Valency-controlled Molecular Spherical Nucleic Acids with Tunable

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Valency-controlled Molecular Spherical Nucleic Acids with Tunable Biosensing Performances Xue Hu, Guoliang Ke, Lu Liu, Xiaoyi Fu, Gezhi Kong, Mengyi Xiong, Mei Chen, and Xiao-Bing Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02614 • Publication Date (Web): 12 Aug 2019 Downloaded from pubs.acs.org on August 13, 2019

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Analytical Chemistry

Valency-controlled Molecular Spherical Nucleic Acids with Tunable Biosensing Performances Xue Hu, Guoliang Ke,* Lu Liu, Xiaoyi Fu, Gezhi Kong, Mengyi Xiong, Mei Chen,* Xiao-Bing Zhang* State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Materials Science and Engineering, Hunan University, Changsha 410082, China *To whom correspondence should be addressed. E-mail: [email protected], [email protected], [email protected] ABSTRACT: Spherical nucleic acids (SNAs) play critical roles in many fields such as molecular diagnostics, disease therapeutics, and materials application. Due to the important role of DNA density on the properties of SNAs, the controlled synthesis of monodisperse SNAs with precise DNA density is an important approach for the structure-function relationship study and finite functions regulation of SNAs. In particular, the construction of monodisperse SNAs in a valency-tunable and site-specific manner is highly important, however, is still challenging. Herein, based on the high controllability, nanometer precision and addressable modification ability of framework nucleic acid (FNA), we develop the concept of valency-controlled framework nucleic acid-based molecular spherical nucleic acids (FNA-mSNAs) with tunable biosensing performances. The FNA-mSNAs consist of a valency-tunable FNA-based DNA nanocube as the core and controlled, precise number of DNA strands per core. By simple alternating the binding site number for shell DNA strands on the DNA nanocube, homogeneous FNA-mSNAs with different valencies were easily designed, which enabled the molecular level study of the effect of valency on their properties such as nuclease stability and cellular uptake. Furthermore, taking advantage of the addressable modification ability of FNA, the first heterogeneous molecular SNAs with tunable valency was demonstrated. Importantly, the valency of heterogeneous FNA-mSNAs was able to tune their biosensing performance, such as response dynamics, detection sensitivity and response range. With these remarkable features, FNA-mSNAs provide new research methods for the development of functional SNAs at the molecular level for a wide range of biological applications. . Spherical nucleic acids (SNAs) are polyvalent nanostructures consisting of a highly oriented and packed nucleic acid shell on a nanoparticle core.1 From the first SNAs based on gold nanoparticle core reported in 1996,2 the core compositions of SNAs have been explored to a wide range of nanoparticles including inorganic particles (e.g. silica, quantum dot, metal oxide), organic particles (e.g. liposomes, proteins), hybrid structures (e.g. metal-organic frameworks), and even hollow core without any material.3-15 Comparing to traditional linear nucleic acids, SNAs exhibit several distinct DNA density-dependent properties including improved nuclease resistance, higher cellular uptake, and so on,16-24 which enables the wide applications of SNAs in molecular diagnostics,25-28 disease therapeutics,21,29,30 and materials applications.31-33 However, all above-mentioned SNAs structures are polydisperse systems with imprecise number of DNA strands per core particle, because of the variations in surface nucleic acid density and/or particle size. Thus, rare report demonstrated the study on structure-function relationship of SNAs at the molecular level, although it is highly important for the mechanism study, finite regulation and biological application of SNAs. To address the challenge, Mirkin et al. recently reported the concept of molecular spherical nucleic acids (molecular SNAs) that precise number of DNA strands was quantitative modification on per core. In their system, T8 polyoctahedral silsesquioxane and buckminsterfullerene C60 scaffolds were employed as core with 8 or 12 binding sites for shell DNA strands per core, respectively.34 By this way, quantitative control of the surface densities of shell DNA strands at molecular level was achieved. The development of molecular spherical nucleic acids enables studying the relationship between DNA surface densities and the properties of SNAs, including nuclease resistance, cellular uptake, and gene regulation capabilities. Despite of the achievement, it is difficult to arbitrarily tune the number of binding sites on a same core of SNAs, since the binding site of current molecular SNAs core is immutable (8 or 12 only). Moreover, because each binding site of

the core is equal, the construction of molecular SNAs with site-specific modification of different shell DNA strand at different site on a same core is still challenging. In addition, although the valency should logically have important influence on their biosensing performance, rare example was reported related to the biosensing application of molecular SNAs. Therefore, the development and biosensing application of new molecular SNAs in a valency-tunable and site-specific manner is highly demanded, while has not been reported. Framework nucleic acid (FNA),35 the rational design of DNA nanostructures, provides the ability for high controllability and nanometer precision based on the high predictability of Watson-Crick base pairing. FNA has emerged as powerful tools in molecular logic calculation,36-39 molecular device simulation,40-42 molecular motor design43,44 and other applications.45-48 In particular, the addressable FNA-based DNA nanostructures enable the quantitative and site-specific control of the number and position of binding sites for guest objects such as nanoparticles and proteins.42,49-51 Taking advantage of these features, herein we developed the first valency-tunable framework nucleic acid core based molecular spherical nucleic acids (FNA-mSNAs) with tunable biosensing performances. As shown in Scheme 1, the FNA-mSNAs consisted of a valency-tunable FNA-based DNA nanostructure as the core and different density of DNA strands as shell. Based on the high predictable and programmable nature of FNA, the number of binding sites for shell DNA strands on per core (termed as the “valency” for molecular SNAs) could be quantitatively alternated. For example, the valency of DNA nanocube employed here could be tuned from 0 to 8. In this way, the homogeneous FNA-mSNAs (in this case, all the shell DNA strands have same sequence) with different number of shell DNA sequence per core could be successfully designed by simple alternating the valency of FNA core. Subsequently, this design realized the molecular level study of the effect of the valency on the typical properties of molecular SNAs, such as nuclease stability and cellular uptake. Furthermore, the site-specific modifi

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Scheme 1. Schematic illustration of valency-tunable framework nucleic acid core based molecular spherical nucleic acids (FNA-mSNAs). (a) homogeneous FNAmSNAs with same shell DNA strands; (b) heterogeneous FNA-mSNAs with different shell DNA strands.

cation ability of FNA realized the first example of heterogeneous molecular SNAs, whose shell DNA strands have different sequence. Interestingly, the valency of heterogeneous FNAmSNAs was able to tune their biosensing performance, such as response dynamics, detection sensitivity and response range. With these features, FNA-mSNAs were successfully applied for the microRNA imaging in living cells, which indicated the potential application of FNA-mSNAs in biological study.

MATERIALS AND METHODS Preparation of FNA-mSNAs. The preparation of FNAs core for FNA-mSNAs was carried out based on previous reports with slight modification.52 In brief, equal amount of DNA strands for respective design (shown in Table S1 and Table S2) were mixed in 1×TAE/Mg buffer (20 mM Tris, pH 7.4, 12.5 mM MgCl2, 2 mM EDTA). The mixtures were annealed with the as followed: 95℃ for 5 minutes, 80℃ for 3 minutes, then cooled from 80℃ to 60℃ with a rate of 2 min/℃, then annealed to 4℃ with a rate of 3 min/℃. To increase assembly yields of the hairpin strands, they were heattreated at 95℃, followed by slow annealing to room temperature. The hairpin strands and FNAs cores were mixed in 1×TAE/Mg buffer in 37℃ for 30 min, followed by cooling to room temperature. Characterization of FNA-mSNAs. Native polyacrylamide gel electrophoresis (N-PAGE) was carried out to characterize the formation of FNA-mSNAs. 10 μL 100 nM samples were mixed with 2 μL of loading buffer (6×). Then, the mixtures were loaded to 5% N-PAGE and run at 120 V in 1×TAE/Mg buffer, followed by analysis using a FLA-3000G image scanner (Fuji, Tokyo, Japan). To further characterize the hydrodynamic diameter of FNA-mSNAs before and after modification, dynamic light scattering (DLS) analysis of the samples were performed. As for AFM characterization experiments, samples were prepared by depositing onto a cleaved mica. Nuclease Stability Assay. Different kinds of cores were mixed with equal amount (1 μM) of DNA duplex probes consisting of a FAM-labeled DNA sequences and a DABCYLlabeled complementary strand in 1×TAE/Mg buffer. The mixtures were then kept at 37℃ for 30 min, followed by slow cooling to room temperature. After diluting to 20 nM (fluorescein-labeled strand concentration), 50 μL of each sample was transferred to a 96-well black plate. Then 50 μL DNase I in the reaction buffer (20 mM Tris, pH 7.5, 5 mM MgCl2, and 1 mM

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CaCl2) was added to each sample, followed by rapidly mixing. The final concentration of DNase I was 0.08 units per milliliter. The fluorescence intensity in each well (with excitation wavelength at 485 nm and emission wavelength at 525 nm) was recorded at a frequency of once per minute for about 3 h on a microplate reader (TECAN) immediately. Confocal Fluorescence Imaging. HeLa, MCF-7 and HEK293 cells were respectively seeded on cell culture dish at 37℃ for 24 h. The culture medium contains Dulbecco’s modified Eagle’s medium (DMEM) medium with 10% inactivated fetal bovine serum and 1% penicillin. The cells were then washed with DPBS buffer for three times. After that, the cells were incubated with probes at respective equal doses of fluorescent samples. After washing, the cells were characterized using a Nikon confocal laser scanning microscope at excitation wavelength of 488 nm (FAM) or 650 nm (Cy5). Flow Cytometry Analysis. The cells were incubated with different kinds of samples labeled with Cy5 for 4 h. After washing, the cells were transferred to be characterized using a CytoFLEX™ flow cytometer. Data were analyzed with FlowJo 7.6 software. Cytotoxicity. A standard MTT assay was implemented to measure the cytotoxicity of the samples. On a 96-well plate in a cell culture incubator, the cells were seeded at 37℃ with 5% CO2 for 24 h. Then the cells were incubated with 500 nM probes for 6 h. Then, the cells were washed using DPBS buffer for three times. Subsequently, 0.5 mg/mL MTT (final concentration) was added and mixed with the cells, and incubated for 4 h at 37℃. Then 100 µL of dimethyl sulfoxide (DMSO) were added to the wells and incubated at 37℃ for a while until the precipitates dissolve. Finally, the absorbance intensity of samples at 490 nm was recorded on a multimode microplate.

RESULTS AND DISCUSSION Synthesis and Characterization of Homogeneous FNAmSNAs. The FNA-mSNAs consisted of a valency-tunable FNA-based DNA nanocube as the core and different density of DNA strand as shell. Since both the core and shell are made of nucleic acids, FNA-mSNAs could be prepared through the simple sequence-complementary hybridization between the binding sites on DNA nanocube and the shell DNA strands. Moreover, by altering the number of binding sites for shell DNA strands on the DNA nanocube, a series of valencycontrolled homogeneous FNA-mSNAs structures could be easily designed. The formation of DNA nanocube core was first confirmed using a native polyacrylamide gel electrophoresis (N-PAGE). The result (Figure S1) showed that the DNA nanocube core was successfully assembled (lane 4) with high purities. The atomic force microscopy (AFM) also confirmed

Figure 1. (a) 5% native-PAGE for the characterization of FNA-mSNAs with different valencies including the core (lane 1), mono-(lane 2), di- (lane 3), tetra- (lane 4) and octa-valency (lane 5). (b) Dynamic light scattering (DLS) characterization of DNA nanocube core and FNA-mSNAs.


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the successful formation and uniform size of DNA nanocubes (Figure S2). Next, we constructed homogeneous FNA-mSNAs by inserting binding sites with complementary sequences of the shell DNA strand on DNA nanocube core. Importantly, by alternating the number of binding sites, a variety of FNA-mSNAs could be designed with different valency including mono-, di-, tetra- and octa-valency. As shown in Figure 1a, a steady reduction of electrophoretic mobility in PAGE revealed that the configuration of FNA-mSNAs increases gradually with increasing number of shell-strands. Furthermore, DLS was employed to analysis the hydrodynamic diameter of DNA nanocubes before and after modification. Figure 1b indicated that the hydrodynamic diameter of FNA-mSNAs obviously increased after modification of shell DNA strands. These results demonstrated that DNA nanocube core-based FNAmSNAs had been successfully prepared. Besides DNA nanocubes, other shape of DNA nanostructures, such as DNA triangular prism, could also act as the core for the preparation of valency-tunable SNAs (Figure S3), which indicates the powerful ability of DNA nanostructures. Nuclease Stability. The traditional properties of SNAs, such as nuclease stability and cell uptake, were reported to be significantly affected by the surface density of shell DNA on SNAs in previous polydisperse SNAs. To study the relationship between structures and functions at a molecular level, herein we investigated the nuclease stability and cell uptake stability of FNA-mSNAs with different valencies. The effect of valency on nuclease stability at the molecular level was first investigated by incubating different kinds of SNAs (total fluorescent DNA was equal in each system) in the presence of same concentration of nuclease. As a common model for nuclease stability assay, DNase I was employed here. The FNAmSNAs with different valencies were designed by quantitative binding of different numb DNA duplex probes, which consisted of a FAM-labeled (fluorophore) DNA sequences and a DABCYL-labeled (quencher) complementary strand. As shown in Figure 2a, in the presence of DNase I, a significant fluorescence recovery was observed since the probes were degraded, inducing the separation of FAM from DABCYL. To reveal this degradation process, a time-dependent fluorescence analysis was implemented. As shown in Figure 2b, the halflives of FNA-mSNAs were 1.23-1.87 times longer than that of free duplex of the same sequence. Moreover, with the increase of valency, the half-lives of FNA-mSNAs increased, indicating its gradual improvement of nucleases resistant ability with higher valency. This result made agreement with the mechanism of SNAs in previous reports, 18,19 in which the increase of DNA density

Figure 2. (a) Schematic illustration of the nuclease stability assay of FNA-mSNAs in the presence of DNase I. (b) Timedependent percent duplex degraded of FNA-mSNAs with different valencies in the presence of DNase I.

could improve the negative charge density and the local salt concentrations on the surfaces of SNAs. Cellular Uptake. Next，the influence of valency on the cellular uptake of SNAs was studied. HeLa cells were incubated with these FNA-mSNAs modified with Cy5 (0.2 M) in serum-free medium for 4 h (the total concentration of Cy5 in each sample is equivalent). The probes were able to enter cells without any transfection agents, which was confirmed by a zstack image of cells on a confocal microscopy (Figure S4). Moreover, with the increase of valency, a slight increase of fluorescence signal of Cy5 was observed in HeLa cells incubating with different FNA-mSNAs (Figure 3a). The flow cytometry-based quantitative analysis for the cellular uptake efficiencies of different kinds of Cy5-labeled FNA-mSNAs was also carried out. As shown in Figure 3b, the efficiency of cell uptake slightly increased from mono-valency to octavalency of FNA-mSNAs. These results showed that the increase of valence could at a certain extent improve the cellular uptake of FNA-mSNAs, which agreed with previous reports. 17,22

Figure 3. Cellular uptake of FNA-mSNAs with different valencies. (a) The confocal fluorescence microscopy images of the HeLa cells treated with different kinds of Cy5-labeled FNA-mSNAs. (b) The flow cytometric assay for the cellular uptake efficiencies of different kinds of Cy5-labeled FNAmSNAs. The Construction of Heterogeneous FNA-mSNAs. Comparing to previous nanoparticle cores for SNAs, the framework nuclei acids core in this work exhibited the advantage of addressable modification of DNA shell. That is, with well design, each binding site on framework nuclei acids core could be different. In this way, the first example of heterogeneous molecular SNAs with tunable valency was demonstrated here. As the proof of concept, the two hairpin DNA probes (H1 and H2) for catalyzed hairpin assembly (CHA) reaction were modified on the DNA nanocube with different valency and position. For example, as shown in Figure 4a, the DNA nanocube core was modified with equal amount of H2 and tunable numbers of H1 (from 1 to 7). Thus, the valency of H1 in heterogeneous FNA-mSNAs could be tuned from 1 to 7 (the FNAmSNAs were named as A1B1 to A7B1, respectively). As shown in Figure S5, in the CHA reaction, based on multiple steps of toehold-mediated strand displacement reactions, the target (microRNA21 here) can finally catalyze the hybridization between hairpins H1 and H2. Meanwhile, the target microRNA21 could be released to trigger other H1-H2, thus realizes an amplification detection of targets. Both FAM (fluorophore) and DABCYL (quencher) were modified on the stem of hairpin H2. Thus, the probe was quenched at hairpin structure while lighted up when the hairpin H2 hybridized with H1, enabling the fluorescence measurement of the CHA reaction and amplified detection of microRNA21. In this way, the design and construction of heterogeneous FNA-mSNAs with



different valencies enables studying the effect of valency on the biosensing performances of heterogeneous FNA-mSNAs at molecular level. Tunable Biosensing Performances of Heterogeneous FNA-mSNAs. First, we investigated the formation of these FNA-mSNAs through PAGE. With the step-by-step hybridization of four strands, the cores of heterogeneous FNA-mSNAs were successfully assembled with high yield (Figure S6). At the same time, the successful hybridization of H1 strand and single H2 on DNA nanocube core was also confirmed by PAGE (Figure S7). As shown in Figure 4b, as the number of H1 binding sites on the core increased, the mobility of the bands in the glue gradually became slower, indicating the successful hybridization of each kind of FNA-mSNAs with high efficiency. Besides, the heterogeneous FNA-mSNAs with equal amount of H1 and tunable numbers of H2 (from A1B1 to A1B7) was also successfully prepared (Figure S8a and Figure S8b), indicating the addressable ability of FNA-based DNA nanostructures. We then investigated the biosensing performances of different valency of heterogeneous FNA-mSNAs. Their dynamic response to the presence of microRNA21 was first studied through a time-dependent fluorescence analysis. The heterogeneous FNA-mSNAs with equal amount of H2 and tunable numbers of H1 (from A1B1 to A7B1) was first tested. As shown in Figure 4c, with the number of H1 increases, FNAmSNAs exhibited faster reaction rate and higher reaction

Figure 4. Tunable biosensing performances of heterogeneous FNA-mSNAs with different valencies. (a) The construction of heterogeneous FNA-mSNAs with equal amount of H2 (signal probe) and tunable numbers of H1 (from 1 to 7). (b) Gel characterization for the formation of different kinds of FNAmSNAs. (c) Time-dependent fluorescence spectra of different kinds of FNA-mSNAs in response to same concentration of target. (d) The limit of detection was controlled by the valency of heterogeneous FNA-mSNAs. (e) The dynamic range of detection was engineered by tuning the valency of heterogeneous FNA-mSNAs.

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efficiency. The main reason could be the improved local concentration of hairpin H1, which resulted in higher collision frequency and enhanced reaction rate between H1 and H2. At the same time, the heterogeneous FNA-mSNAs with equal amount of H1 and tunable numbers of H2 (from A1B1 to A1B7) was also tested. Similarly, the increase of H2 also improved the reaction rate and efficiency, and achieved maximum at A1B3 (Figure S8c). Subsequently, the heterogeneous FNA-mSNAs with equal amount of H2 and tunable numbers of H1 was selected for further investigation including detection limit (or limit of detection, LOD) and dynamic range. The detection limits of each FNA-mSNAs were investigated by measuring the fluoresce response of each probe to different concentrations of target. The result showed that the detection limit of probes gradually decreased with the increase of H1 number (Figure 4d). In particular, this trend was more significant from free system to heterogeneous FNA-mSNAs with lower valency (such as A1B1 and A3B1), mainly due to their more obvious change on local concentration and reaction efficiency of probes. In addition, the detection of FNA-mSNAs for microRNA21 was proved to be highly selective (Figure S9), suggesting the feasibility of FNA-mSNAs for microRNA21 detection. Moreover, with the increase of H1 number, the dynamic range of probes shifted to lower concentration, indicating its higher detection sensitivity (Figure 4e). There results strongly confirmed that the biosensing performance was tunable through controlling the valency of FNA-mSNAs, which is very important for regulating probes for the application in different situations. Application for microRNA Biosensing in Living Cells. Because of its rapid, sensitive and selective detection features, A7B1 was used for further biosensing miRNA21 in living cells. MicroRNA21 was chosen as the model since it is regarded as a potential biomarker in medical diagnosis.53-55 Referring to previous reports, microRNA21 is overexpressed in in MCF-7 cells and HeLa cells, while is negligible in HEK293 cells.56-58 The cell viability of FNA-mSNAs was first studied by a standard MTT assay, which confirmed that FNA-mSNAs were safe to cells (Figure S10), mainly because they were consist of nucleic acids. Then, three kinds of cell lines were incubated with 0.5 μM of probes for 6 hours respectively, before the confocal imaging. As shown in Figure 5, the green fluorescent signal for microRNA21 in both MCF-7 cells and

Figure 5. Fluorescence image of microRNA21 in MCF-7, HeLa and HEK293 cells treated with FNA-mSNAs. The scale bar is 50 μm.


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HeLa cells were significantly lighted up. However, in the case of HEK293 cells, negligible fluorescence signal was observed. This result is consistent with previous reports, and confirmed that our probe achieves the biosensing of microRNA21 in living cells. The result indicated the potential application of FNA-mSNAs in biological study.

CONCLUSION In summary, we have developed the concept of valencycontrolled molecular spherical nucleic acids based on the high controllability, nanometer precision and addressable modification ability of framework nucleic acid. With precise adjusting the valency, the biosensing performances of SNAs could be well regulated. Compared to previous SNAs, the FNA-mSNAs exhibits several remarkable features. First, because of the controlled valency of FNA core, FNA-mSNAs enable the mechanism study of structure-function relationship at molecular level. Second, benefiting from the addressable modification ability of FNA, the first example of heterogeneous molecular SNAs was developed. Importantly, the performance of heterogeneous FNA-mSNAs could be precisely modulated by tuning the different valencies of DNA ligands, which provides the tunable biosensing ability for different situations. Moreover, FNA-mSNAs can be easily prepared through the simple DNA hybridization rather than complex chemical conjugation, which greatly simplifies the construction of SNAs. Taken together, we believe that FNA-mSNAs with controllable valency can provide a powerful tool for the subsequent research on the development of functional SNAs at the molecular level for future biological applications. 35, 59-61

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Additional characterization data of oligonucleotides sequences used in this study, fluorescence spectra, gel electrophoresis, and cell viability assay. (PDF)

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected], * E-mail: [email protected] * E-mail: [email protected] Author Contributions All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We are grateful for the financial support from National Natural Science Foundation of China (21705038, 21705037, 21890744, 21521063) and Natural Science Foundation of Hunan Province (2018JJ3029, 2018JJ3092).

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