Angewandte Eine Zeitschrift der Gesellschaft Deutscher Chemiker Chemie www.angewandte.de Akzeptierter Artikel Titel: Valence-Engineering of Quantum Dots Using Programmable DNA Scaffolds Autoren: Jianlei Shen, Qian Tang, Li Li, Jiang Li, Xiaolei Zuo, Xiangmeng Qu, Hao Pei, Lihua Wang, and Chunhai Fan Dieser Beitrag wurde nach Begutachtung und Überarbeitung sofort als "akzeptierter Artikel" (Accepted Article; AA) publiziert und kann unter Angabe der unten stehenden Digitalobjekt-Identifizierungsnummer (DOI) zitiert werden. Die deutsche Übersetzung wird gemeinsam mit der endgültigen englischen Fassung erscheinen. Die endgültige englische Fassung (Version of Record) wird ehestmöglich nach dem Redigieren und einem Korrekturgang als Early-View-Beitrag erscheinen und kann sich naturgemäß von der AA-Fassung unterscheiden. Leser sollten daher die endgültige Fassung, sobald sie veröffentlicht ist, verwenden. Für die AA-Fassung trägt der Autor die alleinige Verantwortung. Zitierweise: Angew. Chem. Int. Ed. 10.1002/anie.201710309 Angew. Chem. 10.1002/ange.201710309 Link zur VoR: http://dx.doi.org/10.1002/anie.201710309 http://dx.doi.org/10.1002/ange.201710309 10.1002/ange.201710309 Angewandte Chemie COMMUNICATION Valence-Engineering of Quantum Dots Using Programmable DNA Scaffolds Abstract: Precise control over quantum dots (QDs) valency is critical and fundamental for quantitative imaging in living cells. However, prior approaches on valence control of QDs remain restricted to single types of valences. Here we report a DNAprogrammed general strategy for valence engineering of QDs with high modularity and high yield. By employing a series of programmable DNA scaffolds, we generated QDs with tunable valences in a single step with near-quantitative yield (> 95%). We further demonstrated the use of these valence-engineered QDs to develop 12 types of topologically organized QDs-QDs and QDsAuNPs and 4 types of fluorescent resonance energy transfer (FRET) nanostructures. Quantitative analysis of the FRET nanostructures and live-cell imaging reveal the high potential of these nanoprobes in bioimaging and nanophotonic applications. Quantum dots (QDs) are widely used for probing cell components both in vitro and in vivo due to their strong and stable luminescence. However, the lack of valence control of functionalized QDs hampers their bioimaging abilities since interparticle cross-linking may interfere with internalization, trafficking, motility, and signaling of membrane receptors . There have been many efforts to develop QDs with controlled valency, primarily on monovalent and divalent biofunctionalized QDs. In a straightforward approach, QDs were titrated with monovalent streptavidin (mSA) of different ratios followed by purification using gel electrophoresis to obtain monovalent QD-mSA conjugates. However, the yield of this approach is often low because of the recovery step of the QDs from the gel with typical extraction efficiencies of 30-50%. Improvement of the yield was realized using the principle of [*] steric exclusion which is applicable to both modified nucleic acids and polymers of low dispersity and controlled chemical functionality. In these previous methods, precise control of QD valency in a quantitative manner still remains challenging. Of note, the controllability is highly important for studying multivalent protein–ligand interactions and even tailoring them for many important biological events, including viral infection, immune response, cell signaling, and its regulation. Over the past two decades, DNA nanotechnology has attracted intense interest owing to their unparalleled self-recognition properties that offer flexibility and convenience for the ‘bottomup’ construction of exquisite DNA nanostructures with high controllability and precision. The DNA nanostructures have been shown to possess low cellular toxicity and excellent cell permeability. Given the highly precise and programmable nature, DNA is well suited for creating QDs of customized valency with precise controllability and high yield . More recently, Kelley and co-workers reported a strategy that allows one-step and purification free functionalization of CdTe QDs using a chimeric DNA molecule as a single biomolecular receptor[7c]. The resulting DNA-passivated monovalent QDs exhibited high specific binding to protein, however, quantitative control of multi-valency remained unsolved. It is therefore highly desirable to develop a general approach to produce QDs of customized valency capable of quantitative bio-analysis. Dr. J. Shen[+], Prof. X. Zuo Institute of Molecular Medicine, Renji Hospital, School of Medicine and School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University Shanghai 200127 (China) E-mail: email@example.com Dr. J. Shen[+], Profs. J. Li, L. Wang, X. Zuo, C. Fan Division of Physical Biology and Bioimaging Center, Shanghai Synchrotron Radiation Facility, Shanghai Institute of Applied Physics, Chinese Academy of Sciences Shanghai 201800 (China) Prof. C. Fan School of Life Science and Technology, ShanghaiTech University, Shanghai 201210 (China) Dr. Q. Tang[+], Prof. L. Li[+], Prof. X. Qu, Prof. H. Pei Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering, East China Normal University Shanghai 200241 (China) [+] These authors contributed equally to this work Supporting information for this article is given via a link at the end of the document. Figure 1. Preparation of QDs of customized valency by the principle of steric exclusion and electrostatic repulsion. (a) Schematic showing that the modification of surface chemistry generates monovalent QDs. The neutral mPEG reduces the negative surface charge, favoring the subsequent DNA approaching; whereas ptDNA molecules of appropriate size wrap the QD, preventing the reaction of a second strand owing to steric exclusion. Agarose gel electrophoresis showing QDs at three stages: MPA protected QD (MPAQD), MPA/mPEG co-protected QD (PEG-QD), and monovalent QD (Mono-VQD). The surface zeta potentials confirm the modification of surface chemistry at three stages. (b) DNA scaffolds self-assembled from different sets of chimeric DNA strands for valence-engineered QDs. The phosphorothioate (ps) portion of the sequence serves as a nanocrystal ligand, while the phosphate (po) potion of the sequence remains free for recognition. (c) The agarose gel electrophoresis confirming the formation of multivalent QDs. The DNA: QD This article is protected by copyright. All rights reserved. Accepted Manuscript Jianlei Shen+, Qian Tang+, Li Li+, Jiang Li, Xiaolei Zuo*, Xiangmeng Qu, Hao Pei, Lihua Wang, Chunhai Fan 10.1002/ange.201710309 Angewandte Chemie COMMUNICATION In this work, we report a single-step and purification-free method to create QDs of customized valency with precise control and high yield. The approach relies on the programmable design of DNA scaffolds that exploits a dual steric hindrance and electrostatic repulsion strategy. Four types of functional QDs with four different valencies, including monovalent, divalent, trivalent, and tetravalent, were created in high yield (higher than 96%, 95%, 80%, and 85%, respectively). The resulting functional QDs hold great promise for precise construction of complex nanostructures. Moreover, the highly controllable DNA distributions on QD also provided an unprecedented platform to study the resonant energy transfer from QDs to acceptor dyes which was of great importance and interest for quantitative bioanalysis. Lastly, the monovalent QDs was used for bio-imaging which exhibited reduced background due to minimized oligomerization during QDs-target interaction. First, we prepared monovalent QDs in high yield using surface electrostatic repulsion and steric exclusion effects. The CdSe/ZnS QDs suspended in chloroform (emission wavelength at 590 nm) were transferred into aqueous solution through the modification of mercaptopropionic acid (MPA) (Figure 1a). Then, in order to facilitate the approaching of negatively charged DNA, we reduced the surface charges of MPA protected QDs (MPAQD) by replacing a portion of MPA with neutral 2,5,8,11,14,17,20-heptaoxadocosane-22-thiol (mPEG, with seven repeated units). Since the competitively adsorption of mPEG on QD to form disulfide bond, the ratio of and MPA and mPEG on QD can be tuned by mPEG concentration and reaction time. To implement the steric exclusion strategy, the resulting MPA/mPEG co-protected QDs (PEG-QD) were then incubated with the chimeric DNA containing a 50-adenosine phosphorothioate DNA (ptDNA) fragment that serves as both a nanocrystal ligand and a steric hindrance component . The surface modification process was verified by the surface zeta potential measurements and gel electrophoresis (Figure 1a). The zeta potential was increased from -46.2 mV to -18.6 mV after mPEG replacement process, and was decreased to -48.6 mV after modification with chimeric DNA. The change of surface zeta potential was further validated by the agarose gel electrophoresis results. As shown in Figure 1a, only one new band appeared in the agarose gel electrophoresis for each step, suggesting that nearly all bare QDs were consumed. It should be noted that DNA decorated QDs (Mono-V-QD) migrated slower than MPA protected QDs (MPA-QD) despite their lower surface potential, which can be attributed to the increased resistance force of the extended ssDNA during migration of QDs. We next investigated the effect of salt concentration and pH on the assembly process (Figures S1 and S2). We found that the 0.1 M of sodium citrate buffer and pH 5 resulted in the highest yield of >96%. This assembly strategy can be generalized to QDs with different sizes. As confirmed by the agarose gel electrophoresis (Figure S3), we demonstrated the preparation of monovalent QDs with the size of approximately 4 nm (emission wavelength at 560 nm) to exemplify the generality of this assembly strategy. In order to verify the formation of monovalent QDs, we constructed QD dimers by assembling two Mono-V-QD with complementary strands. Two chimeric DNA strands with the same sequence in ptDNA fragment (50-adenosine) and complementary sticky ends were assembled onto QDs (denoted as QD1 and QD2 in Figure S4, Supporting Information), respectively. After the assembly, suspensions with two complementary monovalent QDs were mixed together without purification. As shown in Figure S4, a new band with slower migration speed appeared in the agarose gel electrophoresis which was mainly composed of QD dimers and negligible higher-order nanostructures, including trimers (<1%) and tetramers (<0.2%) (TEM image in Figure S4). We further explored the effect of the stoichiometric ratio between DNA and QDs on the preparation of monovalent QDs. Figure S5 displays the electrophoresis results of QDs incubated with DNA under different molar ratios ranging from 0:1 to 100:1. As can be clearly seen from digital image and corresponding gray values (quantified by Image J analysis), a new band appeared with adding 0.2-fold DNA; a further increase in DNA ratio only led to the consumption of bare QDs and generation of modified QDs. The positions of newly formed band remained unchanged even with 100-fold DNA. This result suggested that no stoichiometry was needed for the preparation of monovalent QDs, which was of critical importance for the subsequent preparation of multivalent QDs. We further investigated the steric hindrance effect on the preparation of monovalent QDs. The chimeric DNA with different lengths in ptDNA fragment (the total length of ssDNA was kept unchanged) was incubated with QDs at different molar ratios. As shown in Figure S6, the bands of monovalent QD appeared when the number of phosphorothioate adenosine was more than 20; whereas with shorter ptDNA fragment, many bands were observed in gel electrophoresis, indicating that more than one DNA was adsorbed on the surface due to relatively weaker steric exclusion effect. To identify the non-specific adsorption of DNA, ssDNA with the same sequence as that of chimeric DNA but without phosphorothioate modification was also incubated with QDs. Negligible non-specific adsorption was found during the assembly (Figure S7). Furthermore, we monitored the assembly dynamics through agarose gel electrophoresis. The gel results showed that the assembly process can be completed within 40 min (Figure S8). Taken together, these results elucidated that our assembly strategy allowed the one-pot, purification-free, and fast preparation of monovalent QDs with high yield and insensitivity to the stoichiometric ratio of DNA/QDs. The preparation of monovalent QDs with a remarkably high yield can be ascribed to the sufficiently charged surface of QDs. Firstly, it allows only a small amount of DNA to approach the surface sequentially. Once one sulfur atom of ptDNA fragment is anchored on the surface, the whole ssDNA in ptDNA fragment would wrap around QDs (the persistence length of ssDNA is approximately 2 nm and the ptDNA fragment is much longer) and block the remaining active anchor sites, which can impede This article is protected by copyright. All rights reserved. Accepted Manuscript molar ratio was 2:1. Monovalent: Mono-V-QD; divalent QD: Di-V-QD; trivalent QD: Tri-V-QD; tetravalent QDs: Tetra-V-QD. S1: MPA-QD, S2: PEG-QD, S3: DNA-QD. 10.1002/ange.201710309 Angewandte Chemie subsequently approaching ssDNA. Secondly, the surface charge could stabilize QDs under harsh reaction condition where high concentration salt is typically involved. Having successfully prepared monovalent QDs, we next sought to control over the valences of QDs with pre-designed DNA scaffolds. As schematically illustrated in Figure 1b, we can generate divalent QDs (Di-V-QDs) using a linear DNA nanostructure comprising a 30-adenosine ptDNA fragment and two recognition sites at two opposite positions. The assembly process was conducted following the same protocol to prepare monovalent QDs. The electrophoresis results suggested a remarkable assembly efficiency (with yield higher than 95%), which well agreed with above results that ptDNA of more than 20-adenosine was sufficient for achieving high assembly efficiency. Likewise, as shown in Figure 1b, Y-shaped and cross-shaped DNA scaffolds were designed for the fabrication of trivalent and tetravalent QDs, respectively. Specifically, the Yshaped DNA nanostructure comprises a 45-adenosine ptDNA fragment and three recognition sites topologically arranged on the loop; the cross-shaped DNA nanostructure comprises a 48adenosine ptDNA fragment and four recognition sites topologically arranged on the loop. Each DNA scaffold contains a ptDNA domain consisting of a single strand “loop” that could interact with QDs. The gel electrophoresis results confirmed that the Y-shaped DNA nanostructure was successfully prepared with yield of 95.3% (Figure 1c) and its three sticky ends can well hybrid with different complementary DNA strands to produce the trivalent QDs (Figure S9-11). Similarly, the gel electrophoresis results also verified the formation of the cross-shaped DNA nanostructure (yield: 98.7%) and its hybridization capability with four different complementary DNA strands (Figure 1c and Figure S9-11). Significantly, we found that Y-shaped and cross-shaped DNA nanostructures led to the formation of trivalent and tetravalent QDs in high yields of ~80-85%. Figure 2. QDs-QDs and QDs-Au clusters fabricated with multivalent QDs. Assembly of monovalent, divalent, trivalent, and tetravalent QDs with complementary AuNPs of 5, 10, and 15 nm, respectively. The AuNPs used here were fully decorated with complementary thiolated DNA and were added into the QDs solution excessively. Red arrows in figures indicate the position of QDs. The valency control of QDs offers great potential for bottom-up construction of nanostructures with high precision. We first tested our strategy by construction of heterodimers assembled from monovalent QDs with AuNPs of 5, 10, and 15 nm, respectively (Figure 2 and Figure S12). The precise structure of the heterodimers can be visualized in the transmission electron microscopy (TEM, Figure 2). The AuNPs used here were fully modified with complementary ssDNA and added excessively to ensure that the hybridization was dominated by the valences of QDs. To demonstrate the generalizability of nanostructure construction, we further assembled trimers by mixing divalent QDs with two monovalent QDs, or two AuNPs of the same sizes (5 nm and 5 nm) or distinct sizes (5 nm and 10 nm), respectively. As shown in Figure 2 and Figure S13, the decorated two recognition sites were anchored at the opposite positions of QDs, resulting in a “headset”-like configuration. Similarly, we can obtain tetramers by mixing trivalent QDs with three monovalent QDs, or three AuNPs of the same sizes (5, 5, and 5 nm) or distinct sizes (5, 10, and 15 nm), respectively (Figure 2 and Figure S14). Interestingly, all AuNPs were found to lay at one side of QDs, indicating that three DNA arms of Y-shaped DNA nanostructure were extended at the same side after assembly. Based on this, the generated nanostructure of trivalent QD with three AuNPs of different sizes could adopt a tetrahedron configuration with intrinsic chirality, which was confirmed by the circular dichroism (CD) spectrum (Figure S15). The weak CD signal can be ascribed to the slightly uneven distribution of sulfur atoms at two sides (Figure 2). Compared with Y-shaped DNA nanostructure, cross-shaped DNA nanostructure led to less distortion of planar geometry after assembly. As shown in Figure 2 and Figure S16, tetravalent QDs lay in the middle of the nanostructure with QDs or AuNPs surrounding in a cross shape. The difference of the assembly behaviors between Y-shaped and cross-shaped DNA nanostructures was probably due to the difference of the shape of loops at the center of each nanostructure. As described above, the loop in cross-shaped DNA nanostructure has rather larger inscribed circle (with average diameters of approximately 3.7 nm for Y-shaped and 6.4 nm for cross-shaped DNA nanostructures) and weaker rigidity than those of triangular loop in Y-shaped DNA nanostructure. These results substantiate the potential of multivalent QDs for bottom-up construction of heterogeneous oligomeric nanostructures with high precise valency control as atoms and molecules do. The established multivalent QDs provide an unprecedented platform to study the resonant energy transfer between QDs and their acceptors in a quantitative and precise manner. Here, the stoichiometric fluorescence resonance energy transfer (FRET) between QDs (emission wavelength at 530 nm) and discrete number of organic dyes (Cy3) was explored as a test bed. As schematically illustrated in Figure 3a, tetravalent QDs can hybrid with four complementary DNA strands each labeled with Cy3. The introduce of complementary DNA strands enabled FRET from QDs to fluorophore and turned on the fluorescence. By introducing different complementary DNA, the energy of donor was accessible to different number of acceptors. As shown in Figure 3b, the fluorescence signal of Cy3 was found to increase monotonically with increasing number of acceptors. The energy transfer efficiency (EQD-dye) from QDs to Cy3 was calculated based on the following equation: E QD dye FD FD A FD This article is protected by copyright. All rights reserved. Accepted Manuscript COMMUNICATION 10.1002/ange.201710309 Angewandte Chemie COMMUNICATION 6 EQD dye nR0 6 nR0 r 6 where n is the number of acceptors around the donor, r is the real distance between the donor and acceptor, R 0 is the calculated Fӧrster distance, with R0 0.98 103[k 2 n 4QD J ]1 / 6 where k2 is the dipole orientation factor which was usually approximated as 2/3, n is the refractive index of the medium (n=1.33 for water), QD is the quantum yield of QDs and J is designated as the spectral overlap integral. Figure 3c displays the experimental ET efficiency values, which were consistent with theoretical prediction. The minute deviation can be ascribed to the impurities during the DNA assembly since no purification process was conducted after the preparation of tetravalent QDs. Compared with several previously reported FRET systems constructed via stoichiometric addition of dye-labeled protein or ssDNA, valence-engineering of QDs using programmed DNA scaffolds offers precise controllability in the number of acceptor dyes. Figure 3. Quantitative FRET study between QDs and fluorophore Cy3. (a) Schematic demonstration of the fluorescence resonance energy transfer (FRET) between QD and four acceptors around its core with equal distance. The number of the acceptor dyes can be tuned via the hybridization of four types of different DNA sequence (complementary DNA 1-4 in supporting information). (b) The fluorescence spectra with different donor-acceptor ratios. The excitation wavelength at 370 nm was chosen to reduce the direct excitation of Cy3. (c) The experimental (calculated from b) and theoretical energy transfer efficiencies. The table shown as inset lists theoretical parameters for the calculation of energy efficiencies. (d) The agarose gel electrophoresis confirming the surface modification of monovalent QDs. 1 and 0 represent the presence and absence of reagents, which are BSA DNA , decorated primary antibody and QD, respectively (from top to bottom). The optical images of (e) microtubules using monovalent QDs as imaging tag and (f) the control sample without adding secondary antibody. Both images in (e) and (f) were taken under the same conditions. We then employed monovalent QDs as imaging tags to visualize the microtubules in Hela cell. Specifically, monovalent QDs were firstly hybridized with complementary DNA-primary antibody conjugation (with averaged 1.5 to 2 DNA per antibody). Here, monovalent QDs were added in excess during hybridization to ensure that each QD can be labeled with one primary antibody. As shown in Figure 3d, we found that only one new band with larger molecular weight was formed after incubation with primary antibody; whereas no complex structures can be found. The mixture was then incubated with bovine albumin (BSA) solution to reduce the non-specific adsorption during cellular imaging (Figure 3d). The resulting monovalent primary antibody labeled QDs were incubated with the antibody treated sample and the control sample (without secondary antibody), respectively. As shown in Figures 3e and 3f, monovalent QDs produced a clear imaging of microtubules with low oligomerization and negligible non-specific adsorptions. In summary, by combing the surface electrostatic repulsion and steric exclusion effects, as well as DNA nanotechnology, we achieved a precise control over the valences of DNA on QDs. This strategy offers several unprecedented advantages. First, QDs of customized valency can be rapidly and reliably synthesized with high yields, providing a readily available building blocks for bottom-up construction of nanostructures with high precision, and atom-like property. Second, rationally valence engineered QDs provides a versatile platform for studying the FRET effect in a quantitative manner, which is of great importance for FRET based bioassay/bioimaging where the discrete number and topological positions of acceptors are critical influential aspects. We demonstrate the use of monovalent QDs as imaging tags to label microtubules with remarkable specificity. Third, given that the information of assembly was pre-written by DNA nanotechnology, QDs with more complex valences can also be achieved through this method. The method established here presents a simple and effective strategy towards arbitrary decoration of DNA on QDs, which not only provides diverse functional “bricks” towards the fabrication of nanophotonic nanostructures but also offers a new paradigm in designing versatile bio-probes for applications in biological diagnostics and bio-imaging. Acknowledgements This work was ﬁnancially supported by NSFC (Grants 21422508, 31470960, 21390414, 21722502, 21505045, 21705048), Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (20171913), Ministry of Science and Technology of China (2016YFA0201200, 2013CB933802, 2013CB932803), the Chinese Academy of Sciences (QYZDJSSW-SLH031). This article is protected by copyright. All rights reserved. Accepted Manuscript Where FD and FD-A are referred to as the donor fluorescence in the absence and presence of the acceptor, respectively (all signals were extracted after an index weight analysis with a simple customized program written in MATLAB software, see supporting information and Figure S17). The EQD-dye can also be theoretically calculated. 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By employing a series of programmable DNA scaffolds, we generated QDs with tunable valences in a single step with near-quantitative yield (> 95%). We demonstrated the use of these valence-engineered QDs to develop 12 types of topologically organized QDs-QDs and QDs-AuNPs and 4 types of FRET) nanostructures. This article is protected by copyright. All rights reserved.