Multivalent TraptavidinâDNA Conjugates for the Programmable

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Multivalent Traptavidin-DNA Conjugates for Programmable Assembly of Nanostructures Young-Youb Kim, Yongbin Bang, Ah-Hyoung Lee, and Yoon-Kyu Song ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b06170 • Publication Date (Web): 17 Jan 2019 Downloaded from http://pubs.acs.org on January 18, 2019

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Multivalent Traptavidin-DNA Conjugates for Programmable Assembly of Nanostructures Young-Youb Kim†, Yongbin Bang†, Ah-Hyoung Lee†, and Yoon-Kyu Song*, †, ‡

†Graduate School of Convergence Science and Technology, Seoul National University, Seoul 08826, South Korea

‡Advanced

Institutes of Convergence Technology, Suwon, Gyeonggi-do 16229, South

Korea

[* Corresponding author: [email protected]]

ABSTRACT: Here we explore the extended utility of two important functional biomolecules, DNA and protein, by hybridizing them through avidin-biotin conjugation. We report a simple yet scalable technique of successive magnetic separations to synthesize traptavidin-DNA conjugates with four distinct DNA binding sites, which can be

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used as a supramolecular building block for programmable assembly of nanostructures. Using this nano-assembly platform, we fabricate several different plasmonic nanostructures with various metallic as well as semiconductor nanoparticles in predetermined ways. We also use the platform to construct dendrimer nanostructures using valency-controlled traptavidin-DNA conjugates in a programmable manner. These results suggest that our protein-DNA supramolecular building blocks would make a significant contribution to the assembly of multi-component and complex nanostructures for numerous contemporary and future applications from molecular imaging to drug delivery.

KEYWORDS supramolecular building block, traptavidin, DNA nanotechnology, programmable nanoassembly, plasmonic nanostructure, dendrimer nanostructure

DNA and protein are the most extensively studied of nature’s biopolymers – not only because of their critical roles for life but also because of their unique structural and


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functional characteristics.1-5 Their properties of molecular recognition provide the basis for synthesizing biological assemblies with pre-determined structures and anticipated functions. Over the past few decades, synthetic DNA has been extensively used in the field of nano-science due to its predictable base-pairing interactions as well as the simple and cost-effective production through solid-phase synthesis. Many researchers have exploited synthetic DNA to create various self-assembled nanostructures using so-called “DNA origami techniques”,6-11 aiming at a wide range of applications such as drug delivery,12-15 plasmonic engineering,16-20 molecular imaging,21,22 and nano-robotics.23-25 Also, proteins are the central biological building blocks in nature with extremely broad structural diversity and enormous functional versatility.26-28 Hence, they have been used in various applications of protein-based supramolecular structures such as smart nanocarriers,29,30 biological nanoenzymes,31,32 and nano-biosensors.33 While DNA and protein have proven themselves immensely useful in nano-science and nano-technologies, they may work together synergistically to extend their utilities by adjoining unlimited design flexibility of DNA and extreme structural and functional diversities of protein. Although site-specific binding of protein-DNA conjugation is still


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challenging, several chemical conjugation methods have been recently developed to precisely coordinate the molecular components at nanoscale.34 Among them, genetically modified protein with specific chemical handles, such as HUH-tag and Gene-A* proteins, have been successfully employed for highly stoichiometric protein-DNA conjugation.35-37 We anticipate that these efforts could lead to the nanostructures with various functional proteins (e.g., coordinated array of catalysts with other nanomaterials) using protein-DNA supramolecular nano-assembly platform. In this work, we use protein-DNA conjugate platform to synthesize supra-molecular building blocks to construct various nanostructures. We are particularly interested in valency control of multivalent building blocks by conjugating a different DNA at each bonding site, which expands our design capability of multi-component nanostructures through precise stoichiometric control of the constituents. To investigate the semisynthetic DNA-protein conjugate that has an exact valency and structural rigidity, we use an avidin-biotin system, which has been a simple, yet powerful tool in nano-biotechnology for decades.38-40 We combine the structural rigidity of an avidin-based protein core and the multivalent programmable bonds of biotinylated DNAs. Also, a tetrameric protein


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traptavidin (TAv), a mutant variant of streptavidin (SAv), is used to greatly improve the chemical and mechanical stabilities of the nanostructure as well as to enhance its affinity to biotin molecules.41 Each domain of the identical tetramer has a biotin binding-site where a biotinylated DNA is specifically attached via one of the strongest noncovalent bonds known in nature. However, the identical biotin binding sites of avidin could raise some issues regarding random association and oligomerization of biotinylated molecules due to indistinguishable nature of the interactions at the four binding sites. Nonstoichiometric bindings among the avidins and biotins may cause detrimental effects on the valency control of the avidin-DNA conjugates and thus limit the rational utility as DNAbased molecular building blocks. Thus, we propose a simple method to control the valency of TAv by passivating all four binding sites of the tetramer with four individually programmed biotinylated DNAs, rather than modifying the valency of the tetrameric protein itself. The hybrid molecule with four different sticky ends acts as a supramolecular building block with independently controllable binding sites through sequence-specific base-paring interactions of DNAs. To synthesize multivalent Traptavidin-DNA Conjugates (mTDCs), we developed a simple


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technique based on successive magnetic separation steps that enables us to have high purity molecules in an efficient and scalable manner without difficulty.42

RESULTS AND DISCUSSION

Design of Multivalent Traptavidin-DNA Conjugates

Figure 1 illustrates the process of synthesis for mTDCs through four stepwise magnetic separations. The basic idea of making an mTDC is straightforward – we employ four binary pathways to filter mTDCs with distinct tetravalency from a random mixture of four different types of biotinylated DNAs and TAv through sequence specific hybridization between DNAs on TAv and capture DNAs on magnetic beads (MBs). For each bifurcation step, specific DNA-decorated magnetic beads are required to recognize and capture the target DNAs in the mTDCs. The capture DNAs are complementary to the target DNAs and bound to the surface of the MBs via highly stable covalent bonds based on maleimide-thiol chemistry.43 The mixture interacts with the capture (DNA decorated) MBs at each step, and the TDCs with the target DNAs proceed to the next separation step,


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while those without target DNAs are discarded before the mixture being thermally dehybridized. During the first magnetic separation step, the mixture is “filtered” using A’MBs (DNA with base-sequence complimentary to A attached to MBs), and thus TDCs with A DNA (DNA with base-sequence A) are collected while those without A DNA are discarded. The second magnetic separation step also runs along the same pathway, and TDCs including A DNA and B DNA proceed to the next step while those without B DNA are removed. After four subsequent magnetic separation steps are completed, only TDCs with all four kinds of DNAs – A, B, C, and D, remain in the mixture. Although the successive magnetic separation technique offers a simple route to control the “valency” of the TDCs, the biotin dissociation may greatly affect the purification yield and the longterm reliability of the final products. While a popular alternative SAv shows the problems associated with biotin dissociation, TAv demonstrates much enhanced reliability or stability, since it has about a ten-fold lower biotin dissociation rate than that of SAv. In the case of SAv, biotinylated DNAs begin to dissociate from binding sockets at 50 °C and detach significantly above 70 °C, whereas in the case of TAv, most biotinylated DNAs remain attached under the same condition. TAv is also known to have improved


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thermostability and increased mechanical strength over its non-mutant or other mutant siblings. 41

PAGE Analysis of mTDCs.

Figure 2a shows the distinct band images of the TAv and biotinylated DNA complexes with various mixing ratios from native polyacrylamide gel electrophoresis (PAGE). The I, II, III and IV bands indicate the monovalent, divalent, trivalent and tetravalent complexes, respectively. The PAGE results are consistent with our hypothesis that excess TAv helps the biotinylated DNAs to preferably bind to only one of the four binding sites, showing the dominance of the monovalent complexes with high mobility in lane 1. As the molar ratio of the biotinylated DNA increases from lane 1 to lane 6, the dominance of monovalent complexes diminishes gradually, and the increasing dominance of the tetravalent complexes is clearly observed in lane 6. In lane 7, we utilized the same sample as that used in lane 6 after four successive magnetic separation steps, as described in Figure 1. Compared with lane 6, the band of trivalent complexes disappears in lane 7, indicating


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the successful purification of the TDCs with distinct tetravalency. We also confirmed the PAGE results for each magnetic separation step after mixing TAv and biotinylated DNA (Figure 2b). Lane 1 is the result of mixing biotinylated DNA and TAv at the molar ratio of 1:3. The monovalent, divalent, and trivalent complexes were removed after each magnetic separation step until the fourth magnetic separation process, as shown in lanes 2 through 5. To calculate the purification yield of the mTDCs, we use the peak of UV spectral absorbance (A260/280 nm ratios) of 20 bps biotinylated DNA-to-TAv. TAv and four different biotinylated DNAs were initially mixed at the molar ratio of 1:5 to fully conjugate DNAs at four binding sites of TAv forming tetravalent complexes. The mixture is filtered by four successive magnetic separation steps. The peak values of A260nm/280 nm absorbance ratios are taken from previous study, which are 1.06, 1.28, 1.39, and 1.44 for monovalent, divalent, trivalent, and tetravalent complexes, respectively.24 After four magnetic separation steps, only the tetravalent complexes remain in the mixture, showing the A260/280 nm ratio of 1.44 (Figure S1). The concentration of four DNAs measured at 260 nm absorbance in tetravalent complexes is divided by four to get the concentration of TAv. Then, the final purification yield is determined as a percentage of the measured


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concentration of TAv in the purified tetravalent complexes to the initial concentration of TAv in the mixture. We find the yield of TAv based complexes to be 8.1 % of the initial volume (Figure 2d), as compared with 3.5 % in the case of SAv (Figure 2c). We attribute strong affinity between biotinylated DNA and TAv to the high purification yield (Figure S2).

FRET Analysis of mTDCs.

We conduct cascade Förster resonance energy transfer (FRET) experiments to investigate the structural properties of the TDCs with independently programmable binding sites. We place Alexa488, Cy3, Cy5 and Alexa750 dyes near four biotin binding sockets of TDCs, in order to maximize the consecutive spectral overlaps in the FRET cascade while keeping non-consecutive overlaps relatively small (Figure 3a and Figure S3). Each four dyes are attached to the 5’ end (proximal to the TAv core) of the singlestranded DNAs (ssDNAs) and biotin molecules are synthesized to the thymine residue of the first base sequence from 5’ end of ssDNAs (Figure S4). We also use a mixture of four dye-conjugated DNAs as a reference sample. When the samples are excited with 470 nm


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wavelength light, TDCs sample show emission peaks at 520 nm, 570 nm, 670 nm and 775 nm wavelengths while reference sample shows only one peak at 520 nm. Under 540 nm excitation, three peaks emerge in the emission spectrum of the TDCs sample as compared to only one peak in the emission from the reference sample. Similarly, with 640 nm excitation, two peaks emerge in the spectrum of the TDCs, but only one is shown in the reference spectrum. Under 740 nm excitation, both TDCs and reference spectra show only one peak, as expected (Figure 3b). Although there are multiple factors contributing to the spectral characteristics of the cascade FRET, such as non-consecutive energy transfers44 and multiple isomers – 6 isomers in TAv with asymmetric separations in biotin binding sites (Figure S4), the distinct tetravalency of TDCs primarily accounts for the downstream energy transfer characteristics, as shown in the series of emission spectra at different excitation energies (Figure 3a and Figure 3b). These results, therefore, indicate that the TDCs indeed have four distinct binding sites for sequence specific pairwise interaction of DNAs. Fabrication of Gold Dimer Nanoparticles with Two-step Magnetic Separations.


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Four independently programmable DNA recognition sites of TDCs enabled us to assemble DNA-decorated gold nanoparticles (AuNPs) to form various plasmonic nanostructures. TDCs offer programmability as well as structural rigidity in the plasmonic nanoparticle assemblies. Based on the TDCs, Au dimers were assembled in two stepwise magnetic separation processes, involving exchange release rather than thermal release, to filter out monomers and other multi-unit clusters. Based on the TDC motifs, Au dimers were synthesized in two stepwise magnetic separations to purify the Au dimers (Figure 4a). In the first assembly step, A, B-TDCs were selectively hybridized with A’ and X’ DNAmodified gold nanoparticles (A’X’-AuNPs) and B’Y’-AuNPs at a molar ratio of 1:2 with each DNA probes of the TDC. (1) To eliminate non-hybridized AuNPs, X-MBs were first allowed to capture A’X’-AuNPs and Au dimers by magnetic particles (2) before the captured samples were washed out with PBS buffer to remove non-hybridized B’Y’AuNPs (3) When the Au dimers is thermally released from the surface of MBs, the double strand DNA(dsDNA) could be dehybridized above melting temperature or the dissociation of biotinylated DNA from TAv could decrease the yield of the Au dimers. We then used DNA displacement reaction to release captured AuNPs and Au dimers with selective


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dehybridization at room temperature.45 The A’X’-AuNPs and Au dimer were released from the X-MBs by exchanging 15 bps DNA modified AuNPs to 23 bps exchange DNA on MBs. As 23 bps dsDNA interaction is more energetically favorable than 15 bps dsDNA, the captured AuNPs were easily released at room temperature with exchange DNA 1. (4) Next, the Au dimers were captured by Y-MBs and released from Y-MBs in the same manner. (5)(6) Figure 4b shows TEM images of the 10 nm/10 nm Au dimers before and after magnetic separation. Nonhybridized Au monomers were removed after magnetic separation, resulting in a high yield of Au dimer. The representative assembly yields were calculated as 81 % in Au dimers, 7 % in Au monomer, and 12 % in AuNP clusters equal or larger than trimers (Figure 4c). The dynamic light scattering (DLS) analysis revealed the average hydrodynamic diameters (Dh) of the Au monomers and Au dimers: 18.7 nm and 29.6 nm, respectively (Figure 4d). Also, compared with no separation or magnetic separation with thermal release, the magnetic separation with exchange release enabled us to assemble Au dimers easily without denaturing the dsDNA or dissociating the biotinylated DNAs from the TAv during the magnetic separation process (Figure S5 and


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S6). We note that the exchange release of DNAs from magnetic beads at room temperature preserves the integrity of double strand DNAs in the nanostructures, thus the same method can be applied to prevent thermally unstable proteins from unwanted temperature-triggered denaturation during the DNA release process. We also assembled asymmetric plasmonic nanostructures as well as multicomponent nanostructures through programmability of the TDCs.

Fabrication of Various Plasmonic Nanostructures.

Using four different recognition probes of multivalent TDC motifs, we fabricated the plasmonic nanostructures with pre-programmed DNA modified nanoparticles. The TDC motifs offer the selectivity and rigidity of DNA bonding to construct desired plasmonic nanostructures. Figure 5 shows TEM images of various plasmonic nanostructures. The distinguishable tetravalent TDCs enabled us to make Au trimer, tetramer with specific spatial arrangements. Both trimers and tetramers underwent multiple magnetic separation steps with the DNA exchange release scheme, the same as the one used in


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Au dimer synthesis, and showed progressively lower yields of 57%, 43%, respectively (Figure S7 and S8). We attribute the progressive reduction of the yields in dimers-trimerstetramers to electrostatic repulsion and physical interference among AuNPs due to surface bound DNAs. The 10 nm/20 nm Au heterodimers were easily assembled with two different pre-encoded DNAs on AuNPs with two different sizes. The multicomponent nano-assemblies composed of SAv coated quantum dot (QD) and AuNPs were also constructed, and 5nm silver coated 10nm/10nm Au dimers were synthesized by chemical reduction method.46

Synthesis and Characterization of Dendrimer Nanostructures Dendrimers are synthetic polymers with branch structures in layered architecture. By manipulating their designs, one can precisely control their molecular weight and chemical composition, hence attain predictable tuning of their biochemical characteristics, such as biocompatibility and pharmacokinetics.47 We have applied our mTDC building blocks to construct dendrimer nanostructures using layer-by-layer assembly scheme, similar to that used in surface-bound DNA-Streptavidin dendrimers (Figure 6a).48 We keep a simple


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architecture that uses bis-biotinylated DNA to passivate two adjacent biotin binding sockets of TAv before conjugating two identical biotinylated DNAs to the other binding sites. This type of trivalent mTDC has flip or mirror symmetry in the structure and thus does not introduce structural complication related to the multiple isomers as appears in mTDC with four distinct tetravalency. Figure 6b shows the band images of the TAv and biotinylated DNA complexes in PAGE. Lane 1 indicates the mixtures of biotinylated DNAs and TAv at the molar ratio of 1:3, showing 3 bands presumably correspond to mTDCs with 2-4 DNAs. The band in lanes 2 is the dendritic monomers after two magnetic separation steps, showing that the successful purification of the dendritic monomers with symmetric trivalency. The lane 3 is the band of mTDCs after four magnetic separation steps. The dendritic monomers are designed to have one bis-biotinylated ssDNA that can hybridize with a pre-generation dendrimer and two biotinylated ssDNAs that can hybridize with post-generation monomers (Figure 6c), except an initial monomer (with XAAA DNAs) that has three ssDNA sticky-ends for post-generation monomers (Figure S9). With these stoichiometrically

controlled

starting

monomers,

dendrimer

nanostructures

are

assembled in stepwise layer-by-layer reactions without further purification step (Figure


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6c). For the first-generation dendrimers (G1), dendritic monomers with bis-A’BB DNAs are injected to initial monomers (G0). Next, dendritic monomers with bis-B’CC are injected to the first-generation dendrimers (G1) to form the second-generation dendrimers (G2). Then, dendritic monomers with bis-C’DD are injected to the second-generation dendrimers (G2) to form the third-generation dendrimers (G3). Each generation (Gn) molecules are mixed with post-generation monomers at the molar ratio of 1:3 (n = 0) or 1:2 (n  1) for approximately 180 minutes of reaction time to ensure complete interaction. We characterize the size dependent mobility of four dendrimer generations in 1% agarose gel electrophoresis (Figure 6d), and also find average sizes of G0, G1, G2, and G3 dendrimers being 5 nm, 11 nm, 19 nm, and 41 nm, respectively in DLS measurements (Figure 6e). These results are consistent with our observation by transmission electron microscope (TEM). For example, G3 dendrimers have 1, 3, 6, 12 TDC molecules at each layer, giving approximately 40 nm in diameter. In Figure 6f, we visualize G3 dendrimers by TEM with 2% aqueous uranyl formate negative staining.

CONCLUSION


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In summary, we developed a supramolecular building block based on protein-DNA hybrid molecules using avidin-biotin conjugation system and simple successive magnetic separation technique. The high binding affinity between the TAv and biotinylated DNAs enabled us to maintain the structural integrity of the hybrid conjugates during the thermal release of dsDNA, ensuring a high yield of the purification process. The successive magnetic separation technique is not only simple, but also easily scalable to produce materials in large quantity, which is considered important in many commercial or industrial applications. It is also noteworthy that we could avoid the non-stoichiometric binding issue of TAv-biotinylated DNA in this platform by attaching four different DNAs on four binding sites of TAv. The distinct tetravalency has been characterized by PAGE and cascade FRET experiments. Using these four independently programmable DNAs with distinct sequence specific selectivity, we could easily fabricate various multi-component plasmonic nanostructures with metal as well as semiconductor nanoparticles in both homogeneous and heterogeneous configurations. Also, we have applied the TDC platform with bis-biotinylated DNAs to synthesize dendrimer nanostructures without isomer related issues due to flip symmetry of the building blocks.


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From these results, we conclude that our protein-DNA based supramolecular platform has a significant potential in rationally designing nano-architectures for various contemporary applications, such as smart drug delivery systems,49,50 nano-biosensors,5153

bio-responsive materials,54,55 and molecular theragnosis.56-58


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EXPERIMENTAL SECTION

Materials.

The

chemical

reagents

[Gold

nanoparticles(AuNP),

silver

nanoparticles(AgNP), dithiothreitol(DDT), anhydrous Dimethyl sulfoxide(DMSO), AgNO3, polyvinylpyrrol-idone(MW 40000), (+)-sodium L-ascorbate, sodium dodecyl sulfate] were purchased from Sigma-Aldrich. (St. Louis, MO, USA) HPLC-purified thiolated and biotin modified DNAs were purchased from IDT & Bioneer Inc. (USA, South Korea) The reduced DNAs were then purified through a desalting NAP-5 column (Sephadex G-25 medium, DNA grade) NANOpure H2O (18.0 MΩ), purified using a Milli-Q water purification system. Streptavidin (SAv) coated CdSe/ZnS quantum dots(QDs) and Dynabeads M-270 amine were purchased from Invitrogen. Succinimidyl-4-(p-maleimidophenyl)-butyrate (SMPB), Sulfosuccinimidyl acetate(Sulfo-NHS-acetate) and NHS-ester polyethylene glycol(PEG), MW 333 Da were purchased from Thermo Fisher Scientific (USA). The carbon coated copper grid (Ted Pella, Inc. Redding, CA, USA).

Preparation of traptavidin. In order to produce and purify traptavidin protein, we purchased the plasmid encoded TAv sequence at Addgene Corp. The plasmid is encoded


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with TAv protein DNA sequence and Ampicillin resistance gene sequence. In order to select single colony from Luria Broth (LB) Agar Stab format, we streaked the bacteria with a sterilized micro-pipette tip onto LB Agar plate containing 100 μl/ml ampicillin. The streaked LB Agar plate was incubated for 16 hours at 37 °C. When the single bacterial colony was observed on the plate, the single colony was picked with a sterilized micropipette tip. Then, we put this tip into 15 ml conical tube, which contained LB media and 100 μg/ml ampicillin. It was incubated for 12 hours at 225 rpm and 37 °C. The conical tube cap was slightly open for air to come into the tube for bacterial growth. After the incubation, TAv plasmid was purified from the bacteria by using DNA-spinTM plasmid DNA purification Kit (iNtRON biotechnology). The purified DNA plasmid of Traptavidin was sent to Ab FRONTIER Company for protein expression and purification followed by the same method.41

Synthesis of DNA modified magnetic beads. DNAs modified magnetic beads (MBs, Invitrogen Dynabeads M-270 Amine) were synthesized from the previous developed procedures.59 Amin-functionalized MBs were placed on a magnetic stand and discarded


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supernatant and washed three times with 1.5 ml of anhydrous DMSO. After the washing step, the MBs were resuspended in 50 mg SMPB in 1 ml anhydrous DMSO and incubated with gentle mixing for 4 hours. Next, the MBs were washed with anhydrous DNA for three times and then with coupling buffer (0.1 M sodium phosphate buffer, pH 7.0 with 0.2 M NaCl) two times. The MBs were resuspended in coupling buffer with thiol-modified DNAs (25 nmole) after disulfide bond of DNAs were cleaved. The DNAs and MBs were incubated with gentle mixing for 4 hours at room temperature (RT) wrapped with foil and parafilm. After incubation, the supernatant was removed and MBs were washed with coupling buffer for three times and passivation buffer (0.15 M sodium phosphate buffer, pH 8.0 with 0.15 M NaCl) for two times. Next, And the MBs were resuspended in 100 mg sulfo-NHS-acetate in 35 ml of passivation buffer for 60 minutes at RT. After passivation, the MBs were washed three times with 20 ml passivation buffer, and resuspended to a final concentration of 10 mg/ml.

Synthesis of the multivalent traptavidin-DNA conjugates. Four different biotinylated A, B, C, D DNAs (5 μM, 75 μl) are randomly mixed with four binding sites of TAv (16.7 μM,18


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μl) in PBS buffer for 6 hours at RT. To separate the multivalent traptavidin-DNA conjugates (mTDCs) among all randomly mixed samples, stepwise purification methods are conducted by DNA modified magnetic beads in which DNA sequences are complementary with A, B, C and D biotinylated DNAs on TAv surface. In the first magnetic separation step, the complexes including A DNA of randomly distributed samples in PBS buffer hybridized with A’ DNA modified MBs (1 mg/ml) in PBS buffer for 6 hours at RT. After the complexes bounded to MBs are captured by an external magnetic field, the complexes without complementary DNA were removed. The collected complexes were washed with PBS buffer three times to remove unbound residues and released from the MBs in PBS buffer by heating at 60 °C for 5 min. Next, the complexes were redispersed to B’-DNA modified MBs in PBS buffer and conducted to the identical magnetic separation step with C’-DNA and D’-DNA modified MBs in consecutive order.

Native 15% Polyacrylamide gel electrophoresis analysis (PAGE) of traptavidin-DNA conjugates at various ratio. Native PAGE analyses for the mTDCs at various ratio were performed in 15 % PAGE (37.5:1, acrylamide/bisacrylamide) in 45 mM Tris, 45 mM boric


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acid, and 0.5 mM EDTA (0.5X TBE buffer solution). TAv and biotinylated DNA(b-DNA) solution was mixed with an equivalent amount (10 μl) of loading buffer containing 0.03 % bromophenol blue, and 0.03 % xylene cyanol FF and then loaded onto each gel. Loading concentration: [TAv] = 1 μM, [b-DNA] = 0.25, 0.5, 1, 1.5, 3, 4 μM. (from lane 1 to 6, respectively), lane 7 = mTDCs after 4 magnetic separation steps). After running (37.5 V/cm, constant voltage) for 2 hours, the gel was stained with GelStar, and each band was confirmed with Gel Doc Ez system.

Fluorescence experiments for four channel cascade FRET. To synthesize Alexa 488, Cy3, Cy5, and Alexa 750 dye FRET probe to traptavidins, four different biotinylated A, B, C, D DNAs (10 μM,15 μl) are randomly mixed with TAv (10 μM,10 μl) in PBS buffer for 3 hours at RT, followed by four magnetic separation process (Figure 1). We implemented four-dye Forster resonance energy transfer (FRET) in mTDCs. Each four DNA probes were synthesized to Alexa 488, Cy3, Cy5, and Alexa 750 dye at the 5’ end and were added sequentially to a biotin-dT residue for binding to traptavidin. Four channel FRET was excitated at 490 nm, 540 nm, 640 nm and 740nm sequentially. Fluorescence spectra


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of the probes (100 nM) were measured in PBS buffer, using a quartz cuvette with a 1 cm path length at RT. The excitation and emission bandwidths were 1.5 nm. The spectra were recorded using a FluoroMate FS-2 (SCINCO). The ﬂuorescence measurements were conducted at RT in PBS buffer and measured within 24 hours.

Synthesis of DNA-modified gold nanoparticle. DNA modified AuNPs (10nm, 20nm and 40nm) were synthesized from the previous procedures.55 AuNPs 1 ml (OD 1) stabilized suspension in citrate buffer solution was washed by centrifugation for 60 minutes at appropriate rpm according to the size of AuNPs before discarding supernatants and resuspend in 1 ml of 10 mM phosphate buffer (PB) at pH 7.0. Lyophilized DNAs were redispersed in a cleave solution (0.1 M dithiothreitol in 0.17 mM PB at pH 8.0) to cleave the 5'-thiol-modified C6 S-S DNAs. After incubating 2 hours, the sulfhydryl formed DNAs were purified by an NAP-5 column. The cleaved DNAs (4 nmol) in nanopure water (500 μl) were added to AuNP in PB (1 ml). The solution was incubated with gentle mixing for 16 hours before salt aging step. The DNAs and AuNPs solution were mixed with a final concentration of 10 mM PB at pH 7.0 and 0.1 % SDS. The solution was allowed to


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equilibrate for 30 minutes to reach a final NaCl concentration to 0.3 M over in 6 steps. The solution was sonicated for 10 seconds during each salt aging steps. After salt aging steps, the AuNPs were incubated with gentle mixing for 40 hours at room temperature (RT). Finally, to remove unreacted DNAs the solution was washed out by centrifugation for four times and discard supernatant and resuspend PBS buffer (pH 7.4, 150 mM NaCl).

Fabrication of Au dimer, trimer, and tetramer nanostructures through stepwise magnetic separations. To synthesize Au dimers, in the first step (1), A’ and X’ DNA modified AuNPs (A’X’-AuNPs, 10 nM, 200 μl) and 15 bps B’ and Y’ DNA modified AuNPs (B’Y’-AuNPs, 10 nM, 200 μl) were selectively hybridized to 15 bps A and B DNA of the mTDCs (10 nM, 100 μl) in PBS buffer. To conjugate one AuNP to one site of the mTDCs, the molar ratio between the binding site of the mTDC and AuNPs was 1:2. These mixtures were heated up from RT to 60 °C for 10 minutes, cooled down to RT for 6 hours. Next (2) nonhybridized AuNPs was removed to purify Au dimers through two magnetic separation steps. First, 23 bps X DNA modified MBs (X-MBs, 0.5 mg/ml) were mixed to A’X’-AuNPs and Au dimers. The captured samples were washed out with PBS buffer three times to remove


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uncaptured B’Y’-AuNPs. Next, we released the dimer clusters by exchanging short 15 bps DNA to long 23 bps complementary DNA (10 μM, 200 μl) from X-MBs. This process is based on strand displacement mechanism of DNAs45, and plays an important role to release target DNAs in supramolecular nano-assemblies when temperature cycles compromise the integrity of constituent materials (e.g., thermally unstable proteins) or weak chemical bonds (e.g., hybridized DNAs) in the nanostructures (Figure 4a). After the first releasing step, (3) A’X’- AuNP and Au dimers were captured with 23bps Y DNA modified MBs (Y-MBs). A’X’-AuNPs were eliminated from Y-MBs in the same manner and the final products were released by long 23bp Y DNAs as exchange release agents. The two-step magnetic separation ensures the final product to have both X’ and Y’ DNAs in the nanostructure, thus high purity AuNP dimers are obtained. For Au trimers, the molar ratio between the mTDC and AuNPs was 1:3 and the mixtures underwent the identical magnetic separation with exchange release three times. For Au tetramer, the molar ratio between the mTDC and AuNPs was 1:4 and the mixtures underwent four magnetic separation and exchange release steps.


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Fabrication of QD-Au hybrid dimers and Au@Ag core-shell dimers. To fabricate single QD and AuNP hybrid structure, SAv coated QDs (1 μM, 1 μl) were mixed with biotinylated A’ DNA (1 μM, 5 μl) for 15 minutes at RT considering with approximately 5 to 10 SAv per one QDs. Next, 10 nm B’- AuNPs (1 nM, 1 ml) and A’- QDs were conjugated to A and B DNAs of the mTDCs (0.1 μM, 10 μl) for 3 hours at RT. In order to synthesize Au@Ag core-shell dimers, we used the chemical reduction method. The mTDCs (10 nM, 10 μl) were incubated with A’ and B’ DNA modified AuNPs (1 nM, 100 μl) for 3 hours at RT. Next, 14.6 μL of PVP, 6.5 μL of 0.1 M L-sodium ascorbate and 43.8 μL of 1 mM silver nitrate were added gently and sequentially for 12 hours at RT. Next, the Au@Ag coreshell dimers were centrifuged three times at 12000 rpm for 30 minutes and redispersed in PBS buffer.

Self-assembly of dendrimer nanostructure. The initial monomer (XAAA) was synthesized by two stepwise magnetic separations. TAv (10 μM, 100 μl) and biotinylated X-DNA (1 μM, 200 μl) were mixed at the ratio of 5:1 in PBS buffer to make desired monovalent complexes. The mixture was heated at 70 °C for 10 min and then cooled down to RT for


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20 min. To isolate the monovalent complexes, we added X’-MBs in the mixture in PBS buffer at RT, mixed them for 3 hours, and placed on a magnetic stand to discard the supernatant of unreacted TAv. After washing three times with PBS buffer, the monovalent complexes were released from X’-MBs at 60 °C for 5 min. Next, the monovalent complexes were mixed with 25bps A-DNAs (10 μM, 100 μl) to passivate unreacted biotin binding sites of TAv complexes at RT for 3 hrs. The mixture went through an identical magnetic separation step with X’-MBs to remove unreacted A-DNAs. Dendritic monomers (A’BB, B’CC, and C’DD) were also synthesized by two stepwise magnetic separations. TAv (10 μM, 100 μl) and bis-biotinylated A’-DNA (1 μM, 250 μl) were mixed at the ratio of 4:1 in PBS buffer to make monovalent complexes. The mixture was heated at 70 °C for 10 min and then cooled down to RT for 20 min and hybridized with A-MBs. After mixing the monovalent complexes with A-MBs for 3 hours, A-MBs were washed three times with PBS buffer. The monovalent complexes were released from A-MBs at 60 °C for 5 min. Next, 25bps B-DNAs (10 μM, 75 μl) were added to the monovalent complexes to passivate unreacted biotin binding sites at RT for 3 hrs. The mixture went through the


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identical magnetic separation step with A-MB to remove unreacted B-DNAs and purify dendritic monomers.


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ASSOCIATED CONTENT

Supporting Information Supporting Information (SI) is available in the online version of the paper. SI contains more information on absorption spectra of mTDC, thermal stability of mTDC, absorption and emission spectral data of four dyes, geometrical structure of mTDCs, statistical analysis of plasmonic nanostructures (AuNP dimers, AuNP trimers, AuNP tetramers), schematics of initial dendritic monomer, and detailed oligonucleotide sequences.

Financial Interest Statement The authors declare no competing financial interest.

AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected]

Present Addresses


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†Program in Nano Science and Technology Seoul National University Seoul 08826, South Korea ‡Advanced Institutes of Convergence Technology 864-1 Iui-dong, Yeongtong-gu, Suwon-si, Gyeonggi-do 16229, South Korea Author Contributions Y.-K. Song, Y.-Y. Kim and Y. Bang initiated the project. Y.-Y. Kim synthesized DNA-modified magnetic beads, traptavidin-DNA conjugates, and plasmonic nanoparticles. Y. Bang and Y.-Y. Kim purified traptavidin proteins and performed PAGE measurement. Y.-Y. Kim performed TEM measurement. A. -H. Lee and Y.-Y. Kim performed PL and DLS measurements. All the authors discussed the results. Y. -Y. Kim drafted the manuscript and Y.-K. Song revised the contents. All authors contributed to the final version. Y.-K. Song contributed to and directed all aspects of this work. Funding Sources This work was supported by the Brain Research Program (2016M3C7A1904987) and the Biomedical Research Program (2017M3A9E2062706) of the National Research Foundation (NRF) funded by the Ministry of Science and ICT of Korea. ACKNOWLEDGMENT This research was supported by the Brain Research Program and the Biomedical Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2016M3C7A1904987 and 2017M3A9E22062706).


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ACS Nano

Figure 1. Scheme illustrating the four magnetic separation steps for mTDCs: (1) randomly mixing TAv with four distinct biotinylated DNAs, (2) separating the complexes including A-DNA using A’-DNAmodified MBs, (3) separating the complexes including A, B-DNAs using B’-DNA-modified MBs, (4) separating the complexes with A, B, C-DNA using C’-DNA-modified MBs, and (5) separating the complexes with A, B, C, D-DNA using D’-DNA-modified MBs. 425x500mm (300 x 300 DPI)


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Figure 2. (a) PAGE analysis of the mTDCs at various molar ratios - 4:1, 2:1, 1:1, 1:2, 1:3, 1:4 (TAv : DNA) for the lane 1 through 6. The sample in the lane 7 is the same as the sample in the lane 6, except it is purified by four successive magnetic separation steps. (b) PAGE analysis of the random mixture of TAv and biotinylated DNAs during four magnetic separation steps. Lane 1 indicates the band image of the initially mixed sample at a molar ratio of 1:3. The lanes from 2 to 5 show the bands after each magnetic separation steps. (c) Four magnetic separation yield of the SAv-DNA conjugates is about 3.5 %. (d) Four magnetic separation yield of the TAv-DNA conjugates is about 8.1 %. 425x599mm (300 x 300 DPI)


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ACS Nano

Figure 3. (a) Experimental scheme of the cascade FRET on TDC with distinct tetravalency (b) Emission spectra for the cascade FRET based on the TDCs conjugated with four different fluorophores – Alexa 488, Cy3, Cy5 and Alexa 750. Solid lines are emission spectra of TDCs with four different dyes, while dashed lines are spectra of a reference sample, a mixture of four dye-conjugated DNAs. Numerical labels indicate different excitation wavelengths – I for 490 nm, II for 540 nm, III for 640 nm, and IV for 740 nm. 425x500mm (300 x 300 DPI)


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Figure 4. Schematics for Au dimers via two stepwise magnetic separation steps with the mTDCs. (a) (1) conjugating A’X’-AuNPs and B’Y’-AuNPs to mTDCs, (2) hybridizing Au dimers and A’X’-AuNPs to XMBs, (3) removing non-conjugated B’Y’-AuNPs, (4) releasing Au dimers and A’X’-AuNPs from X’-MB by substituting the exchange DNA 1, (5) hybridizing Au dimers to Y-MBs, (6) removing non-conjugated B’Y’AuNPs after release of Au dimers from Y-MBs by substituting the exchange DNA 2. (b) TEM images of the Au dimers before magnetic separation and after magnetic separation. Scale bar = 200 nm. (c) Statistics of the Au monomer, Au dimer and Au clusters before and after magnetic separation. (d) DLS data of the Au monomer (black line) and Au dimer (red line). 379x729mm (300 x 300 DPI)


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ACS Nano

Figure 5. TEM images of various plasmonic nanostructures assembled with TDCs with distinct tetravalency. (a) 20 nm/10 nm asymmetric Au dimers, (b) 20 nm/10 nm/10 nm asymmetric Au trimers, (c) 10 nm/10 nm/10 nm Au trimers, (d) 10 nm/10 nm/10 nm/10 nm Au tetramers, (e) 10 nm/10 nm Au-QD heterodimers, (f) 10 nm/10 nm Au @ 5 nm Ag core-shell dimers. Scale bar = 20 nm. 425x350mm (300 x 300 DPI)


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Figure 6. Design and characterizations for dendrimer nanostructures using the TDCs. (a) Synthesizing dendritic monomers via two stepwise magnetic separation steps (1) mixing A’-bis-biotinylated DNAs and TAvs at the molar ratio of 1:4, (2) hybridizing A’-bis-biotinylated DNA tethered TAvs to A-MBs, (3) removing non-hybridized TAvs, (4) releasing A’-bis-biotinylated DNA tethered TAvs from A-MB from thermal releasing at 60 °C for 5 min, (5) mixing A’-bis-biotinylated DNA tethered TAvs and B-biotinylated DNAs at the molar ratio of 1:3, (6) hybridizing A’-bis-biotinylated DNA tethered TAvs to A-MBs, (7) removing nonhybridized B-biotinylated DNAs, (8) releasing dendritic monomer from A-MBs (b) PAGE analysis of dendritic monomer . Lane 1 is the sample of biotinylated DNAs and TAv at the molar ratio of 1:3. The lanes 2 is the band of dendritic monomers after two magnetic separation steps. The lane 3 is the band of mTDCs after four magnetic separation steps. (c) Scheme for synthesizing dendrimer nanostructure via step-by-step assembly of dendritic TDCs. (d) Analysis of agarose gel electrophoresis. Lane 1 is a dendritic monomer of TDCs, lane 2 is G1 dendrimers, lane 3 is G2 dendrimers, and lane 4 is G3 dendrimers respectively. (e) DLS data of G0, G1, G2, and G3 dendrimer nanostructures. (f) Negatively-stained TEM images of G3 dendrimer nanostructures.


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TOC Figure 329x178mm (300 x 300 DPI)


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Multivalent TraptavidinâDNA Conjugates for the Programmable

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