistryTsinghua UniversityBeijing 100084, ChinaE-mail: zyang@mail.tsinghua.edu.cn; liudongsheng@tsinghua.edu.cn

Dr. Y. Yang, C. Zhou, Dr. E. Cheng National Center for Nanoscience and TechnologyBeijing 100190, China

T. Zhang Department of ChemistryRenmin University of ChinaBeijing 100872, China

Different from duplex DNA, the most unique thing about tetraplex DNA (four-strand-helix) geometry is its four free ends, one on each of its poles. This fact enables the ends of the tetraplex to reach out in four directions (positive and nega-tive) in the XY plane, perpendicular to the Z axis of the helix, and be available for 3D assembly. Unlike the G-quadruplex, which has several different possible orientation combina-tions, the i-motif, with its unique topology, turns out to be the best candidate of this work. The i-motif is a special DNA secondary structure formed at slightly acidic solutions by DNA strands with repeated cytosines (Scheme 1A).[9] This structure can fold and unfold with pH as the trigger, and has potential for fabricating DNA machines and devices.[10] In this work, two strands, A and B, both containing five repeated cytosines (C5), are designed. A random sequence is linked to the 3′-end of the C5 in strand A, while its complementary sequence is linked to the 5′-end of the C5 in strand B. Under acidic conditions, both of them can assemble into a tetramer, named here blocks A and B, containing an i-motif core and four free tails (Scheme 1B), and the tails of blocks A and B are fully complemented. Consequently, after mixing the two preformed blocks together, they can further assemble into long pillars in the Z direction with tetraplex backbones and duplex branches in parallel XY planes (Scheme 1C). The two sequences can be presented as A: 5′-TC5ANn-3′ and B: 5′-NnAC5T-3′. A thymine (T) and an adenine (A) are intro-duced at the end and the corner positions of the sequences to avoid continued growth of the C-wires and to offer appro-priate flexibility for the arms.

Our first set of experiments is to achieve the two building block structures and examine their assembly ability. Ini-tially, the blocks with 9 base tails were used. Sequence A1: 5′-TCCCCCAGTACTCAGA-3′ (200 μm) and B1: 5′-TCT-GAGTACACCCCCT-3′ (200 μm) in pH 5 buffer containing 50 mm 2-(N-morpholino) ethanesulfonic acid monohydrate (MES) and 200 mm NaCl were slowly annealed from 95 to 4 °C at a rate of 0.5 °C min−1, respectively. Then, they were mixed together at the ratio of 1:1, and incubated at 4 °C for 12 h. The resulting product was testified via circular dichr-oism (CD) spectroscopy and 8% native polyacrylamide gel electrophoresis (PAGE). In Figure 1A, Block A1, Block B1, and their mixture at pH 5 exhibit a positive peak at 285 nm, which is a typical signature for an i-motif structure, showing a clear difference from the samples studied at pH 8. This was also determined by the gel image (Figure 1B), in which Blocks A1 and B1 gave a single band that was slower than

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Stem and Duplex Branch DNA Pillars

Scheme 1. Model of the DNA self-assembly process: A) i-motif structure formed by multicytosine sequences at acidic pH; B) blocks A and B with 5-cytosine bodies and complementary tails self-assemble to form the building blocks; C) mixing of the blocks A and B gives the DNA pillar.

the band of the denatured A1. Additionally, lane 5 for the mixture of Blocks A1 and B1 presented ladder-shaped DNA bands, indicating that a series of assembly structures have

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Figure 1. A) CD spectroscopy of sequences A1,B1 and also of their mixturlong term stability, with 8% native gel under 260 V for 3 h in 50 mm MES sA1 prepared with pH 8 MES buffer and mixed with denatured loading buf

been formed, including a monomer (unreacted A1 and B1), dimer, trimer, and tetramer which, from the best resolu-tion of the gel image, was the longest of the assemblies. The

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es A1B1 at pH 5 and 8; B) electrophoretic image of the assembly and its olution at pH 5. As a negative control, Lane 1 was loaded with the sample fer.

Y. Yang et al.

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Figure 2. PAGE images of: A) comparison of the assembly level of A2B2 and A1B1, and B) A2B2 assembly at 1:1 ratio at different concentrations.

duration of assembly was also examined: from lane 5 to 8, it is shown that, after incubation of the mixtures of Blocks A1 and B1 from one day to even seven days, there is no obvious change to the ratio for the different assemblies, suggesting that an equilibrium state existed between the assembly and disassembly processes.

In order to inhibit the disassembly process, in other words, to achieve longer assemblies, the most efficient way is to pro-long the block tails in order to enhance the stability of the branches. Therefore, we chose the sequence A2: 5′-TCC CCC AGT ACT CAG AAT CG-3′ and B2: 5′-CGA TTC TGA GTA CAC CCC CT-3′, which had tails 4 bases longer than A1 and B1. As expected, longer assemblies were observed in the gel image, Figure 2A. Note that these assemblies composed of an odd number of monomers have two types, for example, ABA or BAB types (Figure 2B), appearing as two adjacent bands, probably attributable to the different molecular weights of A2 and B2, thus leading to different electrophoretic migrations. However, as the number of building blocks (n) is increased, for example, above four, only a single band appeared due to the shorter migration distances and the smaller fractions of molecular weight, as compared to the whole polymer weight (W), [4·|WA–WB|/(WA+WB)·4n]. It is interesting that assem-blies with odd numbers of building blocks have a darker band than their neighbors. This phenomenon may be attributed to an asymmetric structure of the ABA or BAB, however, the mechanism is still unclear.

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We also studied the influence of concentration of building blocks. At a 1:1 ratio, it was observed that, as the concentra-tion was varied from 50 to 200 μm, the bands of assemblies on the gel showed almost similar trends (Figure 2B). As the ratio changed, in the presence of excessive A2, more and larger assemblies appeared, whereas less assemblies formed when B2 was in excess (see Supporting Information (SI), Figure S1).

In recent studies, DNA-modified Au nanoparticles[11] have shown advantages in the precise directive assembly of 1D, 2D, and 3D nanostructures. Substantial efforts have been devoted to the fabrication of DNA-modified AuNPs and to improve assembly efficiency.[12] In this study, with these assemblies in hand, we sought to employ the duplex branches to direct the assembly of Au nanoparticles. We used a 3′-SH- modified B2 mixed with A2 to form DNA pillars, then an excessive amount of 5 nm Au nanoparticles was added. Incu-bation for 12 h resulted in assembled AuNPs. Transmission electron microscopy (TEM) images in Figure 3a–c show that parallel lines of AuNPs were formed, and the possible struc-ture is illustrated in Figure 3d. It was also observed that a few assemblies exhibited a twisted shape. This could be explained by the intrinsic property of the i-motif structure, where the distance between the two nearest ends of the branches is less than 5 nm (SI, Figure S2), leading to two attaching strate-gies (SI, Figure S3). Therefore, two neighboring branches had to share one Au nanoparticle, and form parallel or twisted

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Stem and Duplex Branch DNA Pillars

Figure 3. TEM images of DNA pillar-directed AuNP assemblies. a–c) TEM images focussed on different regions at different magnifications. d) Schematic of assembled structures.

AuNP lines. Up to eight continuous Au nanoparticles were observed, which implies the existence of assemblies of 16 constructed from building blocks, which is in good agreement with the PAGE result.

In summary, we have successfully developed a new strategy to build DNA pillars from i-motif stems and duplex branches. The assembled pillar-shaped structure is stabilized by both C·C+ pairs from i-motif stems and Watson–Crick base pairs from duplex branches. These obtained DNA pillars can direct the assembly of Au nanoparticles into two parallel lines or helical structures. This brand new construction would potentially participate in the self-assembly of other systems to construct controllable and functional nanostructures.

Experimental Section

Materials: Regular chemicals, including all the buffer contents, were of reagent grade or better, and purchased from Sigma-Aldrich

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Co., Ltd. Water used in all experiments was Millipore Milli-Q deion-ized (18.2 MΩ cm). All the oligonucleotides were purchased from TaKaRa Biotechnology (Dalian) Co., Ltd.

Circular Dichroism: CD spectra were recorded on a J-810 Spec-trometer (DHS Instruments Co., Ltd. Dalian) with a scan range from 220 to 350 nm.

Gel Electrophoresis: The 8% native polyacrylamide gel was used for all the electrophoresis analysis. The buffer for both gel preparation and gel running was 50 mm MES solution titrated to pH 5 with NaOH. The electrophoresis was operated with 12 V cm−1 for 3 h, and the gel was soaked in freshly prepared Stains All solution for 1 h before imaging.

5 nm Au Nanoparticles Synthesis: Add 6.25 mL, 1% HAuCl4 solution, and 5.8 mL of 0.1 m K2CO3 to 500 mL deionized water. Mix well. Dilute 1 part of the saturated phosphorus solution with 4 parts of diethyl ether. Add 4.16 mL of the diluted phosphorus solution to the chloroauric acid/carbonate solution with mixing. React at room temperature for 15 min. Reflux until the suspension turns from brown to red.

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Y. Yang et al.communications

Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements

The authors thank National Basic Research Program of China (973 program, No. 2011CB935701), the National Natural Science Foundation of China (No. 21121004 & 91027046), and the NSFC-DFG joint project TRR61 for financial support.

This Communication is part of the Special Issue on Multilevel Molecular Assemblies: Structure, Dynamics, and Functions, featuring contributions from the Transregional Collaborative Research Center (TRR 61).

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Received: September 30, 2011 Revised: November 11, 2011 Published online: January 30, 2012

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