Three-Dimensional Molecular Transfer from DNA Nanocages to Inner

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Three-Dimensional Molecular Transfer from DNA Nanocages to Inner Gold Nanoparticle Surfaces Nuli Xie, Shiyuan Liu, Hongmei Fang, Yanjing Yang, Ke Quan, Jing Li, Xiaohai Yang, Kemin Wang, and Jin Huang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b09147 • Publication Date (Web): 04 Apr 2019 Downloaded from http://pubs.acs.org on April 4, 2019

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Three-Dimensional Molecular Transfer from DNA Nanocages to Inner Gold Nanoparticle Surfaces Nuli Xie†, Shiyuan Liu†, Hongmei Fang, Yanjing Yang, Ke Quan, Jing Li, Xiaohai Yang, Kemin Wang and Jin Huang*

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082, P. R. China *To whom correspondence should be addressed:

[email protected].

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Abstract: It is of great interest to construct DNA-functionalized gold nanoparticles (DNA-AuNPs) with a controllable number of DNA strands and relative orientations. Herein, we describe a three-dimensional (3D) molecular transfer strategy, in which a pattern of DNA strands can be transferred from a DNA icosahedron cage (I-Cage) to the wrapped AuNP surface. The results show that DNA-AuNPs produced by this method inherit DNA pattern information encoded in the transient I-Cage template with high fidelity. Controllable numbers and positions of DNA on the surface of AuNPs can be simultaneously realized by direct “printing” of a DNA pattern from the nanoshell (I-Cage) to the nanocore (AuNP), further expanding the applications of DNA nanotechnology to nanolithography. Prospectively, the customized DNA-printed nanoparticles (DPNPs) possess great potential for constructing programmable architectures for optoelectronic devices as well as smart biosensors for biomedical applications. Keywords: 3D molecular transfer, gold nanoparticles, DNA icosahedron cage, DNA nanotechnology, lithography

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DNA can be utilized as functional ligands to interact with various nanoparticles.1-4 Among these nanoparticles, DNA-functionalized gold nanoparticles (DNA-AuNPs) have attracted tremendous attention in past decades and have been applied in the fields of nanofabrication,5-7 optoelectronic devices,8 biological analysis,9-11 and disease diagnosis.12-14 Benefitting from specific recognition, strict Watson-Crick base pairing and structural programmability, these functionalized DNA molecules can not only act as sensitive noses to detect target molecules15 but also serve as robust hands to bottom-up fabricate complex nanoparticle architectures.16,17 In many cases, it is significant to customize DNA-AuNPs with a controlled number of DNA strands and placements, especially while being used for an accurate diagnosis and controlled assembly. However, it is a fascinating challenge and becomes the focus of ongoing efforts because conjugated DNA by gold-thiol bonds has no selectivity under salt-aging conditions.18-21 Traditionally, the AuNPs modified with a prescribed number of DNA strands (mainly limited to one, two or three strands) could be obtained by isolation methods, such as gel electrophoresis22 and anion-exchange HPLC.23 Recently, a postsynthetic approach based on polymeric nanoparticle precursors was used to prepare monovalent AuNPs during one-pot synthesis.24 On the other hand, some regioselective modification strategies have been proposed concerning controlling orientation.25-27 For example, Mirkin et al. applied magnetic beads as geometric restriction templates to prepare asymmetric functionalization of oligonucleotides on AuNP surfaces.26 Tang et al. reported Janus modification of AuNPs with the help of silica colloids.27 However, despite the great progress, it is very difficult to simultaneously control both the number and orientation of DNA strands on AuNP surfaces. Interestingly, the invention of DNA origami technology provides an indirect but viable 3


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approach to restrain the number and position of AuNPs in DNA-based nanoparticle assemblies. Various DNA origami nanostructures have been used as the breadboards to endow nanoparticles with structural information and specific arrangement, constructing one-dimensional (1D), two-dimensional (2D) and three-dimensional (3D) architectures.28-30 Nevertheless, the fabrication of DNA origami nanostructures requires many oligonucleotides, and AuNP architectures cannot stabilize independently without DNA origami. Additionally, AuNPs may have great limitation in function and application because they are passive guests in assemblies. Compared with complex DNA origami, simple DNA frames manage without large consumption of DNA strands. This method was demonstrated to be capable of controlling DNA-strand motifs on AuNP surfaces. For example, Suzuki et al. described a method to modify a certain number of DNA strands with controlled placement using a 1D double-stranded DNA template.31 Sleiman et al. developed a strategy to transfer DNA patterns from the surface-conjugated DNA nanocubes to the AuNP surface that can inherit DNA molecular information encoded in the template with high fidelity.32,33 Size-tunable DNA polyhedron nanostructures provide excellent flexibility to match different nanoparticles, and their cavities are dependent on external DNA skeleton. Few studies have focused on the cavity of the DNA polyhedron. Herein, we describe a 3D molecular transfer strategy to prepare DNA-printed nanoparticles (DPNPs) that can control the number and spatial position of DNA strands on the whole surface of AuNPs. The idea is to encapsulate AuNPs in a minimal DNA nanocage, in which molecular information (including the number of DNA strands and their relative placements) is intently transferred to the inner AuNP surface. Here, a DNA icosahedron cage (I-Cage) is chosen as a scaffold to 4


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load DNA pattern information. The 3D molecular transfer process can be divided into two steps. As shown in Figure 1, two half icosahedra with predesigned overhangs (dithiol-labeled 10-nt polythymine (T10) sequence domains, red color) are first assembled into an intact I-Cage and encapsulate one AuNP in it. These overhangs extended from component DNA strands of the nanocage can covalently conjugate to the AuNP via Au-S bond. The next procedure is to remove the external I-Cage template and retain the DNA pattern on the AuNP surface. Through this strategy, the customized DNA patterns can be designed on the I-Cage and transferred to the AuNP surface with high fidelity. To our knowledge, this approach of printing molecular information from the nanoshell to the nanocore has not been examined. Moreover, it is an exemplification of merging DNA nanotechnology with lithography at the nanoscale.34-36

RESULTS AND DISCUSSION Modular assembly of the I-Cage. The prerequisite of molecular transfer on the surface of the nanoparticle is to construct a minimal cage that fits the nanoparticle. Many polyhedron nanostructures can be used as cages, such as cubes,37 tetrahedrons,38,39 octahedrons,40 and dodecahedrons.41 However, several criteria should be considered: entire encapsulation with a maximized capacity factor, high operability, structural symmetry and easy removal after transferring patterns. We analyzed three polyhedral models, including tetrahedron, octahedron and icosahedron models (Figure S1). Considering all other conditions, DNA icosahedrons had a maximum use ratio of interior cavity as high as 0.829, compared with 0.302 for tetrahedrons and 0.605 for octahedrons. Additionally, thirty DNA edges can be used to design 5


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overhang sites, implying high operability for DNA pattern design. Therefore, DNA icosahedrons are employed as nanocages to encapsulate gold nanoparticles. The approach to assemble the I-Cage is a modular bottom-up assembly method (Figure 2A), initially reported by the Krishnan group.42,43 Briefly, three types of five-end-junction motifs (5JMs), namely, X, Y and Z, were the first-step products (DNA sequence information shown in Table S1). Each end of a 5JM had a sticky segment, which could hybridize with the complementary segment of the other 5JMs. Meanwhile, an overhang could be added from a sticky end through sequence extension. Half-icosahedrons were assembled by combining one X with five Ys or Zs. The sphere-like I-Cage was assembled by annealing two half-icosahedra (XY5 and XZ5) together. Polyacrylamide gel electrophoresis (PAGE) was used to identify the product of each step and formation of the I-Cage. The gel bands gradually slowed along with the increase in the molecular weight and size from 5JMs to the I-Cage (Figure 2B-D). Gel mobility of Y in lane 2 and Z in lane 3 ran distinctly slower than X in lane 1. The results may contribute to the formation of quintuple structures Y5 and Z5 under annealing conditions. The 10% native PAGE confirmed that I-Cage was successfully produced through stepwise assembly, with a yield of 61.8% by ImageJ analysis (lane 3, Figure 2D). Next, we used dynamic light scattering (DLS) to evaluate the hydrodynamic radius of the I-Cage, which was determined at 24.2 ± 4.5 nm (Figure S2). Considering the hydration effect, it was basically consistent with a mathematic model. In this model (Figure S3), 26 base-pair double-helical edges were calculated to be lines of 8.8 nm in length (one base pair corresponds to approximately 3.4 Å in length). The height of the I-Cage was 18.8 nm, and the lateral width was 16.5 nm. The volume of its inscribed void sphere was calculated to 1232.23 nm3, larger 6


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than the volume of a 13-nm AuNP (1150.35 nm3). Thus, the I-Cage could encapsulate an AuNP as large as 13 nm into its cavity. Some specimens were subsequently characterized by transmission electron microscopy (TEM) with 1% uranyl acetate negative staining (Figure S4). From the images, we could observe quasi-hexagonal features with the proper size, further confirming the successful formation of I-Cages. Encapsulation of AuNP into I-Cages. The sodium citrate reduction method was used to synthesized 13-nm AuNPs.12 The particles had a sharp absorption peak at 518 nm in the UV-vis spectrum, and the DLS analysis showed a good size distribution, with an average size of 13.49 nm (Figure S5). TEM was also applied to verify the shape and monodispersity (Figure S6), indicating that most of the particles could be encapsulated. Prior to use, AuNPs were passivated with bis-(p-sulfonatophenyl) phenylphosphine (BSPP) overnight at room temperature.44 BSPP-coated AuNPs can further stabilize against aggregation, especially under high concentration. Importantly, the size of AuNPs would not change after monolayer BSPP passivation (Figure S7). For encapsulation, two half-icosahedra were mixed equivalently in the presence of an excess of highly condensed AuNPs. In principle, an excessive amount of passivated AuNPs can guarantee to a maximum extent that each volume unit of two half-icosahedrons exist a nanoparticle in aqueous solution. The mixture solution was slowly annealed and kept at 4°C for 2 days to generate AuNP-encapsulated nanocages (AuNP@I-Cage). Gel electrophoresis was first used to study the encapsulation results (Figure 3A). Almost no new band was displayed in lane 2, indicating that AuNPs could not be encapsulated by single half-icosahedrons. Compared with lanes 1 and 2, several new bands with slower gel mobility appeared in lane 3. Thus, the I-Cage can successfully encapsulate a 7


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gold nanoparticle in its cavity. Notably, AuNP@I-Cage was not the only product; some side products bands were shown in the gel images, including the I-Cage without AuNP and AuNP attached with multiple DNA nanostructures. To perform the next steps, it is important to purify the AuNP@I-Cage complexes. Moreover, a 4-nm redshift from 518 nm to 522 nm in the UV-vis absorbance spectrum was presented to the purified complexes compared with the BSPP-coated AuNPs (Figure 3B). DLS measurement is also an important tool to analyze size evolution. Our studies indicated that AuNP@I-Cage showed a hydrodynamic radius of 25.3 ± 2.6 nm (Figure 3C), which was similar to that of the I-Cage, indicating that the encapsulation did not greatly change the dimensions of the I-Cage. To visualize this structure, an aliquot of the purified AuNP@I-Cage after negative staining with 1% aqueous uranyl acetate was used for TEM imaging. It revealed some gray nanoparticles with a highly electron dense core under low magnification (Figure S8). In the enlarged views, obvious DNA coronas could be observed outside the highly electron dense core of AuNPs, similar to the appearance of poached eggs (Figure 3E). The average size of the high-electron core in TEM images was coincident with BSPP-coated AuNPs. Therefore, it was confirmed that the I-Cage can successfully encapsulate AuNP in its cavity. Transfer of one DNA strand from the I-Cage. We proceeded to investigate the ability to imprint DNA strands onto AuNPs. For feasibility, we first transferred one strand to the inner AuNP. The working process is shown in Figure 3D. T10-extended AN-X1 (sequences were listed in Table S1) was substituted for X1 in 5JMs X (zoom 1 in Figure 3F). The T10 domain (red color) could provide fine-tuned flexibility to ensure the conjugation. A cyclic disulfide moiety (green color) was labeled at the end of T10 domain. It was reported that 8


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multiple thiols had more efficient and faster binding kinetics than a single thiol.45 The longer alkyl chain may also help to reduce electrostatic repulsion between the DNA strands and AuNP.46 After stepwise assembly similar to the I-Cage, the exposed T10 domain became a DNA overhang inside the I-Cage (red color, zoom 2 in Figure 3F). The free overhang could gently conjugate to the surface of inner AuNP through Au-S bond during annealing period. Next, the I-Cage template was removed under denaturing conditions (3 M urea); only AN-X1 could be retained to achieve monovalent DPNPs (AuNP-1DNA). No evidence indicated that it would destroy the gold-thiol covalent bond when using urea to disrupt hydrogen bonds.32 Before purification, the sample was processed with carboxyl-terminated PEG acid disulfide (OEG) at room temperature for 30 min to inactivate the remaining surface. It is a necessary procedure to maintain the stability and accuracy of the DPNPs. The electrostatic interaction from the PEG strain may also prevent the DNA strands from folding or aggregating. Finally, to test whether AuNP bears one DNA molecule, DNA-functionalized 6-nm AuNPs complementary to AN-X1 were introduced as addressable indicator probes (Figure 3G).

Related size analysis and TEM images of the small AuNPs (Figure S9) showed an average diameter of 6.84 ± 2.3 nm. AuNP-1DNA was incubated with small particles at a molar ratio of 1:3 at room temperature to assemble AuNP satellite structures. AuNP satellite nanostructures are indirect but effective ways to show DNA number and placement for each pattern. After removing redundant small particles, the sample was prepared for microscopy

analysis. From TEM images (Figure 3H and Figure S10), we observed many combination structures, in which one large AuNP was accompanied by one small AuNP. The presence of “large-small” dimers proved successful transfer of one DNA molecule from the I-Cage to 9


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AuNP. Customized 3D transfer of DNA patterns. By generalizing this strategy, we tried to obtain more DPNPs with different numbers and spatial positioning of DNA strands, showing number controllability from one strand to more strands and tunable positioning from 1D to 3D. Thus, three types of DNA patterns were designed (detail modules are shown in Table S2). To be specific, AuNP-2DNA1 (Figure 4B) and AuNP-2DNA2 (Figure 4C) are two-leg DPNPs. Two printed DNA strands of AuNP-2DNA1 were AN-X1 and AN-X2, which came from the same hemisphere. By contrast, AuNP-2DNA2 acquired one strand (AN-X1) from XY5 and the other strand (AN-X1) from XZ5. Two patterns can show distinct dihedral angles between two printed strands. Likewise, three-leg AuNP-3DNAs (Figure 4D) were designed, where two DNA strands were from XY5 and the third strand was from XZ5. The relative geometrical models of these patterns are given in Figure S11. To visualize these DPNPs, multiple DNA-functionalized AuNPs were still used as addressable probes to assemble the AuNPs satellite structures via complementary hybridization. AuNPs (6 nm) were respectively functionalized with X1’ and X2’. As shown in Figure 4B-D and Figure S12-S14, the morphologies were basically consistent with the theoretical models, which revealed high-fidelity transfer of molecular information. Importantly, AuNP-2DNA2 showed a larger dihedral angle than AuNP-2DNA1, suggesting that spatial positioning and angle regulation were well reflected in the transfer process. Nevertheless, side products also appeared in microscopy analysis, including defective nanostructures (such as large AuNPs only and insufficient satellites) and oversaturated satellite nanostructures. By counting at least one hundred nanostructures from different images and different TEM grid positions, the effective 10


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yield of the satellite structures of each DPNP was calculated. The yields of AuNP-1DNA, AuNP-2DNA1, AuNP-2DNA2 and AuNP-3DNA were 28.84%, 25.45%, 18.63%, and 16.69%, respectively. Obviously, the yield decreased as the printed DNA strand increased from one strand to three strands and the pattern complexity increased (Figure S15). Some factors should account for this tendency, such as product loss during purification and crosslinking clusters between polyvalent particles. Additionally, unsaturated hybridization or insufficient small nanoparticles may lead to defective product. Furthermore, to validate the probability of higher valency, we tested four-leg and five-leg DPNPs. AuNP-4DNA contained two strands with a larger angle in the top hemisphere and two strands with a smaller angle in bottom hemisphere. AuNP-5DNA contained five DNA strands in one hemisphere. Similarly, the DPNPs were respectively incubated with an excess of multiple 6-nm DNA-functionalized nanoparticles to produce the satellite structures. We could still observe some desired products dispersed in TEM images despite a poor yield (Figure 4E and F). Overall, our design proved the flexible transfer of DNA patterns could be realized. Next, DLS measurement was applied to study the size evolution of diverse DPNPs. AuNP-1DNA, AuNP-2DNA1, AuNP-2DNA2, and AuNP-5DNA were measured to compare the hydrodynamic diameters. All the samples were filtered using a 0.45-µm nylon syringe filter before DLS measurement. As a result, the data exhibited a gradually increasing tendency when more DNA strands were conjugated (Figure 5A). Although AuNP-2DNA2 had the same amount of DNA strands as AuNP-2DNA1, it had a larger average diameter of 24.39 nm than 19.88 nm of AuNP-2DNA1 (Figure 5B). The increase in its hydrodynamic diameter may be ascribed to the opposite spatial orientation of two DNA strands on AuNPs. 11


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The positioning change from 1D to 3D induced the increase in the hydrodynamic diameter. Additionally, agarose gel electrophoresis of AuNP-1DNA, AuNP-2DNA1, and AuNP-3DNA was performed. Gel mobility of DPNPs slightly decreased as the number of DNA strands increased from one to three, indicating the change in the DNA strand number (Figure S16). Furthermore, to probe the number fidelity and sequence identity, fluorescence assays were employed

using

AuNP-1DNA,

AuNP-2DNA1,

and

AuNP-3DNA.

6-Carboxyfluorescein-labeled DNA probe X1’ (FAM-X1’) complementary to AN-X1 and cyanine 3-labeled X2’ (Cy3-X2’) complementary to AN-X2 were incubated with three types of DPNPs (Figure 5C). After removing redundant probes, the fluorescence intensity of FAM at 520 nm and Cy3 580 nm were measured. For FAM fluorescence, AuNP-3DNA showed almost twice

the intensity than AuNP-1DNA and AuNP-2DNA2 (Figure 5D), consistent with the number of AN-X1. Similarly, AuNP-2DNA1 showed the same Cy3 fluorescence intensity with AuNP-3DNA (Figure 5E) because both nanoparticles had only one AN-X2 strand. The data supported good controllability of the DNA number through I-Cage molecular transfer. Significantly, the retained sequences can still hybridize with other DNA molecules, and sequence specificity was reserved well in the process, which was crucial to building high-order nanoparticle assemblies or construct smart biosensing platforms.

CONCLUSION In summary, we demonstrate a strategy of transferring customized DNA 3D patterns to AuNPs through the I-Cage. DNA molecular information can be “printed” on the surface of AuNPs. This method is an exemplification of merging DNA nanotechnology with lithography 12


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at the nanoscale. The I-Cage provides flexible compilation of DNA patterns for AuNPs, with a controlled number of DNA strands and relative orientations. Importantly, DPNPs inherit a DNA sequence profile derived from the template with high fidelity. With this strategy, we envision that many of our designed DNA patterns can be realized, although the yield needs more improvement. On the one hand, diverse DPNPs can be considered “atom equivalents”, offering a great potential for constructing optoelectronic devices and chiral plasmonic nanomaterials.48 On the other hand, DPNPs can be further developed for DNA computation, disease diagnosis and targeted cancer therapy. This approach has the power to bear targeting moieties (such as aptamers) with designed events, offering a reat potential in logic sensors, polyvalent receptor recognition and targeted therapy in the future.

METHODS Materials All HPLC-purified oligonucleotides were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China), and all the sequences are shown in Table S1. Bis-(p-sulfonatophenyl) phenylphosphine (BSPP) and PEG acid disulfide (OEG) were purchased from Sigma-Aldrich (Milwaukee, USA). Chloroauric acid was obtained from Sinopharm Chemical Reagent Company (Shanghai, China). All other reagents were of analytical grade. The 200-mesh carbon-coated and glow discharged grids were purchased from ZXBAIRUI Company (Beijing, China). The ultrafiltration centrifugation tubes and gel purification kit were obtained from Sangon Biotechnology Co., Ltd. (Shanghai, China). The 1× PB buffer (pH=6.0) comprised 10 mM phosphate salt, 1 mM magnesium chloride and 100 mM sodium 13


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chloride, and 1× TBE buffer (pH 8.3) comprised 90 mM Tris, 90 mM boric acid and 2 mM EDTA. All aqueous solutions were prepared using ultrapure water (≥18 MΩ; Milli-Q water purification system, Millipore).

Instruments DNA quantification measurements were performed by UV absorbance at 260 nm using a Biospec-nano microvolume UV-vis spectrophotometer from Shimadzu. The other absorption spectra were obtained using a UV2600 UV-vis spectrophotometer from Shimadzu. The fluorescence spectra were obtained on a Hitachi F-7000 fluorescence spectrometer (Japan). Gel images were captured using an Azure C600 gel imaging system (Azure Biosystems, USA). Size analysis was performed by dynamic light scattering (DLS) using the Zetasizer Nano ZS instrument (Malvern, USA). The transmission electron microscopic (TEM) images were obtained on a JEM-3010 transmission electron microscope (JEOL, Japan) and Tecnai F20 transmission electron microscope (FEI, USA). Modular assembly of the I-Cage (a) Assembly of three 5JMs: According to the reported protocol,42 five HPLC-purified oligonucleotides, 50 µM, were mixed in equimolar quantities in PB buffer (pH 6), heated to 95°C in a thermostatic water tank for 15 minutes (min) and then were slowly cooled at the rate of 0.33°C/min to 20°C. The samples were incubated at 20°C for 2 hour (h) and then were equilibrated at 4°C for 3 days to form 5JMs. The formation of 5JM components was characterized by 15% native polyacrylamide gel electrophoresis (PAGE). For each 5JM variant reported in the manuscript, different combinations of DNA strands were used as 14


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shown in Table S2. (b) Assembly of XY5 and XZ5: a 1:5 ratio of X: (Y or Z) (0.83 μM) was mixed adequately and then heated to 45°C for 4 h before annealing at the rate of 0.33°C/min to 20°C. The samples were finally incubated at 20°C for 2 h and equilibrated at 4°C for 3 days. The formation of XY5 and XZ5 was characterized by 10% native PAGE. (c) Assembly of the I-Cage: XY5 and XZ5 were mixed in equimolar ratios, heated to 45°C for 4 h and annealed at the rate of 0.33°C /min to 20°C. The samples were incubated at 20°C for 2 h and then equilibrated at 4°C for 2 days. For each I-Cage variant reported in the manuscript, the detail is shown in Table S2. The I-Cage was characterized and purified by 10% native PAGE.

Preparation of AuNPs The 13-nm AuNPs were synthesized using the standard sodium citrate reduction method.12 Before the procedure, all glassware was cleaned in aqua regia (HCl: HNO3=3:1), rinsed fully with deionized water and then oven-dried. Next, 0.01% HAuCl4 (100 mL) was heated to boiling with vigorous stirring, followed by the addition of 3.5 mL of trisodium citrate (1%) under stirring. The color of the solution turned from pale yellow to colorless and finally to burgundy. After cooling to room temperature, it was filtered using a 0.45-µm Millipore syringe filter. The size distribution was analyzed by TEM and DLS. The 6-nm AuNPs were synthesized using this method.47 First, 100 mL of 0.01% HAuCl4 solution was gently stirred for 3 min. Next, 2 mL of 38.8 mM sodium citrate was added, and the mixture was stirred for several minutes. Thereafter, 1 mL of fresh 0.075% NaBH4 in 38.8 15


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mM sodium citrate was quickly added, and the reaction mixture was stirred for 5 min. The solution color turned from pale yellow to bright red. For further use, it was filtered using a 0.22-µm Millipore syringe filter. The size distribution was analyzed by TEM and DLS.

Molecular transfer of DNA patterns Before encapsulation, AuNPs were subjected to BSPP surface passivation.44 AuNPs were quantified by the UV-vis absorbance at 520 nm. Highly concentrated BSPP-coated AuNPs were incubated with a mixture of DNA half-icosahedron scaffolds in 10 mM PB buffer. The mixture solution comprised 40 nM XY5 and XZ5 in a 1:1 ratio. The resulting solution was heated at 45°C for 4 h, and then the temperature was decreased at the rate of 1°C/3 min until room temperature and finally equilibrated at 4°C for 2 days. In this process, it was necessary to add some sodium solution. To remove the I-Cage scaffold, the sample was soaked with 3 M urea in buffer for 10 min, followed by centrifugation and washing with ultrapure water. This process was repeated at least four times. Then, the solution was incubated with OEG at room temperature for 30 min to inactivate the surface, followed by centrifugation at least three times (12,000 rpm) at 4°C to remove the supernatant and resuspension in fresh buffer. Next, DPNPs were separated and purified by ultrafiltration centrifugation (50 kDa MWCO) (13000 rpm) several times and gel electrophoresis purification. Finally, the sample was resuspended in PBS buffer and quantified by UV-vis absorption. To visualize the I-Cage and AuNP@I-Cage complex, 5 µL of each was adsorbed on a 200-mesh carbon-coated copper grid by flotation for 30 min. The excess solution was removed. TEM analysis was carried out under a 120-kV operating voltage using a JEM-3010 16


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transmission electron microscope (JEOL, Japan) after the samples were negatively stained using 1% uranyl acetate solution.

Preparation of AuNP satellite structures To prepare six types of the DPNP satellite structures from AuNP-1DNA to AuNP-5DNA, 6 nm polyvalent DNA-functionalized AuNPs were used. Four different polyvalent AuNPs were generated, functionalized with thiol-modified DNA strands (X1’, X2’, X4’ and Y3’), respectively, using salt-aging method. Typically, TCEP-processed oligonucleotides were added into the AuNPs and kept at room temperature in PBS buffer for at least 16 h. Sodium chloride solution (2 M) was added dropwise into the mixture each 8 h over a one-day period. Next, the particles were centrifuged (14,000 rpm, 30 min) three times and resuspended in buffer.

To

assemble

the

satellite

structures,

three-fold

excess

of

each

small

DNA-functionalized AuNPs was incubated overnight with DPNPs in a tube. The products were then concentrated and purified by agarose gel electrophoresis for subsequent TEM analysis. To visualize the satellite nanostructures, 5 µL of the sample droplet was adsorbed onto the carbon-coated surface of a 200-mesh copper grid by flotation for 25 min. Next, three gentle washes with 10 µL of ultrapure water were performed, followed by immediate removal. The samples were placed under a vacuum before microscopy. TEM analysis was performed under a 200-kV operating voltage using a Tecnai F20 transmission electron microscope (FEI, USA).

Fluorescence analysis 17


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DNA sequences X1’ labeled with FAM and X2’ labeled with Cy3 were used in this experiment. AuNP-1DNA, AuNP-2DNA1 and AuNP-3DNA were chosen as representatives to incubate with an excess of fluorescence dye-labeled DNA strands (FAM-X1’ and Cy3-X2’) for 3 h at room temperature. After centrifugation (13,000 rpm), the supernatant was removed, and a volume of buffer was added. This process was repeated 3 times to remove any unbound dye-labeled DNA strands. Next, these samples were measured using an F-7000 fluorescence spectrometer with an excitation wavelength at 488 nm for FAM and 550 nm for Cy3. The fluorescence from 510 to 650 nm was collected as the excitation spectrum of FAM, and the fluorescence from 560 to 650 nm was collected as the excitation spectrum of Cy3.

ASSOCIATED CONTENT The authors declare no competing financial interests.

Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org. Detailed experimental methods, relevant simulation analysis, characterization of samples, and supplementary results (PDF) Author contribution †

These authors contributed equally to this work.

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Acknowledgements This work was supported by the National Natural Science Foundation of China (21735002, 21874036).

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Figure 1. Schematic overview of the 3D transfer of DNA patterns onto AuNPs via the DNA icosahedron cage (I-Cage). Two half-icosahedra, which are designed with dithiol-labeled overhangs (red), assemble into an intact DNA icosahedron and encapsulate an AuNP in it. The DNA patterns, which comprise different combinations of overhangs that are extended from component DNA strands of the I-Cage, covalently conjugate on the surface of AuNP by a gold-thiol bond. After removing the nanocage template, DNA overhang strands are retained on AuNPs, rendering an identical pattern from the I-Cage. Various DNA patterns can be designed and transferred, indicating number controllability and site addressability.

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Figure 2. Formation of the I-Cage by modular assembly. (A) Illustration of the modular assembly strategy of DNA icosahedron cages. 5JM X has five sticky ends with the same sequence (yellow color domain). In 5JMs X and Y, the same color means that they are complementary segments. (B) 15% Native PAGE analysis for the first-step assembly of 5JMs. Lane 1: X1, lane 2: X12, lane 3: X123, lane 4: X1234, lane 5: 5JMs X. (C) 10% Native PAGE analysis for the second-step assembly of the half-icosahedron. Lane 1: X, lane 2: Y, lane 3: Z, lane 4: XY5, lane 5: XZ5. (D) 10% Native PAGE analysis for the third-step assembly of the I-Cage. Lane 1: XY5, lane 2: XZ5, lane 3: I-Cage.

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Figure 3. Molecular transfer process by the I-Cage to prepare DPNPs. (A) 0.8% Agarose gel analysis to verify the encapsulation of AuNP into the I-Cage. Lane 1: BSPP-coated AuNPs, lane 2: XY5+AuNPs, lane 3: XY5+XZ5+AuNPs. (B) Comparison of the UV-vis spectrum of AuNPs before and after encapsulation. (C) Comparison of DLS measurements of AuNPs before and after encapsulation. (D) Principle of transferring one DNA strand. Step i is to design XY5 with a DNA overhang. Step ii is to encapsulate one AuNP by XY5-1DNA and XZ5. Step iii is to remove the I-Cage template. (E) Enlarged TEM images of AuNP@I-Cage stained negatively with 1% uranyl acetate, under a 120-kV operating voltage. (F) Enlarged view of the overhang in 5JM X-1DNA and the I-Cage. (G) Illustration to assemble dimer assemblies using monovalent AuNPs with polyvalent 6 nm AuNPs. (H) Enlarged TEM images of “large-small” AuNPs dimers under a 200-kV operating voltage. 25


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Figure 4. I-Cage models of six types of DPNPs, as well as TEM images of corresponding AuNP satellite nanostructures. The self-assembled I-Cage has thirty nick sites in total, and each nick site can be a position to extend with an overhang. The dithiol-labeled overhang can conjugate onto the surface of AuNP through the Au-S bond and can be retained after removing the DNA template. By generalizing this strategy, six I-Cage variants carrying prescribed patterns (A-F) were constructed to produce diverse DPNPs, showing number controllability from one strand to five strands and geometry-specific patterns from 1D to 3D. The right panel shows enlarged TEM images of satellite nanostructures, which were assembled by parent DPNPs and multiple DNA-functionalized small AuNPs by complementary hybridization. The scale bar is 20 nm. All images were obtained under a 200-kV operating voltage using a Tecnai F20 transmission electron microscope (FEI, USA). The scale bar is 20 nm.

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Figure 5. Size evolution and fluorescence studies of DPNPs. (A) DLS measurements of four types of DPNPs, including AuNP-1DNA, AuNP-2DNA1, AuNP-2DNA2 and AuNP-5DNA. (B) Histogram of the average size of four DPNPs, calculated from DLS measurements. (C) Scheme showing the binding process of fluorescent probes on AuNPs (AuNP-1DNA, AuNP-2DNA1, AuNP-3DNA) using FAM-labeled X1’ and Cy3-labeled X2’, showing successful inheritance of sequence specificity. Normalized FAM (D) and Cy3 (E) fluorescence histogram of three DPNPs, by taking the intensity of AuNP-3DNA as 1.0. BSPP-coated AuNPs were used as a control. The fluorescence spectra are given in Figure S17.

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Three-Dimensional Molecular Transfer from DNA Nanocages to Inner

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