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Self-assembly of large DNA origami with custom-designed scaffolds Xiaoxing Chen, Qian Wang, Jin Peng, Qipeng Long, Hanyang Yu, and Zhe Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09222 • Publication Date (Web): 10 Jul 2018 Downloaded from http://pubs.acs.org on July 11, 2018

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ACS Applied Materials & Interfaces

Self-assembly of large DNA origami with custom-designed scaffolds

Xiaoxing Chen, a

a, 1

Yu* and Zhe Li* a

Qian Wang,

a, 1

a

a

Jin Peng , Qipeng Long , Hanyang

a

Department of Biomedical Engineering, College of Engineering and

Applied Sciences, Nanjing University, Nanjing, Jiangsu 210093, P. R. China 1

These authors contributed equally to this work.

*Corresponding authors

Keywords: DNA origami; self-assembly; nanotechnology; structural DNA nanotechnology; molecular biology

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Abstract As a milestone in DNA self-assembly, DNA origami has demonstrated powerful applications in many fields. However, the scarce availability of long single-stranded DNA (ssDNA) limits the size and sequences of DNA origami nanostructures, which in turn impedes the further development. In this study, we present a robust strategy to produce long circular ssDNA scaffold strands with custom-tailored lengths and sequences. These ssDNA products were then used as scaffolds for constructing various DNA origami nanostructures. This scalable method produces ssDNA at low cost with high purity and high yield, which can enable production of custom-designed DNA origami for various applications.

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The important goal of nanotechnology is to build functional structures or materials with nanometer precision. Bottom-up self-assembly

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represents a

promising route to build such well-ordered nanostructures. Among a variety of materials for self-assembly, DNA has gained much attention largely because of its unprecedented predictability and programmability. By using Watson-Crick basepairing rule, DNA self-assembly is capable of constructing designer structures with different geometries at nanometer precision.2-4 As a milestone in structural DNA nanotechnology, DNA origami5 emerged as a powerful self-assembly approach that enabled construction of two-dimensional (2D) and three-dimensional (3D) nanostructures with diverse sizes and complexities.6-11 In DNA origami, a long singlestranded DNA (ssDNA) scaffold is folded into designed patterns guided by many short ssDNA staples. Owing to its unique spatial addressability, DNA origami nanostructures have been widely employed for applications in various fields such as nanomedicine,12-14 cell biology,15,16 biosensing,17-19 nanoplasmonics20 and metal nanoparticle synthesis.21-23 However, since the natural source of long ssDNA is scarce, the large majority of DNA origami are made from M13 bacteriophage genomic DNA, which severely limits the size and sequences of these nanostructures. To create larger origami structures, different approaches have been explored. One strategy uses double-stranded DNA (dsDNA) as the scaffold to replace the ssDNA,24,25 although it requires complicated annealing conditions and suffers from mixtures of misfolded contaminants. In another strategy, preformed origami structures are linked with each other or with another ssDNA scaffold (phiX174) to form larger structures.26,27,28 Nonetheless, since 3



the final structures are assembled from the same units, the unique addressability of DNA origami is diminished. Facing such challenges, some researchers have attempted to construct alternative long scaffold strands. For example, Pound and co-workers29 used polymerase chain reaction (PCR) amplification followed by ssDNA separation using streptavidin-coated magnetic beads to assemble several letter shaped origami structures. In another study, a 26 kilobases ssDNA was obtained using PCR amplification and asymmetric enzymatic digestion.30 Aside from strategies based on PCR amplification, a λ/M13 hybrid virus phagemid system was reported to produce a 51,466-nucleotide circular ssDNA .31 The former two strategies suffer from low yield of ssDNA due to the tedious separation steps, while the last one can only generate a ssDNA with a specific sequence. To address this issue, we developed a method to produce long ssDNA with custom-tailored lengths and sequences: by using Gibson assembly technique, different dsDNA fragments including the replication origin of M13 were seamlessly assembled into a circular recombinant phagemid, which was then transformed into E. coli cells. ssDNAs were efficiently produced in the presence of helper phages. The obtained ssDNAs were then used as scaffolds to self-assemble into various nanostructures, whose dimensions were larger than the ones folded from M13mp18. We also demonstrated that origami structures with different shapes were able to assemble in a single reaction simultaneously, as long as their corresponding scaffolds were designed with distinct sequences.

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Figure 1. Schematic illustration of the production of custom-tailored ssDNA scaffolds. The black color represents the replication origin of M13, and the other colors represent the dsDNA fragments. Custom-designed dsDNA fragments and the replication origin of M13 were seamlessly assembled into a circular recombinant phagemid, which was then transformed into E. coli cells for amplification. Helper phage infection yielded numerous in-vivo packaged phage particles. The corresponding ssDNA was obtained by harvesting and purification of these phages, and then was used as scaffold to assemble DNA origami nanostructures.

Figure 1 illustrates the overall experimental process. Custom-designed dsDNA fragments, the replication origin of pBR332 vector (589 bp), as well as the replication origin of M13 phage (510 bp) are amplified by PCR. Each fragment contains a 30 bp overlap sequences, which facilitates seamless assembly using Gibson assembly. This recombinant hybrid phagemid was then transformed to E.coli XL-1 blue cells to 5



achieve a high copy number. After infection with the helper phage M13KO7, the recombinant phagemid was replicated in a single-stranded form via rolling circle replication and packaged into a new phage. To test the feasibility of the method, we first designed a 10,563-nt ssDNA scaffold. Four DNA fragments were assembled into a recombinant phagemid. After sequencing verification, the phagemid was packed into its single-stranded form by helper phage. The ssDNA was then extracted from the phage and purified by ethanol precipitation. Approximately 1 mg ssDNA was finally produced from 1 L cell culture, comparable to previous reports.32,33 Agarose gel electrophoresis was employed to confirm the correct ssDNA production. The bands of the individual DNA fragments, the recombinant plasmid, and the ssDNA product were all at the predicted positions (Figure 2A). The purity of ssDNA was 86%, as measured by the band intensity. To further verify that the ssDNA could be served as the scaffold for DNA origami self-assembly, we designed a monolayer DNA rectangular origami structure using this ssDNA. After proper annealing procedure, the assembled nanostructures were examined by Atomic Force Microscopy (AFM). Rectangle nanostructures with dimensions of 100 nm X 83 nm were observed, which was consistent with our design (Figure 2C and Figure S-11). Some defectives in the nanostructures were observed, which might be caused by DNA fragmentation, nonoptimized annealing conditions, and/or tip perturbation during AFM scanning. It is noteworthy that our method has no restrictions on DNA sequences except for the replication origins of M13 and pBR332 vector (1,099 nt), and thus can be readily 6


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modified to meet user needs. To investigate the versatility of this method, we designed another ssDNA scaffold with a similar length but a totally different sequence. We then used this ssDNA as the scaffold to design a triangular origami nanostructure. The gel electrophoresis result (Figure 2B) confirmed the correct size of the 10,782-nt ssDNA, and also indicated that the purity of the ssDNA was 94%. The triangle nanostructures with designed dimensions were formed as expected, as shown in AFM images (Figure 2D and Figure S-12). Since the two 10k ssDNA scaffolds hold distinct sequences, the two sets of staple strands should have little interference with each other, and therefore the two origami nanostructures could be, in principle, assembled simultaneously in one reaction system. To test this hypothesis, two sets of scaffold and staple strands was mixed and annealed in one tube. The assembled structures were inspected under AFM. As shown in Figure 2E, both triangle and rectangle structures were clearly visualized. The results demonstrate that origami nanostructures with different shapes can be assembled in a single reaction, when scaffolds with different sequences are provided.

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Figure 2. Characterization of the 10k-nt ssDNA. (A) Gel electrophoresis analysis of the 10,563-nt ssDNA preparation. Lanes 1-4: four individual dsDNA fragments (5,181 bp, 1,812 bp, 3,279 bp, and 411 bp respectively). Lane 5: recombinant phagemid. Lane 6: 10,563-nt ssDNA. Lane 7: M13mp18 ssDNA as control. (B) Gel electrophoresis analysis of the 10,782-nt ssDNA preparation. Lanes 1 and 2: two dsDNA fragments (2,781 bp and 8,001 bp, respectively). Lane 3: recombinant phagemid. Lane 4: 10,782-nt ssDNA. Lane 5: M13mp18 ssDNA as control. (C) AFM image of the rectangular DNA origami, which was assembled using the 10,563-nt ssDNA as the scaffold. (D) AFM image of the triangle DNA origami, which was assembled using the 10,782-nt ssDNA as the scaffold. (E) Simultaneous assembly of two origami structures in a single reaction system. The rectangle structure was assembled using the 10,563 nt scaffold, while the triangle structure was assembled using the 10,782 nt scaffold. Scale bar: 200 nm. Two different visual fields are shown here.

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We further expanded the size of the recombinant phagemid to obtain longer ssDNA scaffolds. A 21,261-nt was produced by this method, and a rectangular nanostructure with a dimension of 167 nm X 100 nm was designed by using this ssDNA as scaffold. Moreover, a 31,274-nt ssDNA was produced and used to construct two structures with different geometries: a rectangular origami with a dimension 235 nm X 100 nm, and a triangular origami with the side length of 270 nm. Similarly, gel electrophoresis was employed to confirm the lengths and purities of the ssDNA products (Figure 3A and C). It was found that as the length increased, the ssDNA purity decreased from 82% (20k) to 70% (30k). The main impurities were resulted from ssDNA fragmentation. The successful assembly of the origami nanostructures was verified by AFM (Figure 3B, D and E; Figure S-13, S-14, and S-15). Compared with the classic rectangle origami nanostructure with M13mp18 as the scaffold,5 whose dimensions are 90 nm X 60 nm, our design has expanded the sizes by 3 times.

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Figure 3. Characterization of the 20k-nt and 30k-nt ssDNA. (A) Gel electrophoresis analysis of the 21,261-nt ssDNA. Lanes 1 and 2: the two dsDNA fragments (10,996 bp and 10,307 bp, respectively). Lane 3: recombinant phagemid. Lane 4: 21,261-nt ssDNA. Lane 5: M13mp18 ssDNA. (B) AFM image of the rectangular DNA origami, which was assembled using the 21,261-nt ssDNA as the scaffold; scale bar: 200 nm. (C) Gel electrophoresis analysis of the 31,274-nt ssDNA. Lanes 1-3: the three dsDNA fragments (10,031 bp, 10,337 bp and 10,996 bp, respectively). Lane 4: recombinant phagemid. Lane 5: 31,274-nt ssDNA. Lane 6: M13mp18 ssDNA. (D and E) AFM images of the rectangular and triangle DNA origami, which was assembled using the 31,274-nt ssDNA as the scaffold; scale bar: 200 nm.

All the recombinant phagemids we constructed contained two replication origins: one from pBR322 to regulate phagemid replication in E. coli cells, and one 10


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from M13 to pack ssDNA efficiently in phage cells. The high copy number of pBR322 and the highly efficient replication of the phagemid allowed the production of large amount of ssDNA, while the low error rate of phagemid replication guaranteed the high fidelity of these ssDNA synthesis. It is worth noting that this amplification is readily scalable. The final yield of the ssDNA is proportional to the culture volume. The main cost of the method comes from the production of the hybrid phagemid, including the reagents of PCR reaction and Gibson assembly. Once the phagemid is obtained, the remaining cost is just from the bacterial media cultures, which is much less expensive. The length of the ssDNA produced by this method will be limited by two factors: the recombination capability of Gibson assembly and the package capability of the helper phage. Gibson and colleagues34 have shown that their in vitro recombination system can be used to assemble dsDNA molecules of hundreds of kilobases. This approach goes beyond the limitations of traditional cloning methods which rely on the availability of endonuclease restriction sites, and allows for rapid seamless joining of several pieces of DNA molecules together. The package efficiency of the helper phage is related to phage species and bacterial host strain. In this study, we use a common commercial helper phage M13KO7 to infect E. coli cells. To the best of our knowledge, other types of helper phage which can recognize M13 replication origin may also be used, such as VCSM13 and R408.29 The optimized helper phage-host bacteria system is still under study. Nevertheless, the method we proposed here shows that ssDNA with more than 30k nucleotide, which is three times longer than the routinely used M13mp18, could be able to be produced with high efficiency. 11



In summary, we have developed a strategy to produce a series of ssDNA with custom-tailored lengths and sequences, which can serve as scaffolds to assemble origami structures with larger dimensions than the ones constructed from M13mp18. Specifically, we produced four long ssDNAs with the lengths of 10,563, 10,782, 21,261 and 31,274 nt respectively. Using the 30k-nt ssDNA, we assembled a 235 nm X 100 nm rectangle origami nanostructure, which was 3 times larger than the one assembled from M13mp18. We also demonstrated that different origami structures can be assembled in a single reaction simultaneously, when distinct ssDNA scaffolds and corresponding staples were mixed together. Our approach will open up a new avenue for a rich diversity source of scaffolds for origami on a broad level of applications. This method produces ssDNA at low cost with high yield and high purity, which would also find more broad applications in many other fields, such as synthetic biology.

Author information Corresponding Authors *E-mail: [email protected] *E-mail: [email protected]

Supporting information Materials, methods, design of DNA origami nanostructures, DNA sequences, and AFM cross-sectional profiles are supplied as Supporting Information. 12


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Acknowledgements We are grateful to the financial support from National Key Research and Development Program of China (2016YFA0502600), and National Natural Science Foundation of China (21603100 and 21708018) , and Fundamental Research Funds for the Central Universities. Z.L. and H.Y. thank the Thousand Young Talents Plan of China.

Conflicts of interest The authors have filed a provisional patent on this technology.

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