DNA-Based Nanotemplate Directed In Situ Synthesis of Silver

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DNA-based Nanotemplate Directed in-situ Synthesis of Silver Nanoclusters with Specific Fluorescent Emission: Surface-guided Chemical Reactions Zhen-Gang Wang, Qing Liu, Na Li, and Baoquan Ding Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.6b04150 • Publication Date (Web): 02 Nov 2016 Downloaded from http://pubs.acs.org on November 2, 2016

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Chemistry of Materials

DNA-based Nanotemplate Directed in-situ Synthesis of Silver Nanoclusters with Specific Fluorescent Emission: Surfaceguided Chemical Reactions Zhen-Gang Wang,† Qing Liu,† Na Li, Baoquan Ding* CAS Key Laboratory of Nanosystem and Hierarchial Fabrication, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190 (P.R.China) ABSTRACT:DNA-hosted silver nanoclusters (AgNCs) are a set of metallic fluorescent nanodots that possess high quantum yields and photostability. Here, we show the in-situ synthesis of AgNCs within single-stranded DNA host strands pre-organized on the self-assembled DNA nanostructure templates, which results in the site-specific formation of AgNCs with the specific fluorescence wavelength. The excitation/emission properties of AgNCs were tuned by adjusting the distance between nucleation site and the template, the template configuration and the location of the nucleation site on the template. Mass spectra analysis of AgNC products was performed to study the cluster sizes. The 5’ and 3’ ends of freely diffusing and template-supported host strands were labeled with a donor and an acceptor, and the FRET efficiency was evaluated to reveal the conformations of the host strands and their complexes with Ag+. It is indicated that the rigid template guided the synthetic pathway towards the preferential synthesis of AgNCs with a specific size distribution via a steric effect on the Ag+ adsorption to the host strands, which produces the specifically emissive AgNCs.

INTRODUCTION Chemical reactions on surfaces (e.g. at solid-liquid or solidvacuum interfaces) have attracted intensive attentions.1,2 In contrast to homogeneous systems, a solid surface can exert a variety of effects (e.g. steric, morphological or chemical) on the reaction pathways and products. However, it remains challenging to unravel the mechanisms of surface reactions and to regulate the pathways, due to the complexity in tuning the surface properties. Chemical self-assembly can provide a programmable platform for investigating and regulating surface-activated chemical processes. DNA nanotechnology, based on bottom-up self-assembly, has been demonstrated as a simple and robust methodology for creating DNA nanostructures with arbitrary shapes and patterns.3-9 The nucleicacid strands can be selectively functionalized with various components, which enable DNA nanostructures to behave as the templates for immobilizing a variety of functional nanoscale objects,10-13 fabricating metallic self-assemblies and patterns,14-19and directing chemical reactions.20-22The surface addressability and the precise control over the nanoscale features of the DNA-based templates allow the role of surface activation in chemical pathways to be microscopically resolved and flexibly tuned, which has rarely been reported. Metal nanoclusters with size approaching the Fermi wavelength of electrons exhibit molecule-like behaviors, such asdiscreteelectronic energy levels, redox behavior,intrinsic magnetism and molecular chirality.23-25Water-soluble silver nanoclusters (AgNCs),composed of a few Ag atoms, have shown great promise in biological labeling, biosensing and

information storage,23,26-29 due to high fluorescence quantum yield, photostability, and low toxicity. The formation of AgNCs using a DNA oligomer as the host is a time-saving and cost-effective method,30-32 which facilitated the in-situ synthesis of the AgNCs on the DNA superstructures which assembled with the host strands.33-35The positioned organization of the molecule-like AgNCs enables the correlation of the chemophysical characteristics of DNA-hosted AgNCs with the surface properties of the DNA-based nanostructured templates, which can act as a model platform for unraveling the mechanism of the surface-activated synthetic pathways and open a new avenue for tuning the surface effects. In this study, with single-stranded DNA of different sequences as the host strands, AgNCs were site-specifically synthesized on the structured DNA templates. It was revealed for the first time that the rigid template guided the synthetic pathways towards the formation of AgNCs with a specific size distribution and fluorescent emission properties, via a surface steric effect on the affinity of the reactant Ag+ to the host strands.

EXPERIMENTAL SECTION Materials. M13mp18 single strand DNA (7249 nucleotides)was purchased from New England Biolabs. All other DNA strands were purchased from Life Technologies Corporation with PAGE purity and then dissolved in deionized water to 100µM. Trizma® hydrochloride ( ≥99.9%), Trizma® base ( ≥99%),silver nitrate (AgNO3, >99%),Sodium borohydride (NaBH4, ≥99.9%), ammonium acetate (NH4OAc, ≥98%), magnesium acetate tetrahydrate(Mg(OAc)2• 4H2O, ≥99%), ethidium bromide (~95%), agarose (Vetec™, reagent grade),

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boric acid (≥99.5%),ethylenediaminetetraacetic acid (EDTA, ≥98%)were purchased from Sigma-Aldrich. Uranyl acetate (> 98%) was purchased from Structure Probe, Inc. Synthesis of AgNCs with ssDNA as the host strands.To synthesize AgNCs with ssDNA (N1, N2) as the host strand, 10 µL ssDNA (100µM) were mixed with 6 µL of 1 mM AgNO3 in 40 mM NH4OAc buffer (pH 7.0). The final concentrations of ssDNA and AgNO3 were 10 µM and 60 µM, respectively. After incubated for 3 hours at 4oC, 1.2µL of 10 mM NaBH4 was added to the mixture. The reaction was kept at 4°C for 24 h. Notably, the NaBH4 needed to be dissolved in precooled deionized water and used within 30s. Preparation of triangular DNA origami template. Hoststrand N1 or N2 was introduced to the 3’ end of 65 staple strands that formed one arm of the triangular DNA template. As a common assembly procedure, M13mp18 single strand DNA (5 nM) was mixed with 10 fold excess of staple strands. The assembly was conducted in 40 mM NH4OAc/12.5mM Mg(OAc)2 buffer by slowly cooling from 65°C to 45°C at the rate of 2°C per 10 min and then cooling from 45°C to room temperature over 30 min. Then, the products were purified with Amicon Ultra centrifugal filters (Ultracel-100 K, Merck Millipore) to remove excessive staple strands and the solution was changed to 40 mM NH4OAc buffer (pH 7.0). In the experiments that explored the influence of Mg2+ on the fluorescence of AgNCs, 40 mM NH4OAc /12.5mMMg(OAc)2was used as the washing buffer. For investigation of the template surface effect on the fluorescence of AgNCs, staple strands and template strands (molar ratio: 1/2), the two of which contained the complementary spacer sequences, were mixed with the staple strand of the other two arms and the M13mp18 strands, to assemble into triangular origami nanostructure. The purification and buffer exchange processes were the same as above. In situ synthesis of AgNCs on the DNA origami template.AgNO3 was added to the purified DNA origaminanostructure containing the host strands N1 or N2. After incubated for 3 hours at 4oC, NaBH4 was added to the mixture. The final concentrations of origami template, AgNO3 and NaBH4 were 5 nM, 1.95µM, and 3.9 µM, respectively. The reaction was kept at 4°C for 24 hours. Prior to fluorescence measurement, the DNA origami-AgNCs conjugates were concentrated with Amicon Ultra centrifugal filters to a concentration of 20 nM. Seeded growth of AgNCs on the DNA origami template. Mg (OAc)2 (100 mM), AgNO3 (1 mM) and freshly prepared Tris-HCl solution (pH 7.0, 1 M) were successively added to the purified DNA origami-AgNCs solution and the final concentrations were as follows: 0.5 nM DNA origami-AgNCs conjugates, 12.5 mM Mg (OAc)2, 9.75µM AgNO3 and 100 mMTris-HCl. The mixture was kept at room temperature for at least 12 h. Gel electrophoresis.The DNA origami-AgNCs conjugates were analyzed by agarose gel electrophoresis. The conditions of electrophoresis were as follows: 0.8% Ethidium Bromide stained agarose gel; running buffer, 0.5×TBE buffer; voltage, 15 V/cm; running time, 40 minutes.

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Fluorescence and mass spectral characterization. Fluorescence spectra were measured at room temperature in a 1 cm ultra-micro cuvette (LS55 Luminescence Spectrometers, Perkin-ElmerInstrumentsCorporation). Mass spectra were collected on a Bruker APEXII FT-ICR mass spectrometer (Bruker Daltonics Inc., Germany) in negative-ion mode. The samples were mixed with methanol immediately before the measurement to promote desolvation. This did not significantly affect fluorescence spectra. Time-correlated single photon counting measurements for decay time were done on a DeltaFlex TCSPC system (HORIBA, Japan). The excitation wavelength was set to 479 nm and the emission wavelength was set to 518 nm. The fluorescence decays were fitted with a tri-exponential function using DAS6 Decay Analysis Software. Atomic force microscope (AFM) imaging. Freshly cleaved mica surface (Grade V1, Structure Probe, Inc)was treated with 12.5 mM Mg (OAc)2 solution prior to sample deposition. 2µLDNA origami-AgNCs solution was diluted using 40mMNH4OAc/12.5mM Mg (OAc)2 buffer and left to adsorb on mica surface for 7 minutes. Then 200µL 40mMNH4OAc /12.5mM Mg (OAc)2 buffer was added to the liquid cell. All samples were scanned in liquid mode on a MultiMode 8 AFM (Bruker) in ScanAsyst mode with SNL-10 tips (Bruker). The AFM images were analyzed using NanoScope Analysis software. Transmission electron microscope (TEM) imaging and analysis. The samples for TEM imaging were prepared by placing 5 µL of the sample solution on a carbon-coated copper grid (400 meshes, Ted Pella). Before depositing the sample solution, the grids were first glow discharged using Emitech K100X machine in order to increase its hydrophilicity. After 10 minute deposition, the unbound sample was wicked from the grid using filter paper. To remove the excess salt, the grid was touched with a drop of water and the excess water was wicked away using filter paper. For negative staining, the grid was touched with a drop of 2% uranyl acetate solution for a few seconds and excess solution was wicked away with a filter paper. Again the grid was touched with a second drop of the uranyl acetate solution for 40 seconds, and the excess solution was removed with a filter paper.The grid was kept at room temperature to evaporate excess solution. The TEM characterization and energy dispersive X-ray spectroscopy (EDS)were conducted using a Tecnei G2-20S TWIN system, operated at 200 kV with a bright field mode.

RESULTS AND DISCUSSION The host strands, which extended from corresponding staple strands were assembled with other 208 staple strands (in grey) and a ~7000-base DNA scaffold strand (circular M13 in grey) into the triangular origami template. Two sequences of the host strands, N1 (sequence: CCCTAACTCCCC) and N2 (sequence: TGCCTATGGGGGACGGATA) were chosen since they were reported to host the growth of AgNCs with distinct and reproducible fluorescences36,37. The size of each segment of the template is ca. 120 nm, which will allow the growth of AgNCs to be imaged under microcopies. The host strands were designed as extensions (in red) of the staple strands that took part in the assembly of one arm of the triangular origami, so that the functional host sequences were enriched in the des-


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ignated region and located adjacently to the origami surface (schematically shown in Figure 1A). The origami with protruding host strands was mixed successively with AgNO3andNaBH4, for the reduction of nucleotide-complexing Ag+ to AgNCs.

assembled onto the nanotemplate. (C) Fluorescence excitation and emission spectra of free N2-hosted AgNCs (left) and origami N1-hosted AgNCs (right). λex2/λem2 of AgNCs appeared when the host strands were assembled onto the nanotemplate. (D)

We first investigated the fluorescence properties of the AgNCshosted by free-diffusing N1 or N2.As shown in Figure 1B (left), the free N1-hosted AgNCs (in solution without the rigid template) exhibited two distinct maximum fluorescence excitation (λex)/emission (λem) features: 634nm/698nm and 570nm/630nm, indicating that N1 produced a mixture of emissive AgNCs.29,31 When N1 was pre-assembled on the origami template surface, only the peak at 570nm/630nmremained, showing that specific AgNCs were obtained (Figure 1B, right). The reaction products hosted by single-stranded N2 exhibited λex/λem at 509nm/573nm (Figure 1C, left), whereas the N2integrated origami yielded AgNCs that had two λex/λem: 509 nm/573 nm and 570nm/630 nm (Figure 1C, right). The fluorescent origami was subsequently analyzed by agarose gel electrophoresis (Figure S1), AFM and TEM (Figure 2 and Figure S2). The results indicated that the structural integrity of the origami template was maintained after the hosted synthesis of AgNCs. In particular, no morphological difference was found between the template without AgNCs and that with AgNCs, indicating the AgNCswere smaller than the AFM and TEM resolution limit and were composed of a few Ag atoms.

The spectral results indicate that the DNA origami templatesupported synthesis resulted in the fluorescence change of the AgNCs in a sequence-dependent manner, with N1 (rich in cytosine bases) or N2 (rich in guanine bases) as the host strand. Interestingly, the hosted synthesis of AgNCs by the origami template-assembled N1 and N2 both yielded the products that exhibitedλex and λem at 570 nm and 630 nm, illustrated in Figures 1B and 1C, right.In addition, we introduced another template N3 (sequence: CCCTTAATCCCC)37 that can also template the synthesis of fluorescent AgNCs, but with differentmaximum excitation/emission wavelength. With the free-state N3, AgNCs exhibited λex/λem at 600 nm/666 nm (Figure 1D, left), while the immobilization of N3 onto the origami shifted λex/λemof AgNCs to 570 nm/630 nm (Figure 1D, right), characteristic fluorescence λex/λemof N1-origami and N2-origami.To the best of our knowledge, such a template effect on the fluorescence properties of in-situ synthesized metal nanoclusters has not been reported. To investigate the site-selective formation of AgNCs, further growth of the nanoclusters were implemented. The clustered Ag0 acted as the seeds for the reduction of Ag+ by the mildly reducing agent (Tris), which resulted in the self-enlargement.38 The AFM images demonstrated that Ag nanoparticles (AgNPs) were formed on the N1-immoblized arm of the triangle template, and the height of AgNPs is ~2.0-3.0 nm (Figure 2A, (iii)). No height contrast change was observed when theAgNCs-free template was treated with the Ag+/Tris mixture (Figure 2A, (iv)), which confirm that only nanocluster seeds could result in the observed Ag nanoparticles. TEM was also used to verify the site-specific synthesis of AgNCs. After uranyl acetate staining, the origami arm that supported the nanoparticles and the other regions exhibited a significant contrast, demonstrating that AgNCs were selectively deposited on the N1-assembled arm (Figure 2B, (ii)). Energy-dispersive X-ray spectroscopy (EDX) further confirmed the deposition of Ag0 on the DNA origami template (Figure 2C). The siteselective formation of AgNCs and AgNPson the N2-origami template is illustrated in Figure S2. No fluorescence was observed for the synthesis of AgNCs in the presence of the host strands (N1 or N2)-free origami templates, which indicated the nucleation role of the host strands.

Figure1.(A) Schematic illustration of the assembly of DNA triangular origami template containing DNA N1 strand, the site-specific synthesis of fluorescent AgNCs, and the enlargement of AgNCs by reduction of Ag+ with Tris reagent. (B) Fluorescence excitation and emission spectra of free N1hosted AgNCs (left) and origami N1-hosted AgNCs (right). λex2/λem2 of AgNCs disappeared when the host strands were



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Figure 2. (A) AFM images and cross-section analysis of (i) template N1, (ii) template N1 hosting AgNCs, (iii) template N1 hosting AgNCs after treatment with tris/Ag+ and (iv) template N1 after treatment with tris/Ag+. Scale bar: 200 nm. (B) TEM images of negatively stained AgNCs-immobilized origami before (i) and after (ii) treatment with Tris/Ag+. (C) EDX spectrum of the enlarged AgNCs on the DNA origami template. Cu peak comes from the copper grid. The change in the fluorescence properties of the AgNCs emitters may be attributed to several factors. First, on the origami template, the high density of negative charges around the host strands may interfere with the electrostatic complexation of Ag+ to the bases of the host strands, thus affecting the number of deposited Ag0 atoms. However, after the addition of Mg2+, which could shield the negatively charged ions on the DNA backbones, the AgNCs with emission around 630 nm were still preferentially generated (Figure S3); thus the charge effect was not the dominant contribution. Second, the host strands that protruded out of the origami template were adjacent. The cross-talk between the host strands may affect the production of AgNCs, because one Ag+ can complex with two bases.39,40 Therefore, we varied the density of the N1 host strands to adjust the inter-strand distance. When the density of the host strands was reduced, he λex/λemof the immobilized AgNCs shifted from 570 nm/ 630 nm to 560 nm/ 625 nm, while λex/λem around 634 nm/ 698 nm was still not observed(Figure S4). It indicated that the host strands density did not affect the λex/λem of the immobilized AgNCs significantly.

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Figure 3. Spacer effect on the fluorescence properties of AgNCs synthesized on origami template-supported host strands. (A) Schematic showing the insertion of a duplex spacer (black) between the host strand (red) and the origami template, and the site-specific synthesis of AgNCs on the host strands-assembled arm. (B) Fluorescence excitation and emission of AgNCs synthesized on the spacer N1-immobilized origami template. (C) Fluorescence excitation and emission of AgNCs synthesized on the spacer N2-immobilized origami template. The spacer strands placed the host strand either far away from (left) or closes to (right) the template. Third, the steric hindrance of the nanostructured template, which dominates many organic reactions,1,2,41 may affect the AgNCs synthesis. We introduced a 14bp duplex spacer between the host sequences (N1 or N2) and the origami template so that the growth of AgNCs would not be significantly affected by the template (Figure 3A; for the design scheme of the template, see Figure S5). As shown in Figure 3B,λex/λemat 570 nm/630 nmfor the spacer-N1-AgNCs was still observed, while that at 634nm/698 nm reappeared; the latter is a photophysicalcharacteristic of free N1-AgNCs (Figure 1B, left). The spacer between N2 and the template significantly reduced the characteristic excitation/emission of AgNCs at 570 nm/630 nm, as indicated by Figure 3C (left). These results indicate that (i) the placement of the duplex spacers partly restored the environment of the template-supported synthesis of AgNCs to the environmentof free host, and (ii) the origami template still exerted an effect on the photophysical features of AgNCs, but the effect was weaker than that of the spacer-free template. This result likely occurred because of the insufficient contact between the host strands and the origami surface, which was indicated by Figures S6A (for origami-spacer-N1) and S6B (for origami-spacer-N2).In these AFM images, the height of the origami arm with spacers was approximately double that of the other arms, which indicated that some of the duplexes lied flat rather than completely stood up on the template.38 We also investigated the fluorescence of AgNCs hosted by the


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freely diffusing N1- or N2-spacer complex and found that such spacer sequences did not affect the emission wavelength of AgNCs (Figure S7). This finding further demonstrated the fluorescence changes were due to the distance increase between the nucleation site of AgNCs and the template caused by spacer strands. The site-specific synthesis of N1 or N2hostedAgNCs on the spacer-localized arm is evidenced by the AFM and TEM results (Figure S6). To further study the spacer effect, the host strands were placed on the other end of the spacer duplex, where they remained close to the template surface (schematically shown in Figure S8). The results (Figures 3B and 3C) showed that the hostedAgNCs on this template had fluorescence properties that were almost identical to those on the spacer-free template. These investigations indicated the significant role of template steric hindrance in the emissive properties of AgNCs.

Figure 4. Template configuration-dependent fluorescence of N1 (left)- or N2 (right) hosted AgNCs. The host strand was assembled on (A) TX template, (B) the central site and (C) the end site of the duplex template. Subsequently, we investigated the dependence of the AgNCs fluorescence on the template size, which can provide further evidence for the steric effect of the template. We chose a DNA triple crossover (TX) structure as the model of a smaller template, which was composed of 3 helical bundles (~16 nm × 7 nm, schematically shown in Figure S9). For comparison, each triangular origami arm contained nine helical bundles (~24 nm). On TX template, one host strand (N1 or N2) was placed in the center through self-assembly and protruded out of the surface. The ratio of emission intensity at 698 nm to 630 nm (F698 nm/F630 nm) for TX N1-hosted AgNCs is 0.15 (Figure 4A, left), which is much smaller than that of free N1 (3.33). The ratio of emission intensity at 630 nm to 573 nm of TX N2-hosted AgNCs (F630 nm/F573 nm: 0.3) (Figure 4A, right) was smaller than that of origami templated AgNCs (F630 nm/F573 nm: 0.7) (Figure 1C, right). These results indicated that the TX template exerted a marked steric effect on the fluorescence properties of AgNCs, but this effect was weaker than that of origami template.

To further investigate the size effect of the template, we used a DNA duplex with the host strand assembled in the center of the template. The fluorescence λex and λem of N1-hosted AgNCs were closer to the AgNCs on free diffusing N1 than to the TX-N1 (Figure 4B, left). The fluorescence λex and λemof N2-hosted AgNCs were identical to free N2-AgNCs (Figure 4B, right). This finding indicated that the template with one helix had a significantlysmaller steric effect. However, when the host strand (either N1 or N2) was placed to the duplex end, the fluorescence λex/λem of the hosted AgNCs was almost recovered to those of free-diffusing clusters (Figure 4C). These results illustrate the dependence of the steric effect on both the template size and the spatial location of the host strands, which further clarifies the origin of the template-induced fluorescence changes of AgNCs. The sequences of the duplex templates are schematically shown in Figure S9. To determine if the the steric hindrance of the template could affect the spectra properties of pre-synthesized AgNCs that may differ in their locations or conformations,42,43 AgNCs hosted by free N1 or N2 were assembled on the origami template for investigation the effect of the template on the fluorescence spectra (schematically shown in Figure S10A). The nucleation of AgNCs could only selectively occur on the host sequence site of a nucleic acid strand.44,45According to this knowledge, a strand composed of the host sequence and (T)19 was designed ((T)19-N1 or (T)19-N2), and the (T)19 segment could only hybridize with the capture strandswith sequence (A)19(i.e. (A)19 was conjugated to the desired staple strands) on the origami.36After the formation of AgNCs on the host regions, the (T)19-N1 or (T)19-N2 strands hybridized to the origami template and theAgNCs were located close to the surface. The post-immobilization of AgNCs did not change the fluorescence λex/λem(Figure S10B). The hybridization of the host strands to the origami template was verified by AFM images (Figure S10C). Then, with the TX structure as the template, we disassembledthe AgNCs-hosted strand (N1 or N2) via strand displacement and observed no change in the fluorescence λex/λem (Figure S11). These results showed that the structured template did not affect the fluorescence wavelengths of the pre-synthesized AgNCs. The steric effect of the template may also be correlated to the size (or atom number) of AgNCs, which is generally considered to largely determine the fluorescent properties of metallic nanoclusters.24,29,46,47Therefore, we analyzed the host strands– stabilized AgNCs, using mass spectroscopy (MS). The peaks were correlated to the number of silver atoms based on the reported work.30,36To clearly present the abundance ratio between the cluster species, the peaks for the bare host strands (i.e. Ag0) were not shown. It should be noted that the initial M/Z for TX-N2 (or N1) was much higher than that for free N2 (or N1), since TX-N2 (or N1) contains DNA sequence that took part in TX assembly. We compared the AgNC species synthesized on the free-diffusing and TX-assembled host strands. Herein, the TX template can also be considered a part of the origami structure, since TX has a similar arrangement of DNA double helices to but smaller size than the origami structure. Prior to measurement by mass spectroscopy, the DNAhosted AgNCs were purified from the TX template via strand displacement, biotin-streptavidin recognition and magnetic separation (Figure S12). The MS results showed the size dis-



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tribution of the immobilized AgNCs on the structured template differed remarkably from that of the free AgNC species. Compared to the free N1-hosted AgNCs, the relative abundance of Ag4 and Ag5 increased markedly, but Ag6 disappeared (Figure S13). When the N2 template was used, the abundances of Ag2, Ag3, Ag4 and Ag5 species were significantly higher than the Ag1abundance, these values could then be compared to free-diffusing N2-hosted AgNCs (Figure S14). The different emission wavelength of the AgNCs reflected their different size distributions.29,36Thefluorescence changes generated by steric hindrance were attributed to the variations in the size distribution of Ag2, Ag3, Ag4,and Ag6.

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randomly-coiled N1 or N2 was stretched when linked to the TX template. In particular, afterthe Ag+complexing, enhanced FRET efficiency was observed for both of the free host strands, which indicated greater compactness, due to the inherent molecular flexibility and the formation of the Ag+nucleobases complex. As a control experiment, incubation of FAM-labeled N1 or N2 with Ag+ in the ratio in the previous experiments resulted in only a subtle change in the decay time τ. The significant Ag+-induced conformational changes were also shown by CD spectra (Figure S16). In comparison, for the host strands on the templates, the FRET efficiency changed only slightly, which indicated that the stretched states were largely retained. This finding was probably due to the electrostatic repulsion between the host strands and the templates, which hindered the stacking of the bases of the host strands andthe adsorption of Ag+.48,49The conformational flexibility of the host strands in the freely diffusing state and immobilized on the structured template was illustrated schematically in Figure 5B. These results show that the structured templates constrained the N1 or N2 strand into an energetically-favoredstretched state via steric hindrance, which guided the synthetic pathways towards the production of AgNCs with specific size distribution. However, the mechanism of the guided formation of the specific emitter at 630 nm on different host strands is still unclear. Further efforts are expected to make the quantitative correlation between the conformations of the host strands and their effects on the thermodynamics and the kinetics of the Ag+ adsorption, the structures of the host strands/Ag+ complexes and the sizes of the AgNCs.

CONCLUSION

Figure5. Conformational flexibility of the host strands in freely diffusing state and immobilized on the structured template. (A) FAM decay times τ and FRET efficiency for the different systems. N: N1 or N2 strand. F: FAM (donor). Q: the black hole quencher (BHQ1, acceptor). TX: the triplex template. (B) The hypothesized conformations of the host strands according to the FRET efficiency between the donor (FAM) and the acceptor (Black hole quencher) labeling the 5’ and 3’ end of the host strands before and after Ag+ adsorption. The template stretches and constrains the host strands, which affects the Ag+ adsorption and the sizes of the produced AgNCs. The growth of AgNCs involves the adsorption of Ag+onto the host strands and the reduction of Ag+ for in-situ clustering. During this process, the affinity of the Ag+-DNA bases can be affected by the conformational flexibility of the host strands,48,49 which arose fromthe steric effect of the template and resulted in the size distribution of the product species. Therefore, the conformations of the host strands, before and after Ag+ adsorption, were investigated using FRET between the fluorophore FAM (donor) and a black hole quencher (acceptor) that labeled the 3’- and 5’- ends, respectively. The FRET efficiency η is strongly distance-dependent and can also be calculated based on the donor decay time τ(Figure 5A), according to equation 1 (Figure S15). FRET efficiencies were reduced for both TX template-supported N1 and N2, compared to the freely diffusing strands, which showed that the

In summary, we reported the DNA-hosted synthesis of fluorescent AgNCs on DNA nanostructured templates and the surface effect on the fluorescence. The host strands were designed to protrude out of their designated positions on DNA nanostructures, which enabled the site-specific synthesis of AgNCs on the template. Nucleic acidstrandswithtwo different sequences hosted the formation of AgNCs with distinctfluorescence emission wavelengths. However, the integration of either host strands on the structured templates led to the production of AgNCs with specific fluorescence wavelengths, which showed the rigid template was capable of affecting the fluorescence propertiesof the in-situ synthesizedAgNCs. The mechanism for the fluorescence-specific synthesisis ascribed to the steric effect of the template on the conformational flexibility of the host strands, which resulted in the preferential synthesis of a fluorescent AgNC mixture with a specific size distribution.This workpresents a facile methodfor fabricating shape-controlled metallic nanostructures via control over the spatial locations of the host strands and provides the insight into the mechanisms of the steric effects on surface chemical reactions. Our work promises a programmable self-assemblybased platform that can tailor the surface-activated chemical processes through control of the surface steric hindrance at the reaction site. Furthermore, our results also suggest that the rigid template should be considered an important factor that affects the DNA-directed single-molecule or enzymecatalyzed reactions,20,50,51 which may be sterically enhanced or hindered.

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Supporting Information. Gel electrophoresis images, extra AFM and TEM images and fluorescence spectra and MS data. All DNA sequences that were used to assemble the nanostructured template and support the synthesis of AgNCs. The extra schemes for the assembly and disassembly of AgNCs-hosted or AgNCsfree templates. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected]

Author Contributions †These authors contributed equally to this work. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT The authors are grateful for the financial support from National Science Foundation China (21273052, 91127021, and 21573051), the Beijing Natural Science Foundation (L140008), Beijing Municipal Science & Technology Commission (No. Z161100000116036), CAS Interdisciplinary Innovation Team, CAS Key Laboratory of Nanosystem and Hierarchical Fabrication, Youth Innovation Promotion Association CAS.

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DNA-Based Nanotemplate Directed In Situ Synthesis of Silver

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