DNA Framework-Encoded Mineralization of Calcium Phosphate
Calcium phosphate (CaP) nanocrystals with prescribed geometric structures are constructed using various DNA frameworks as templates. Synchrotron small angle X-ray scattering (SAXS) data reveal the initial crystallization process by DNA frameworks. Further experimental studies and theoretical calculations demonstrate that by encoding DNA frameworks via sequence design, one can rationally control the size and shape of CaP nanostructures. These preliminary findings would enlighten the design and applications of DNA framework-encoded biomimetic mineralization and organic-inorganic composite materials.
INTRODUCTION<!>The Bigger Picture<!>DNA Tetrahedron-Templated CaP Crystallization<!>SAXS and MD Analysis of the Mechanism for CaP Crystallization<!>3À<!>Generality of DNA Framework-Guided CaP Crystallization<!>Mineralization-Protected Functional Nucleic Acids<!>Conclusions<!>EXPERIMENTAL PROCEDURES Materials<!>Gel Electrophoresis<!>TEM Characterization<!>STEM Characterization<!>AFM Characterization<!>SAXS Characterization<!>Statistical Analysis<!>MTT Assays
<p>Biological organics are well known for guiding the mineralization of hierarchical organic-inorganic complex with delicate structures and excellent mechanical properties, [1][2][3] which has inspired the development of biomimetic mineralization with various sizes, shapes, and functions. [4][5][6][7][8] Among diverse biominerals, calcium phosphate (CaP) mineralization is actively explored due to its close relationship to biomedicine as it constitutes the main inorganic component of human bones and teeth. 9,10 However, designed fabrication of well-defined CaP nanostructure remains hampered in both precision and versatility due to its non-classic nucleation process and complex phases. [11][12][13] DNA-templated nanofabrication sheds light on user-defined construction of inorganic materials with prescribed structures. 14,15 The unparalleled programmable ability to create exquisite DNA nanostructures [16][17][18][19][20][21] has led to numerous applications including nanoelectronics, nanophotonics, and nanomedicine. [22][23][24] We were thus motivated to exploit DNA nanotechnology to guide precise nanoscale CaP mineralization. In addition to enhancing the ability for biomineralization, we reason that solidifying DNA nanostructures with biominerals would overcome the vulnerability of pure DNA materials and widen the scope of DNA nanotechnological applications.</p><p>In this work, we developed a DNA framework-templated CaP crystallization strategy to guide the mineralization of CaP nanocrystals (NCs) with prescribed shapes. We synthesized two-and three-dimensional (2D and 3D) DNA frameworks ranging from $10 to $100 nm for this purpose. The growth and crystallization process of</p><!><p>Biological scaffolds offer precise templates for mineralization of delicate structural materials with various sizes, shapes, and functions. Previous bioinspired approaches have led to welldefined synthetic structural materials, e.g., bone and nacre, ranging from micro to macro scales. However, fabrication of calcium phosphate (CaP) nanostructures with user-defined geometric shape is restricted by precision and versatility. A key challenge for nanoscale mineralization is thus the design of artificial scaffolds for precise CaP crystallization. Here, we describe a DNA framework-templated CaP crystallization strategy that guides the mineralization of CaP nanocrystals with prescribed shapes. We demonstrate that CaP mineralization provides a robust means to protect nucleic acids. This work establishes that the structural information of DNA frameworks can be inherited by inorganic crystals, opening a new avenue for nanoscale mineralization and programmable inorganic material synthesis.</p><p>CaP surrounding DNA frameworks can be controlled by the counter-ion concentration and the reaction time. We found that the morphology of thus-produced CaP NCs generally followed the designed topology of DNA frameworks. We further employed synchrotron-based small angle X-ray scattering (SAXS) and molecule dynamics (MD) simulation to elucidate the template crystallization mechanism of CaP.</p><!><p>To demonstrate our strategy, we first employed a common 20 bp DNA tetrahedron (TDN) (edge length: 6.8 nm) to template the mineralization of CaP. Magnesium ions (Mg 2+ ) are often used to help the folding of DNA nanostructures; 25 however, Mg 2+ was previously shown to inhibit CaP crystallization. 26 To avoid this problem, we replaced Mg 2+ with calcium ions (Ca 2+ ) during the DNA self-assembly, which resulted in well-defined nanostructures (Figure S1). Free calcium ions (Ca free ) were removed from the solution by 30 K Millipore centrifugation to ensure fine control of the Ca free concentration. In a typical procedure, a solution of freshly prepared calcium chloride (CaCl 2 , 5 mM) was added dropwise into a phosphorus (Na 2 HPO 4 )-containing TDN solution (20 mM Tris, pH 9.0) at room temperature, generating a final concentration of 4.0 mM TDN, 2.0 mM Ca free , and 1.2 mM PO 4 3À . The sample was further aged for 3 h under 37 C to allow the formation of CaP NCs. The whole process for this DNA framework-templated mineralization was presented in Figure 1.</p><p>We utilized transmission electron microscopy (TEM), low-dose filed-emission scanning electron microscopy (FESEM), and atomic force microscope (AFM) to characterize the size distribution and morphology of as-prepared CaP NCs (Figure 2A). TEM imaging revealed an average diameter of 19.2 G 3.1 nm for TDN-CaP NCs. Subsequently, dynamic light scattering (DLS) measurements indicated that TDN had a hydrodynamic diameter around 13.8 nm (D theo = 8.6 nm), while mineralized TDN was about 36.6 nm (See Figures S2 and S3 for extra AFM and SEM data). The deviation between DLS and TEM characterization was possibly due to the surrounding hydrated layer. Similar phenomena were found in DLS measurement of TDN with different sizes and corresponding TDN-CaP NCs (Figure S4). TEM and FESEM results also revealed triangular or quadrilateral shaped CaP NCs. The morphology differences could be attributed to the varied resting status of each single particle onto the substrate. The projection morphologies were in good accordance with cryo-TEM studies on the TDN structure. 27 The combination of above results confirmed the formation of well-dispersed CaP NCs templated by TDNs.</p><p>To further study the crystal structure of 20 bp TDN-CaP NCs, we performed high-resolution TEM (HRTEM) and fast Fourier transform (FFT) analysis on single TDN-CaP NCs. HRTEM image of a typical 20 bp TDN-CaP NCs (Figure 2B) showed that, from the current projection, the crystal exhibited two triangular domains with three distinct twin grain boundaries (white dash and dot). The boundaries are characteristic of three edges of a tetrahedron particle as templated by the TDN. In addition, FFT image (Figure 2C) showed a typical incoherent twin boundary pattern with two pairs of diffraction spots distributed on a single line. Thus, the $15 nm diameter tetrahedron particle is composed of several correlative single crystals. The diffraction data indicated the sample crystal structure well matched those of synthesized hydroxyapatite (JCPDS no. 09-0432). Several other 20 bp TDN-CaP NCs and 37 bp TDN-CaP NCs were analyzed, which exhibited similar DNA framework-induced structures (Figures S5-S9). By gradually changing the defocus distance, different lattice patterns on the same particle were obtained, while the diffraction lattices derived from FFT remained almost identical (Figure S10), which further demonstrated the 3D tetrahedron structure of mineralized TDN. In addition, solution without TDN or with equivalent effective base concentrations as 1.8 mM 20 bp TDN generated aggregated particles or particle chains (Figure S11). These data accompanied with AFM and SEM results proved the successful mineralization of TDN into tetrahedron-shaped CaP twin crystals.</p><p>Next, we studied how Ca 2+ concentrations affect CaP crystallization. We captured three typical intermediates at different Ca 2+ concentrations, as shown in the UVvis absorption spectra (Figure 2D). PO 4 3À concentrations were maintained at stoichiometric ratio (Ca/P = 1.67). Three representative stages of the mineralization were visualized by TEM, as shown in Figure 2G. The reaction started by formation of very small CaP clusters. Then, with the concentrations of Ca 2+ increased, the clusters aggregated surrounding each TDN and hollow NPs were generated (highlighted with yellow cycle). Finally, the amorphous calcium phosphate (ACP) NPs matured and crystalized to form CaP NCs. Selected area electron diffraction (SAED) graphs revealed the phase transformation from ACP to crystals. Crystalline diffraction spots occurred only at the final stage, as compared to merely amorphous diffraction cycle obtained in the first two stages.</p><!><p>To obtain the in situ structural information of TDN-CaP for understanding the crystallization process, we employed synchrotron-based SAXS to probe the crystallization of TDN-CaP (Figures 2D-2F). SAXS data first suggested an unexpected slight deformation of TDN structures in solution. By fitting the SAXS data with a combination of normal mode analysis (NMA) and fast-SAXS-pro program, 28,29 we found that the revised TDN molecular structure showed a slight twist, which matched well with the experimental results (c 2 = 7.1, Figures 3A and 3B). The average distance  between two base pair increased from 3.40 to 3.55 A ˚, suggesting stretching of DNA molecules due to their intrinsic elasticity. 30 From MD simulations, we also observed similar deformation of TDNs (Figure 3C).</p><p>Further analysis of the SAXS profiles revealed a slight blue shift after adding 0.5 mM calcium ions into TDN solution, suggesting the attachments of CaP clusters onto TDN structures at the initial induction period of crystallization, as confirmed by MD simulation (Figures S12-S14). Through selected time sections of MD simulation, we observed densification of Ca 2+ , PO 4</p><!><p>, and OH À ions onto TDN template within 50 ns, which was mainly originated from the electrostatic interaction (Figure 3C; Video S1). The attachments of CaP clusters onto TDN will increase the total electron density as well as hydrodynamic size of the whole structure. Radius of gyration (Rg) values for theoretical samples before and after reacting with CaP were 39.3 G 0.8 A ånd 58.4 G 1.1 A ˚, which were in line with corresponding experimental one, 43.8 G 0.2 A ˚and 54.1 G 1.7 A ˚. Increasing on Rg and blue shift of the scattering profile from Ca free = 0.0 to 0.5 mM sample group indicated an enlargement of TDN structures without losing the overall tetrahedron shape (Figure 3D). In addition, at higher ion concentrations (Ca free > 1.0 mM), spontaneous nucleation and aggregation would generate inhomogeneous large particles in practice, which resulted in partially aggregated sample that could hardly be analyzed (Figure 2E). 28 Having established the CaP clustering ability of TDN, we next analyzed the effects of DNA framework on CaP crystallization. The nearly perfect monodispersity of TND nanostructures allowed us to design the desired local environment for crystallization. The effective base concentration of TDNs (base numbers of a single TDN multiply its concentration) determine the number of structural calcium ions (Ca struc ) directly bound to the TDNs. Here, we define the total calcium concentration (Ca total ) as the sum of Ca struc and Ca free (free calcium ions in the solution). By taking the 20-bp TDN as an example, we knew that the total base for this structure is 252. Hence, the theoretical maximum number of calcium ions that could adsorb onto a single TDN is 252, which means 4.0 mM TDN would result in approximately 1.0 mM Ca struc . We found that with the following initial ion concentrations: 1.0 mM Ca struc , 0.5 mM Ca free , and 0.9 mM PO 4 3À , after 2 days of culturing, Abs 340 for the sample did not change significantly, excluding the occurrence of precipitation. In the system without TDN, according to precipitation-solution equilibrium (Equation 1):</p><p>where SI indicates the solubility index, I AP indicates the ion activity product, and K sp indicates the chemical activities of the solubility product (e.g., 25 C, K sp = 2.35 3 10 À59 for synthesized hydroxyapatite), a solution with 0.5 mM Ca free and 0.3 mM PO 4 3À (pH 9.00), would already generate a SI of 1.17 at room temperature, which suggests the feasible formation of apatite precipitates at even lower ion concentration without DNA.</p><p>While in the TDN containing solution system, TDN helped the stabilization of CaP clusters, supported by our TEM, UV-vis, SAXS data, and MD simulation.</p><!><p>To examine the generality of DNA framework-encoded CaP mineralization, we carried out mineralization experiment on two typical DNA origami nanostructures: triangle and square (Figures 4A and S15). The successful mineralization of DNA origami was illustrated by the variation of crystal overall morphology and height change (Figures 4B and 4C) and additional TEM images along with energy dispersive spectrometer (EDS) (Figure 4D). Specifically, the resultant bulk structure did not follow the preferred c axis growth direction of CaP 31 but was restricted by the geometric profile of DNA nanostructures. However, the precision of CaP mineralization on DNA origami could not reach that of silica (controllable thickness $1 to 2 nm) in our previous work, which is due to the larger diameter of Posner's cluster ($1 nm, Ca 9 (PO 4 ) 6 ) than that of silica tetrahedron unit ($3.2 A ˚), as well as much faster crystallization speed of CaP at the initial period. 32 We further showed the heterogeneous crystal growth progress and CaP NCs controlled by DNA origami (Figures 4E and 4F). We found that at early stage, ACP clusters tended to aggregate at the tips of the triangle. With the growth of ACP, mineralized area extended along the edges, and finally filled up the hollow center of triangle DNA origami. The crystal structures of triangle CaP NCs were studied through HRTEM analysis. FFT data for the tip (green frame) and center (orange frame) area was distinctly different: the center area showed a polycrystalline pattern, while the tip area showed a uniform single crystalline pattern along the (211) family of crystal planes (See Figures S16-S20 for extra HRTEM analysis and control experiments). In addition, the distance between adjacent base pair of DNA is 3.4 A ˚, which is equal to the typical crystal plane distance of CaP along the c axis (002). 33 This might hint the crystallization restriction ability of DNA on CaP minerals at the atomic scale, both for the abnormal twin-crystal structured tetrahedron and triangular CaP particles.</p><!><p>Having established the DNA framework-guided CaP crystallization strategy, we next sought to explore the use of solidified DNA nanostructures for live-cell delivery of immunostimulatory oligonucleotide. Cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs) is a type of therapeutic nucleic acids with strong immunostimulatory activities; 34 however, its efficiency is limited by low cellular uptake efficiency and poor intracellular stability. 35 TDN can function as nanoscale carrier to improve cellular uptake efficiency of effector molecules and immune response remarkably. 36 Here, we aim to improve the intracellular stability of TDN-CpG using TDN-templated mineralization. Practically, CaP has been proved to be a biocompatible biomedical material. 37 Of note, the TDN-templated CaP NCs have a near-homogeneous particle size distribution that compare favorably with conventional CaP carriers. 38 As shown in Figure 5A, CaP mineralization procedure was applied to CpG bearing TDN (TDN-CpG, with four CpG strands), and was proven successful by particle morphology and size characterizations. AFM height images (Figure 5B) showed that bare TDN-CpG has a typical morphology with a hollow ring and four fluorescence intensities from flow cytometry analysis indeed refer to enhanced cellular uptake efficiency (Figure S24). After entering cells, CaP coated nanoparticles finally reach endolysosomes via microtubule-mediated trafficking, 39 where the CaP shell slowly dissolve in the acidic environment (pH $ 5.5) and release the TDN-CpG. Previous studies have well established approaches for stabilization of DNA structures with oligo-polylysine coating or UV crosslinking. [40][41][42] Our post-dissolution method can protect nucleic acids in the complex intracellular environment, providing a unique and robust means for smart drug delivery with prescribed geometric features and long-term in-vivo bioactivities.</p><!><p>In closing, we have developed a new method to encode mineralization using DNA framework-guided CaP crystallization. Our combined experimental and theoretical studies revealed that DNA framework restricts the crystallization process and affects the crystal morphology of CaP. The inherent affinity of DNA phosphate backbone to calcium ions and the programmability of DNA enables controlled CaP mineralization with predictable structures. Importantly, the CaP shell not only inherits the structural information of DNA framework, but also acts as a protective shell to enhance intracellular delivery of functional nucleic acids. This electrostatic interaction driven strategy is potentially generic and can be extended to crystallize other inorganics such as calcium carbonate and metal oxide with various optical, electrical, magnetic properties, as long as they can be synthesized within the tolerance range of DNA frameworks.</p><!><p>Calcium chloride (CaCl 2 ) and sodium phosphate dibasic dodecahydrate (Na 2 H-PO 4 $12H 2 O) were purchased from Sigma-Aldrich. All DNA short strands without modification were purchased from Jie Li Biology Co., Ltd (Shanghai, China). Cy3modified DNA strands were purchased from Sangon Biotech (Shanghai) Co., Ltd. M13mp18 single-stranded DNA (N4040S) was purchased from New England Biolabs. Other chemicals were purchased from Sinopharm and Sigma-Aldrich. Carbon-coated TEM grids were purchased from Beijing Zhongjingkeyi Technology Co., Ltd (Beijing, china). Amicon Ultra-0.5 mL/15 mL centrifugal filters were purchased from Merck Millipore Ltd. Tullagreen, Carrigtwohill Co., Cork, Ireland. All AFM tips were purchased from Bruker. All the regents were used as received without further purification. Mineralization of DNA Origami on Substrate DNA origami was first absorbed onto substrates (e.g., glow-discharged carbon-filmcoated copper grids and freshly cleaved mica) for 3 min. The substrates were rinsed by Tris-CaCl 2 buffer (20 mM Tris, 2.0 mM CaCl 2 [pH 9.0]) and placed face-down upon the same buffer in an EP tube. Next, Na 2 HPO 4 (5 mM) was added slowly to the solution in the tube, while pH was kept at 9.0 by dropwise addition of 0.01 M NaOH solution. The concentration of final Ca free was kept at $1.0 mM. The samples were then shaken at 300 pm for 30 min before being transferred to incubator under 37 C for 24 h.</p><!><p>For TDN, polyacrylamide gel (8%) in 13TAE-Mg 2+ buffer (40mM Tris, 2mM EDTA, 12.5mM MgAc 2 , pH 8.0) was running on electrophoresis systems using ice-water bath. Gels were stained with 13GelRed before imaging. For DNA origami, 0.5% agarose (BioRad) gel containing 13GelRed in 13TAE-Mg 2+ buffer was running on electrophoresis systems using ice-water bath.</p><!><p>A drop (10 mL) of TDN-CaP dispersion was dripped onto a glow-discharged carbonfilm-coated copper grid. After adsorption for 3 min, the copper grid was rinsed by deionized water and dried under 25 C. For DNA origami, the copper grids were washed and dried in the same way. Imaging was performed on FEI Tecnai G2 F20 S-TWIN TEM (200 kV) and FEI Talos F200X G2 TEM (200kV).</p><!><p>STEM images were obtained by a FEI Magellan 400 SEM. All CaP NCs were loaded directly on to copper grids without coating.</p><!><p>For TDN-CaP NCs, the sample solution (2 mL) was piped onto freshly cleaved mica. After adsorption for 3 min, the mica substrate was washed by deionized water and dried by compressed N 2 gas. For DNA origami, the mica placed upon the reaction solution were washed and dried in the same way. AFM images were obtained either by ''Peak Force QNM in air'' mode with Scanassyst-air tips (Bruker) or ''Peak Force QNM in solution'' mode with fluid+ tips (Bruker), using a Multimode Nanoscope VIII instrument (Bruker).</p><!><p>SAXS experiments were performed on beamline BL19U2 of the National Center for Protein Science Shanghai (NCPSS) at Shanghai Synchrotron Radiation Facility (SSRF). The wavelength (l) of the X-radiation was set at 1.033 A ˚. Scattered X-ray intensities were measured by a Pilatus 1 M detector (DECTRIS Ltd). The sample-to-detector distance was 2,125.0 mm for the current measurements, it was set such that the detecting range of momentum transfer q (q = 4psinq/l, where 2q is the scattering angle) of the SAXS experiments was 0.01-0.5 A ˚À1 , which corresponds to an investigated length scale from 10 to 600 A ˚. The solution temperature was held constant at room temperature, 25 C. The volume of each sample was 100 mL, with TDN concentration of 1-6 mM. To obtain optimal signal-to-noise ratio, 20 frames were recorded for each sample and its buffer. The 2D scattering images were converted to one-dimensional (1D) SAXS curves through azimuthal averaging after solid angle correction then normalized to the intensity of transmitted X-ray beam using BioXTAS RAW 1.2.1. The data were further analyzed by ATASAS 2.7.2 package and Fast-SAXS-Pro. 29 Briefly, the fitting was achieved by initially performing NMA sampling on MD-generated models, and then structural energy minimization, followed by evaluation via goodness-of-fit (denoted by c 2 ) between theoretical and experimental scattering data, where each theoretical SAXS curve was calculated by fast-SAXS-pro, which specially accounts for the hydration characteristic of DNA. The fitting results were also validated by CRYSOL and FoXS. [43][44][45] Cell Culture RAW264.7 macrophage-like cells were grown in RPMI 1640 medium supplemented with 0.15% NaHCO 3 , 10% heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin, 100 mg/mL streptomycin, and 2 mm L-glutamine at 37 C in humidified air containing 5% CO 2 .</p><p>Confocal Microscopic Imaging RAW264.7 cells were seeded on glass coverslips in 24-well culture plates at a density of 5 3 10 5 cells/mL and incubated under 37 C for 24 h. They were then washed twice with phosphate buffer (PBS) and incubated with Cy3-labeled TDN-CpG or TDN-CpG-CaP in fresh RPMI 1640 medium for 6 h at 37 C. Then, cells were washed twice with PBS and fixed with 3% paraformaldehyde-sucrose. The coverslips were mounted on a glass slide. All images were obtained using a laser confocal microscope (Leica TCS SP5). Wavelength sets were 561 nm ex/565-600 nm em.</p><p>Uptake of TDN, TDN-CpG, and TDN-CpG-CaP by RAW264.7 Cells RAW264.7 cells were seeded on 24-well culture plates at a density of 5 3 10 5 cells/mL and cultured for 24 h and then washed twice with PBS. They were incubated with Cy3 labeled TDN-CpG, TDN-CpG-CaP for 6 and 48 h at 37 C, then harvested and washed three times with PBS. The fluorescence intensity of the cells was determined by flow cytometry (FACSCarray; BD Biosciences, San Jose, CA, USA).</p><p>Cytokine Assays RAW264.7 cells were washed twice with 0.5 mL of PBS. Then they were incubated with CaP, TDN, TDN-CaP, and TDN-CpG-CaP at 37 C for 8 h (for TNF-a analysis), and the supernatants were collected and stored at À80 C before use. The levels of TNF-a in the supernatants were determined by enzyme-linked immunosorbent assay (ELISA) using antibody pairs specific to these cytokines (eBioscience, antimouse TNF-R pair, Cat. nos. 14-7,325 and 13-7,326; antimouse IL-6 Pair, Cat. nos. 14-7,061 and 13-7,062; antimouse IL-12 pair, Cat. nos. 14-7,125 and 13-7,123), following protocols recommended by the manufacturer.</p><!><p>Statistical differences of data were evaluated by one-way analysis of variance (ANOVA) followed by the Tukey's post-hoc test for multiple comparisons. A p value of less than 0.05 was considered to be statistically significant.</p><!><p>Cytotoxicity was evaluated by an MTT assay. RAW264.7 cells were seeded in 96-well plates and cultured overnight to reach $ 80% confluency. Fresh media containing CaP or TDN-CpG-CaP were incubated with cells for 24 h. Then, 20 mL of 5 mg/mL thiazolyl blue tetrazolium bromide (MTT, Sigma-Aldrich, USA) solution was added to each well, followed by 4 h incubation at 37 C. Next, cells were lysed with 10% acid SDS solution (pH 2-3). After centrifugation, the absorbance of supernatant was measured at 490 nm using a microplate reader (Bio-Rad 680, USA).</p>