文档一翻译

A Introduction.动物病毒选为药物载体

然而在人体内拥有基因的动物病毒有可能自发突变和自我复制。

Animal viruses具有特异性因为他们能够被受体细胞认出。 that can be naturally recognized by cell receptors of specific types of cells8,23 are of particular interest because this increases the specificity of the nanoparticle itself for uses in biomedical applications or as functional imaging probes. To this end, we have selected alphaviruses as a platform for nanoparticle delivery.

α病毒Alphaviruses are small, icosahedral, enveloped viruses that cause disease in humans and domestic animals.24类核病毒粒子的离体自组装能力使其具有良好的生物纳米科技方面的应用。

With respect to the viruses discussed by Singh et al.,8 α病毒从介质受体的胞饮（endocytosis）作用开始，在细胞表面与受体成键的病毒被带入（细胞内部）（endosomes）细胞内部表现出来的较低的ph值有助于病毒表面蛋白质的溶解。The ensuing fusion between the viral and the endosomal membranes allows the penetration of the viral core into the cytoplasm through a so-called fusion pore.29 A large number of viruses infect cells by this route (see ref 29 for a review). The endocytotic pathway distinguishes the alphavirus from plant and bacteriophage virus platforms tested for biomedical nanotechnology applicatons.

Cryo-EM imaging and 3D image reconstructions confirmed that alphaviruses contain a lipid bilayer sandwiched between an external glycoprotein membrane and an internal nucleocapsid core. Cryo-EM 成像技术 3D影像技术重建证实了α病毒夹在外面的糖蛋白膜和内部核衣壳之间的脂类双层的存在。The two concentric protein shells are interconnected across the lipid membrane with pseudo-T = 4 icosahedral symmetry.两个蛋白质壳通过脂类膜相互连接。 The nucleocapsid self-assembles from 240 identical protein subunits and measures 39 nm, while the inner cavity encapsulating the genome has a diameter of 33 nm。核酸衣壳由240个相同的蛋白质亚基自组装而成并有39nm，包裹有基因的内腔直径约为33nm。The genomic RNA, 11.7 kb in length, is encapsulated within the nucleocapsid。基因组RNA，长11.7kb，被核衣壳包裹在内。The cDNA sequence of many of these viruses is known, making them ideal for vector expression models for gene/drug delivery and cancer therapy and neuron chemical synapses。许多病毒的cDNA序列已经得知，使得它们成为基因/药物传送和癌症治疗和神经元化学接合的理想表达模型。In this context, alphaviruses represent an ideal system because the nucleocapsid core forms independently from the glycoprotein shell in vivo and the nucleocapsid assembly can be reconstituted in vitro. 本文α病毒代表了一种理想系统：其核衣壳中心可以从活体糖蛋白壳中出来合成的，并且核衣壳可以在体外进行重组。Furthermore, in vivo, alphaviruses can be specifically engineered through site-directed mutagenesis of the glycoprotein E2 to exhibit specific targeting activity toward specific receptors in animal models. 而且在活体内，α病毒能

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够通过糖蛋白E2的位点突变进行特定的设计以显示出对动物模型的特定受体具有特定的选择性。

To make an alphavirus-based molecular container, one has to gather insight from the principles of the native virus assembly. 为制造α病毒基的分子容器，必须将现有病毒组装的各项指标加以考虑。Unfortunately, the exact mechanism of in vitro corelike particle (CLP) formation is still unclear and the preparation of infective virus particles in laboratory still requires host cell involvement。 不幸的是，离体corelike particle (CLP)合成的具体原理还是不够清晰，并且实验室中具有传染性的病毒粒子的制备仍需要宿主细胞的参与。Previous research on CLP assembly has focused on the C-terminal domain of the capsid protein (CP). CLP组装的先前的研究主要集中衣壳蛋白的C末端区。 The N-terminal region is believed to have a random coil organization with the exception of a helical sequence. N端区域被认为除了具有一个螺旋序列外其余是胡乱卷曲的结构。As a marked difference from many other in vitro assembly systems，alphavirus CLPs are believed to form only in presence of single-stranded nucleic acid. α病毒CLPs与其他离体组装体系最显著的区别被认为是只有在单链核酸存在下才能进行。Absence of nucleic acid or ds-DNA inhibits CLP assembly.25 缺少核酸或者ds-DNA会抑制CLP的组装。It should be noted, however, that CLPs can also form with tRNA, suggesting the structure of RNA may have more of a role than the sequence.然而我们应该注意CLPs也可以利用tRNA合成，这也意味着RNA结构有可能不单单是序列的角色。

In this work, we exploit in vitro CLP assembly features to form particles that encapsulate a functionalized gold nanoparticle (GNP). Gold nanoparticles have been chosen to test the idea of encapsulation because of their well-known surface chemistry10,21 and because of their characteristic optical properties.在此项工作中，我们利用离体组装CLP的特征合成包裹功能化金纳米粒GNP的粒子。金纳米粒的表面化学稳定性和典型的光学特性。

Two questions are being addressed: The first question is

whether nonspecific electrostatic interactions plus the rigidity of a spherical nanoparticle template of different sizes is sufficient to bypass the specific interaction requirement for the in vitro assembly of CLPs. 现在有两个问题，第一个是非特异性的静电作用加上不同尺寸的球形纳米粒子模板的刚性是否可以绕过CLPs离体组装需要的特定反应。The nanoparticles were functionalized with different ligands, Figure 1: methoxy PEG ligands, negatively charged phosphane molecules, and specific and unspecific sequences of 5‘ thiol-modified ss-DNA. Reassembly reactions and product analysis were carried out as a function of core size and ligand. 纳米粒子由不同的配体进行官能化，Figure 1: 甲氧基化的聚乙二醇配体，带负电的磷酸盐分子，特异性或非特异性的5-硫醇改性的ss-DNA。重组反映和产品分析作为核心尺寸和配位体的函数实施的。

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Figure 1 Functionalization of Au nanoparticles. (A) phosphane. (B) Phosphane and a single strand of unspecific 30-mer DNA (1 equiv DNA:1 equiv Au). (C) Mixed monolayers of 48-mer DNA and phosphane molecules (2200 equiv DNA:1 equiv Au). (D) Mixed monolayers of 30-mer DNA and OMe-PEG molecules (750 equiv DNA:1 equiv Au).

The second question is whether an increase in stability could be imparted 给予，增加from the interior 内部of the nucleocapsid through the incorporation合并 of a solid spherical template. 第二个问题就是通过固体球形模板合并的核衣壳的内部是否可以达到稳定性的增加。In this context, Forsell et al.48 have suggested that the outer membrane glycoprotein network has a direct role in stabilizing and organizing the structure of the mature alphavirus particle. 在此情况下，Forsell et al.表明膜外糖蛋白网络在稳定和组织成熟的病毒粒子方面起了直接作用。In the absence of membrane and glycoproteins, nucleocapsid core particles are fragile, lacking accurate icosahedral symmetry.28 在缺少膜和糖蛋白的情况下，核衣壳中心粒子是脆弱的，不具有精确的二十面体结构。More stable nucleocapsids should provide a broader range of experimental conditions to choose from upon subsequent（随后的）attempts of adding lipid membranes and glycoproteins. 更稳定的核衣壳应该提供一个更加宽松的的实验条件，并根据随后脂质膜和糖蛋白的添加而加以选择。

The main findings discussed here are: (1) an anionic nanoparticle core is sufficient to promote the association of alphavirus CP with the core, (2) electrostatically driven core−CP association occurs regardless the size of the nanoparticle template, and (3) among the tested ligands, maximum efficiency of encapsulation has been obtained when GNPs were functionalized with a mixture of phosphane and 48-mer ss-DNA. 讨论的主要结论是：（1）一个阴离子NP核可以有效改善病毒CP与核的结合，（2）静电驱动使得core-CP的结合呈现出与NP模板的尺寸无关，（3）在检测的配位体中，包覆效率最大时是当膦与48-mer ss-DNA 官能化GNPs时获得的。In this case, the assembly protocol yielded spherical capsids similar in shape and size to the wild type (wt) CLPs. Moreover, the Au-encapsulating CLPs were more stable over time than wt CLPs. 在这种情况下，形成球状衣壳的组装方法，Interestingly, we found that the presence of free 48-mer ss-DNA in solution significantly increases the yield of Au−CLP production, which may indicate that nucleoprotein precursors have to form prior to CLP self-organization.有趣的是，我们发现在溶液里free 48-mer ss-DNA的存在增加了Au−CLP的产量，这有可能表明核蛋白前驱体不得不在CLP自组织之前合成。

Materials and Methods. 材料和方法I. Synthesis of Functionalized Gold Nanoparticles. 官能化金纳米粒子的合成。GNPs of different sizes were prepared by citrate reduction of HAuCl4.

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不同尺寸的GNPs由HAuCl4 的减少得到的。Exchange reactions with different coatings were performed according to established methods described below.不同膜的交换反应是根据下面介绍的已知的方法进行的。

Exchange of Citrate−Au NPs (14 nm) with Carboxylated PEG Ligand (PEG)−COOH. PEG-coated GNPs通过添加过量的硫醇基 (10 equiv/particle) to citrate−Au NPs solution（10当量）The number of equivalents was estimated by assuming that the occupied surface area by a single thiol molecule is 0.2 nm。混合物搅拌24h。混合物用三体积的四氢呋喃稀释，同时消除未反应的，离心。移去浮在表面的杂质，并将微粒溶解在PH=3的酸性水溶液中，相同条件下进行第二次离心。最终，微粒溶于中性水中，悬浮物超声5min，以确保金络合物能完全分散在溶液中。

Exchange of Citrate−Au NPs (10 nm) with Methoxy PEG ligand (PEG)−Ome。简单步骤：10 equiv PEG/particle加到citrate-stabilized GNPs solution柠檬酸盐稳定化的GNPs溶液中，室温下搅拌20h。加入3体积的THF净化混合物，离心。移去表面物质，将微粒再次溶解于10ml的纯水核30ml的THF中，相同条件下再次离心，再将微粒溶于纯水中，超声5min以使得金络合物完全分散在溶液中。

Exchange of MeO−PEG−Au NPs (10 nm) with 30-mer 5‘ Thiol-Modified ss-DNA. Generation of a mixed layer containing ss-DNA thiolated at their 5‘ end and PEG−OMe on the gold surface was obtained from a small modification of Zhu's protocol.51 A small amount of surface-attached PEG molecules were exchanged with thiolated ss-DNA using 0.5 DNA equiv/1 PEG equiv (750 DNA equiv/particle).51 For simplicity, these molecules will be termed as 30-mer DNA/MeO−PEG−Au NPs.

Exchange of Citrate−Au NPs (8.3−27.4 nm) with Bis-(p-sulfonatophenyl)phenyl Phosphine Dehydrate. Bis-(p-sulfonatophenyl)phenyl phosphine dehydrate ligands (1) were exchanged from citrate−Au NPs according to Loweth's protocol.52 Aqueous solutions (10 mL) of citrate−Au were stirred overnight with a large excess of ligand (20−40 mg) at room temperature. Addition of NaCl induced GNP precipitation followed by separation through centrifugation at 16000g for 40 min and 4 °C. After a second work up, the final pellet was resuspended in a small volume (200 μL) of phosphane solution (25 mg of 1 in 100 mL of water). These particles are termed p−Au nanoparticles.

Exchange of Phosphane−GNPs (10 nm) with a Single Strand of 30-mer 5‘ Thiol-Modified ss-DNA. Exchange reactions of phosphane−Au NPs with a single ss-DNA modified with a thiol group at its 5‘ end HS-(CH2)6OPO3-5‘-GTCTTCCGCTCT CGGCAGAGGTGTGAAGGA-3‘) were performed according to Zanchet's protocol.53 Briefly: DNA/phosphane−Au conjugates were prepared by adding 1.1 DNA equiv per particle to phosphane−Au NPs dispersed in 1 mL of 0.5× TBE buffer. To promote the exchange of ligands, the electrostatic repulsion between DNA and

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phosphane molecules must be lowered by increasing the ionic strength of the reaction mixture (10 μL of 2.5M NaCl) to reach 50 mM. After shaking for a few seconds, the mixture was allowed to stand for 2 h before concentrating the volume for further use. For simplicity, these particles will be named as one 30-mer DNA/p−Au nanoparticles.

Exchange of Phosphane-GNPs (8.3−27.4 nm) with 48-mer 5‘ Thiol-Modified ss-DNA. Generation of a mixed monolayer containing ss-DNA modified with a thiol group at their 5‘ end (HS-(CH2)6OPO3-5‘-CCGTTAATGCATGTCGAGATATAAAGCATAA

GGGACATGCATTAACGG-3‘) and phosphane molecules on gold surface was obtained as described in Parak et al. with the following modifications. Prior to DNA exchange, the ionic strength of the phosphane−Au NPs solution was increased to 50−100 mM by adding 1M NaCl. This initial step is crucial to minimize the electrostatic interactions between the phosphate backbone of DNA and the phosphanes on the nanocrystal surface. At the same time, the ionic strength jump promotes reversible GNP flocculation. Thus, the addition of salt is enough to promote thiol attachment while avoiding irreversible aggregation of the nanoparticles. Addition of a large excess of ss-DNA to phosphane−Au nanoparticles (2200 equiv per particle) was used to ensure saturation coverage in ss-DNA. The number of equiv was calculated by assuming that the occupied surface area by a single thiol molecule is ca. 0.20 nm。

After thiol addition, the reaction mixture was left stirring for 48 h at room temperature, long enough to ensure maximum replacement of oligonucleotides.,55 The unreacted ligands were then removed using ultracentrifugation. Subsequent purification was carried out using microcon ultrafilters of 60 kDa nominal molecular weight cutoff (14000g for 45−75 min). The resulting reddish pellet was redissolved in tris-borate-EDTA buffer (TBE 0.5×). For simplicity, these particles will be named 48-mer DNA/p−Au nanoparticles.

II.

Characterization.表征 Particle (CP or gold particle) concentration and coat

composition were obtained by UV−vis absorption using a Nanodrop ND-1000 UV−vis spectrophotometer. To prevent saturation, 50× diluted aliquots of the master colloidal solutions were used for analysis. The plasmon bands of the differently sized particles (8.3−27.4 nm) were between 520 and 526 nm, as expected.

粒子浓度和包覆结构是由UV−vis absorption获知的。为避免饱和，分析的是50倍稀释溶液，不出所料，不同尺寸的粒子（8.3-27.4）的基因组在520-526nm。

Dynamic light scattering (DLS, Zetasizer NanoS, Malvern) of functionalized GNPs in TBE buffer was performed to determine the hydrodynamic size increase after functionalization and

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after Au−CLP assembly. Prior to DLS measurements, all solutions were filtered using a 0.1 μm syringe filter and sonicated for 30 min to remove large aggregates that would otherwise dominate the scattering, even if in small numbers, due to their large scattering cross section. In the case of DNA/p−Au−CLP, the samples were sonicated in cold water (6 °C) for 30 min and the DLS measurements were taken at 8 °C.

functionalized GNPs的动态光散射（DLS）用来确定官能化核Au-CLP组装后流体力学尺寸的增加，DLS处理前，所有溶液都经过0.1um的过滤器过滤，声波处理30min以移除，大的粒料，不然因为其具有较大的散射截面，会影响粒子的分散，哪怕数量很少。而对于DNA/p−Au−CLP而言，样品先在冷水中（6℃）进行声波处理30min，然后在8℃进行DLS。

Gel electrophoresis was used to evaluate the purity of the DNA/p−Au conjugates. The matrix （基体）was made of 3% agarose gel and EtBr (10 mg/mL) in 1× TBE buffer (0.09 M Tris:0.09 M Boric acid:2 mM EDTA; pH 8.3). To have a clear visualization of the gold bands, use of concentrated samples (100 nM) is required.

凝胶电泳用于评估DNA/p−Au conjugates.的纯度。基体由3%琼脂糖凝胶和溴乙烷（10mg/ml）in 1× TBE 缓冲液(0.09 M Tris:0.09 M Boric acid:2 mM EDTA; pH 8.3).为获得清晰地金基的视图，需要利用浓缩的样品（100nm）。

Transmission electron microscopy (TEM) data were obtained on a JEOL 1010 equipped with a 4k CCD camera operating at 80 kV. For TEM imaging, the samples were unstained or negatively stained with 1% uranyl acetate. Electron micrographs of gold nanoparticles and Au−CLP were taken by transferring 10 uL of solution to a carbon-coated Formvar copper grid (200 mesh). Solution excess was removed after 20 min and stained (in case of Au−CLP) for another 20 min. The stain excess in the grid was removed with filter paper and the grid was visualized by TEM. Au−CLPs diameters and efficiencies were determined using two or more grids independently prepared, with at least 800 particles imaged per grid.

透射电子显微镜法（TEM）数据were obtained on a JEOL 1010 equipped with a 4k CCD camera operating at 80 kV。为TEM成像，样品不进行染色或者用1% uranyl acetate醋酸双氧铀进行负电荷染色。金纳米粒子核Au-CLP的电子显微图是通过转移10ul的溶液到炭包裹Formvar（聚醋酸甲基乙烯脂）的200目(200 mesh)的合金栅格里，20min后移除过量溶液并进行染色(in case of Au−CLP)20min。栅格中过量的染色剂利用滤纸移除，TEM下观察栅格。Au−CLPs的尺寸和效率由利用两个或者更多的栅格independently prepared,每个栅格至少800粒子。

III. Efficiency of the Gold Core Size in Promoting 48-mer DNA/p−Au−CLP Assembly. 金核尺寸在改善DNA/p−Au−CLP 组装方面的效率。

The encapsidation efficiency was calculated using the following equation:

NAu-CLP represents the total number of 48-mer DNA/p−Au−CLPs assembled,

while NAu corresponds to the total number of functionalized GNPs at a constant CLP:Au initial

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ratio of 1:1 equiv. 包覆效率可利用下面这个方程计算NAu-CLP。

Encapsulation efficiency studies were carried out only with 48-mer DNA/p−Au particles because of their good stability and monodispersity after assembly with CP. 包覆效率的研究是通过48-mer DNA/p−Au particles 进行的，因为他们具有良好的稳定性能和CP组装后的单分散性能It is worth noting here that the way efficiency of encapsulation is defined implies that 100% efficiency would be achieved when all the protein would assemble into Au-filled capsids. 值得注意的是，这暗含着在蛋白质组装到Au-filled衣壳应该得到包覆效率100%的效率。The amount of protein that has associated in nonproductive ways (or precipitated) can be estimated from the fraction of Au particles that are not encapsulated in a spherical shell. 和蛋白质的副产品相关的蛋白质数量可以通过未包裹球形壳的Au粒子份数估算得到。

A completely amorphous protein layer adsorption is not expected to show correlations in the encapsulation efficiency with size. However, size-dependent encapsulation can be an indicator of closed-shell formation (the occurrence of encapsulation is expected to decrease when the size of the gold particle exceeds the size of the cavity, for a given T-number). This is why studies of efficiency of encapsulation vs core size have been carried out.

我们并不期望（预计不会出现）完全无定形的蛋白质层表现出尺寸与包裹效率的相关性。然而，与尺寸相关的包裹可用以表明封闭壳的形成。This is why studies of efficiency of encapsulation vs core size have been carried out.

IV.

In Vitro CLP Assembly with 48-mer Single-Stranded Nucleic Acid and

Characterization.

CLP与48-mer Single-Stranded Nucleic Acid一起，离体组装和表征。

In vitro CLP assembly was performed as described before25,28 with slight modifications. Briefly, RRV CP was expressed in pET-29b (+) DNA plasmid (Novagen) in Rosetta2 cells until the OD600 was 0.4−0.6. At that time, cells were induced with 1 mM ITPG and grown at 37 °C for 4 h before being harvested. Cells were lysed by French press, and the clarified lysate was applied to a High Trap SP FF 1 mL column. The protein was eluted with 500 mM NaCl in 20 mM Hepes and 2 mM EDTA, pH 8.0, concentrated, and buffer exchanged into HNE (20 mM HEPES:0.15 M NaCl:0.1 mM EDTA; pH 7.5). Protein concentrations were determined by the Bradford assay technique.

RRV CLPs with 48-mer DNA were formed under conditions described previously25,28 in TNKM assembly buffer (50 mM Tris-HCl:50 mM NaCl:10 mM KCl:5 mM MgCl2; pH 7.4). This buffer was successfully used in the assembly of GNPs with brome mosaic virus (BMV) capsid proteins, and it did not cause nanoparticle aggregation as seen with other assembly buffers. This

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change was not expected to affect the in vitro assembly of alphavirus CLP assembly since it was previously shown that a wide selection of assembly buffers is available for capsid formation.25,28 The formation of CLPs was characterized by gel electrophoresis and TEM as described previously.25 DLS and TEM measurements of RRV CLPs were compared with structural data of wt nucleocapsids.28,30,31,33,40,41,57,58

与48-mer DNA 一起RRV CLPs的合成是在TNKM组装缓冲液（50mM Tris-HCl:50 mM NaCl:10 mM KCl:5 mM MgCl2; pH 7.4）。这种缓冲液成功的利用于在与BMV衣壳蛋白质一起GNP的组装，而且，它不会像其他缓冲液一样引起NP的聚集。由于病毒衣壳合成的所需要的缓冲液具有一个较宽的选择范围，因此这种变化预计不会影响α病毒CLP的离体组装。CLPs的合成由凝胶电泳和TEM进行表征。RRV CLPs的DLS 和TEM测量用以和wt nucleocapsid的结构数据进行比较。

V. Functionalized Au−CLP Assembly and Characterization Protocol. 官能化Au-CLP组装和表征草案

For the self-assembly of CP around functionalized GNPs, we used 240 equiv of capsid protein monomers per functionalized GNP (corresponding to a Τ = 4 nucleocapsid) in TNKM buffer. GNP concentrations were determined by UV−vis spectroscopy, and protein concentrations were determined as mentioned above. Typical protein concentrations were 0.3−0.4 mg/mL and gold nanoparticle solutions were in the 100−800 nM range. Before assembly, the gold conjugate in 0.5× TBE buffer was sonicated for 30 min in cold water (8 °C). The required volume of CP in HNE buffer (described above) was then added to the nanoparticle solution. This step was immediately followed by the addition of the same volume of assembly buffer.

包覆官能化GNPs的衣壳蛋白质的自组装是利用在TNKM缓冲液中每个官能化GNP(corresponding to a Τ = 4 nucleocapsid)上240当量的衣壳蛋白单体。GNP聚集度由紫外可见光谱确定的，蛋白质聚集度的确定方法之前提过了。典型的蛋白质聚集度为0.3−0.4 mg/mL，GNP溶液in the 100−800 nM range。组装之前在0.5× TBE缓冲液中金络合物在8℃冷水中声波处理30min。在HNE缓冲液中CP量添加到了NP溶液中。这个步骤后紧跟着就是相同量的缓冲液的添加。

Reaction mixtures were incubated for 10 min at room temperature. Au−CLP solutions were stored at 4 °C. The formation of Au−CLPs was characterized by negative stain TEM and DLS. Prior to DLS measurements, all solutions were sonicated for 30 min to redisperse all possible large aggregates. TEM diameters were estimated using at least two grids, and DLS values were corroborated with three different preparations in each case.

反应混合物在室温下孕育10min，Au-CLP溶液在4℃下贮存。Au-CLP的结构由经过负染色后TEM和DLS表征。DLS测量之前，所有溶液声波处理30min以使尽可能所有的大的聚集物分散开来。TEM直径是利用至少两种栅格估算，DLS values were corroborated with three different preparations in each case. DLS值由三种不同的制备方法证实。

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Results and Discussion. I. Influence of Electrostatics in Templating Au−CLP Formation. As a control for our nanoparticle assembly protocol, we formed RRV CLPs with 48-mer DNA, as previously described.25,28 The assembly reaction yielded particles of 36.9 ± 5 nm (800 particles), as measured by TEM (Figure 2A), in agreement with the literature.28,30,31,40,41,57,58 For clarity, it is important to note that the reported dimensions for the wt nucleocapsid core range from 3834,57 to 40 nm31,40,41 in diameter while the entire virion measured between 6834,58 and 71 nm31,41 in diameter.

模板合成Au-CLP中静电影响。

作为NP组装草案的一个控制因素， 与48-mer DNA 一起RRV CLPs的合成之前已经介绍过。组装反应产生的（36.9± 5 nm）粒子，由TEM测量(Figure 2A)得的，与文献中的尺寸一致。清楚点说，值得注意的是报道的the wt nucleocapsid core直径在38-40nm之间，

Figure 2 In vitro assembly of RRV CLPs. (A) Negatively stained TEM images of RRV CLPs measured 36.9 ± 5 nm. (B) Scattered light of the nucleocapsids (28 nm) reveals an increment in the hydrodynamic diameter compared to RRV CP and single strands of 48-mer DNA.

The DLS data indicated an average hydrodynamic diameter of 28 nm. The smaller sizes detected by DLS (Figure 2B) compared to TEM could be the result of averaging between intact CLPs, side products from the CP expression system, and protein intermediates unable to self-assemble into CLPs. It is interesting to note that viral particles that have been used in nanotechnology include CPMV, CCMV, CPV, and MS2, all of which have a diameter between 25 and 30 nm3. The significance, if any, of this size consistency is unknown.

To form RRV CLP encapsulating nanoparticles, we used negatively charged, 12 nm diameter, phosphane-coated GNPs (p−Au). The p−Au GNPs were mixed with 1 equiv of RRV CP to prepare p−Au−CLPs.

Negatively stained electron micrographs of p−Au and RRV CP clearly indicate that protein adsorption onto the nanoparticle surface process had occurred, Figure 3A. This is not surprising because RRV CP is basic, especially in the N-terminus region, with the entire protein having a pI > 9.5. The average diameter of the protein shell measured by TEM was 32 ± 4 nm (400 particles). The difference in thickness of the light shell seen around the GNPs before and after the encapsidation protocol indicates the deposition of the positively charged CP around the negatively charged p−Au. However, the average thickness of the CP shell (10 nm) in p−Au−CLPs was

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greater than the expected thickness of the wild type nucleocapsid (5 nm).28,31 The thick protein packing in p−Au−CLP suggests either protein multilayers around gold or a denaturated protein layer, at least in the conditions required for TEM analysis.

Figure 3 Negative staining TEM images of RRV CLPs: (A) 9 nm Au core diameter, P−Au−CLP diameter 32 ± 4 nm; (B) 10 nm Au core size, one 30-mer DNA/phosphane−Au−CLP diameter 29 ± 3.5 nm; (D) 11.5 nm Au core diameter; (C) 10 nm Au core size, 30-mer DNA/OMe−PEG−Au−CLP (diameter: 28.3 ± 7 nm); (D) 48-mer DNA/p−Au−CLP (diameter: 38 ± 3.7 nm).

TEM imaging of 48-mer CLP and p−Au−CLPs showed more irregular CLP formation compared to 48-mer CLPs (Figure 2A vs Figure 3A). This observation suggests possible denaturation caused by hydrophobic direct interactions of the CP with the metallic surface. In addition, DLS experiments on p−Au−CLPs resulted in an average particle diameter of 34 nm (Figure 4). In this case, DLS gave a large diameter particle than TEM, contrary to what was seen with other samples. Compared to the 28 nm diameter obtained for RRV CLPs, this result is consistent with p−Au−CLP having multiple protein layers or the DLS, reflecting the average of multiple species present in the heterogeneous mixture.

Figure 4 DLS size distribution of various functionalized GNPs (9−11.5 nm) after assembly with RRV CP. The hydrodynamic radius of one 30-mer DNA/p−Au−CLP, 48-mer DNA/p−Au−CLP, and p−Au−CLP is shown as a function of the Au surface coating after assembly; 30-mer DNA/p−Au−CLPs measured 29 nm by TEM and DLS, but 48-mer DNA/p−Au−CLPs measured 33 nm by DLS and 30 nm by TEM. p−Au−CLPs measured 32 nm by TEM and 34 nm by DLS.

We deduce that, although p−Au−CLPs are unlikely to be similar to wt nucleocapsids, a first step of electrostatic recruitment of the protein by the anionic core does occur and it results in complexes that have protein multilayers or a disorganized protein shell.

II. Influence of Partial and Complete ssDNA Templating in Au−CLP Formation. To test the hypothesis that CLP formation may be dependent on an initial binding and nucleation event between nucleic acid and capsid protein, a single 30-base ss-DNA modified with a thiol group at its 5‘ end (HS-(CH2)6OPO3-5‘-GTCTTCCGCTCTCGGCAGAGGTGTGAAGGA-3‘)) was coupled to the p−Au particle. The new core is termed here 30-mer DNA/p−Au−CLP. The product

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of the CP association with p−Au conjugates containing only one ss-DNA was a one 30-mer DNA/p−Au−CLP of 29 ± 3.5 nm in average (as measured by TEM and confirmed by DLS) (Figure 3B).

TEM images (Figure 3B) of one 30-mer DNA/p−Au−CLPs reveal the formation of incomplete or deformed CLPs and/or inability of the CP associated with the core to rearrange into a closed shell. To determine if the CP shell symmetry will improve by coating the entire p−Au core with a uniform layer of 30-base ss-DNA instead of a single strand, we have coated the Au particles with a monolayer of 30-base ss-DNA and thiol methoxy PEG (1 Au:748 DNA:748 PEG). These cores are termed 30-mer DNA/OMe−PEG−Au.

Addition of CP to these conjugates resulted in 30-mer DNA/OMe−PEG−Au−CLPs (Figure 3C) measuring 28.3 ± 7 nm (by TEM, 400 particles counted). The white protein ring was around 6 nm thick. This is close to the expected wt protein shell thickness and suggests a single protein layer around the gold surface.

Visually, the 30-mer DNA/OMe−PEG−Au−CLPs were more spherical and the ensemble more homogeneous when compared to the case of one 30-mer DNA/p−Au−CLPs. However, the number of encapsulated particles seen on TEM (40 out of a total of 500 imaged particles) indicated a low efficiency of incorporation, which might suggest that a minimum surface charge density as well as DNA−CP interactions throughout the entire surface capsid/core interface are required for encapsulation. This minimum charge density requirement may not be met by 30-mer DNA/OMe−PEG−Au nanoparticles due to the fact that the thiolated methoxy PEG is not charged and it carries only the role of hydrophilic protectant of the Au surface.

Therefore, the unspecific DNA sequence (30-mer) has been replaced by an alphavirus specific DNA (48-mer) sequence and we used phosphane-coated GNPs instead of pegylated GNPs. Phosphane molecules are smaller than thiolated PEG molecules and therefore offer less steric hindrance when thiolated 48-mer DNA ligands are used for exchange. The anionic charge density is also expected to be higher.

III. DNA-Saturated Nanoparticle Templates. A nanoparticle template was constructed by coating GNPs with 48-base ss-DNAs modified with thiols at the 5‘ end: (HS-(CH2)6OPO3-5‘-CCGTTAATGCATGTCGAGATATAAA-

GCATAAGGGACATGCATTAACGG-3‘), which resembles the alphavirus encapsidation signal

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from the genomic RNA.46 This sequence is predicted to have a stem loop with CP affinity in its secondary structure.46

Addition of RRV CP to these conjugates in solution produced homogeneous 48-mer DNA/p−Au−CLPs (Figure 3D) measuring 38 ± 3.7 nm in diameter (800 particles counted), which is close to the CLP dimensions measured by TEM (Figure 2A). Negatively stained TEM images of 48-mer DNA/p−Au−CLPs show a clear 13 nm white ring assembled around the 48-mer DNA/p−Au, from which 5 nm corresponds to the naked 48-mer DNA/p−Au conjugates. A comparison between the size of these conjugates before and after the assembly protocol suggests that the space occupied by the CP is around 7 nm, which is similar to the wt.28,30,31,33,40,41,57,58 In addition, the DLS measurement gave a diameter of 33 nm for the encapsidated particles compared to 38 ± 3.7 nm from TEM. Smaller hydrodynamic radii are accounted for by the presence of unreacted species. We deduce therefore that both electrostatics and the presence of the encapsidation signal are sufficient for the efficient templated assembly of the Au−CLP.

IV. CLP Stability as a Result of Purification of 48-mer DNA/p−Au. Because the possible persistence of a loosely bound species of DNA at equilibrium with free DNA in solution remaining from the synthesis of 48-mer DNA/p−Au may be a part of the reassembly process, a multistep purification was carried out in order to remove this excess DNA, Figure 5. After three purifications using size exclusion chromatography, all unbound DNA was removed, Figure 5. The outcome of the assembly reaction using 3× purified cores was analyzed by TEM and DLS (Figure 6).

Figure 5 Effect on mobility and size when elimination of unbound DNA from 48-mer DNA/p−Au solution was performed. (A) Gel electrophoresis showed no free DNA present after three purifications. (B) After three purifications, the average diameter decreased 4 nm.

Figure 6 Au−CLP in vitro assembly under covalently bound DNA conditions. Three times purified 11.5 nm gold templates covered with DNA were mixed with stoichiometric amounts of RRV CP to produce Au−CLP, as illustrated in (A). The average diameter of these Au−CLPs was 31.6 ± 4 nm. Assembly with covalently bound DNA on the gold surface was only partially successful. Visible aggregation occurred within 1 h after assembly (B). The DLS of the Au−CLPs showed a broad peak with a mean at 43.82 nm (C).

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Somewhat surprisingly, the 48-mer DNA/p−Au−CLPs assembled with the 3× purified conjugates were not as stable as the unpurified products. Negative staining TEM imaging of the 3× purified solution of 48-mer DNA/p−Au suggested that the purified particles are weak promoters of capsid assembly (Figure 6).

The purified 48-mer DNA/p−Au−CLPs visualized on TEM immediately after synthesis (Figure 6A) conserved spherical symmetry and size with respect to the unpurified 48-mer CLPs. The size distribution of these conjugates had a mean at 32 nm, which is close to the expected 37 nm for the wt. However, in time, a color change of the solution from red to blue was observed, indicating aggregation (Figure 6B,C). However, DLS taken 1 h after assembly (the shortest time possible between an assembly reaction and the DLS experiment) showed an evolving broad peak (Figure 6C), indicating that aggregation was still be taking place at the time of measurement.

Lower efficiencies of encapsulation and stability of Au−CLPs obtained from 3× purified 48-mer DNA/p−Au (Figure 6B) as compared with Au−CLPs, formed when free or loosely bound DNA is available (Figure 3D), seem to suggest that free-DNA may be required for better encapsulation.

A possible explanation could be that the amount of DNA present on the gold surface was not sufficient to prevent detrimental phosphane−CP interactions, which may be responsible for denaturation of the protein coat. When free DNA is present, DLS data indicates that some of it binds to the GNPs in addition to covalently bound DNA, thus decreasing the occurrence of direct phopshane−capsid interactions (Figure 5B).

Another explanation for the smaller number of Au−CLPs formed from purified nanoparticle reactant may be the requirement for a nucleoprotein early intermediate for CLP assembly, possible only when free oligo-DNA is present in the solution. However, to test this hypothesis, further studies are necessary.

DLS was used to compare the stability of the 48-mer DNA/p−Au−CLPs to a native CLP control, Figure 7. The hydrodynamic radii of the 48-mer DNA/p−Au−CLPs remained the same throughout the entire duration of experiment (44 h), while fluctuations and drift toward larger average diameters (aggregation) have been measured for the CLP control. Therefore, nanoparticle-encapsulating CLPs are more stable upon storage than their native counterparts.

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Figure 7 Stability of Au−CLPs and 48-mer CLPs as a function of time, as indicated by changes in the position of the peak of size distribution. The DLS of 1× purified 48-mer DNA/p−Au and 48-mer DNA with RRV CP were measured over a period of 10 min to 44 h after assembly at 8 °C.

V. Influence of the Gold Core Size in Promoting DNA/p−Au−CLP Assembly. Once the 1× purified 48-mer DNA/p−Au ligand was identified as most likely to yield efficient templated capsid growth, different GNP sizes were tested with respect to their encapsulation efficiency.

Six different sizes of functionalized GNPs were prepared using gold cores of 8.3, 11.5, 13.9, 18.7, 23.6, and 27.4 nm. DNA/p−Au conjugates were purified only once for the excess of nucleotide in solution. DNA/p−Au conjugates were then mixed with RRV CP in an assembly buffer. The assembly reaction led to the formation of 48-mer DNA/p−Au−CLPs accompanied, as expected, by an increment in the average particle diameter (Figure 8).

Figure 8 TEM images of encapsidated 48-mer DNA/p−Au particles with different sized Au cores: (A) 8.3 nm core size; (B) 11.5 nm core size; (C) 13.9 nm core size; (D) 18.7 nm core size; (E) 23.6 nm core size; (F) 27.4 nm core size. The equivalent ratios of Au (100−800 nM) to CP (0.3 mg/mL) are 1:240 for all the assemblies.

Negatively stained TEM images of encapsulated 48-mer DNA/p−Au of different sizes show the formation of spherical and homogeneous capsids, Figure 8. The DNA/p−Au−CLP complexes closest in size to the 48-mer CLP are obtained for 18.7 nm cores.

Encapsulation efficiency for each nanoparticle diameter was estimated from TEM images and represented as a function of core size in Figure 9One notes on Figure 9 that, while the size of the CLP complex varied monotonically with the template diameter, the encapsidation efficiency reaches a maximum (62% at 18.7 nm) and then decreases significantly for larger nanoparticle diameters. A similar behavior has been observed for GNP encapsulation in BMV, pointing to a correlation between the T-number and the self-assembly efficiency.16 Because of this correlation, it is possible that closed-shell formation occurs at least for certain core sizes. Among them, the 18.7 nm gold cores provide the highest average encapsulation yield (62%).

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Figure 9 48-mer DNA/p−Au nanoparticle encapsidation efficiencies as a function of GNP core size (vertical bars). Error bars represent standard deviations obtained from ensembles of

800 particles.

Dependence of the T number on the core diameter estimated from the same particle populations.

In addition to estimating efficiencies, the DNA/p−Au−CLP size distribution from TEM was used to compute possible triangulation numbers, T, for CLP polymorphs induced by the different core sizes. The equation proposed by Caspar and Klug59 relates T with the diameter (D) of the icosahedral particle and d, when the center-to-center distance of the basic structural unit (capsomers)is known:60

The alphavirus nucleocapsid diameter measured from structural data has been found to be between 38 and 40 nm,28,30,31,33,40,41,57,58 while the distance d between the capsomers (pentamers of CP) has been calculated to be between 12.04 nm and asuming a pseudo-T = 4 icosahedron.60 The calculated triangulation numbers are plotted in Figure 10 against the core size and 48-mer DNA/p−Au encapsulation efficiencies. There is a plateau observed at T = 4, which also suggests a trend toward a predetermined arrangement of the CP into T = 4 icosahedron.30,33,61 These observations are consistent with 48-mer CLPs and wt nucleocapsids, forming mostly T = 4 sized particles.

Figure 10 Plots of the CLP diameter vs the ligand-coated core diameter (left) and of the CLP and ligand-coated core diameter vs bare GNP diameter (right).

The T number corresponding to 18.7 nm GNPs is also associated with the wt CLP, found from structural data. In contrast, for bigger nanoparticle cores (23 and 27.4 nm), the Au−CLP T number seemed to increase linearly with the gold size, resulting in a possible T = 7 and T = 12, respectively. However, the linear increase may also indicate that CP failed to arrange into a regular quasispherical array.

A comparison between the DLS diameters of the DNA/p−Au particles before and after assembly with the RRV CP shows a linear increment with the size of the nanoparticle, Figure 10. In the case when a layer of protein of constant thickness is added, the slope of the CLP diameter

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vs DNA/Au−p diameter is expected to be 1 because: where: DCLP = CLP

diameter, DGNP = diameter of the nanoparticle core (DNA/Au−p), and t = thickness of the molecular (ligand + protein) layer.

In Figure 10, the slope is 2.0 ± 0.1, which implies that the thickness of the protein plus ligand layer depends linearly on the radius of the core, at least for the size range of 7−30 nm. The question is, does the ligand layer or the protein layer thickness vary with the size of the nanoparticle template? To answer this question, Figure 10 (right) shows how the ligand layer and the protein + ligand layer thicknesses vary with the nanoparticle template diameter. Again, we observe a linear dependence in both cases. However, the linear fit corresponding to the ligand layer has a slope of 1, which implies a constant ligand thickness (of 3 nm), independent of Au core size. Therefore, it is the thickness of the protein shell that varies with the radius of the spherical template. Larger cores seem to induce a thicker protein shell, which raises the question of the nature of the shell. However, structural studies are necessary in order to answer this question. This report provides ways to obtain a large amount of homogeneous sample, therefore opening the door for structural studies and progress toward a complete alphavirus nanoparticle carrier.

Conclusion. We have shown that functionalized GNPs can be used as templates to promote the self-assembly of an animal virus capsid protein shell. A maximum encapsidation yield of 62% has been obtained for core nanoparticles having a diameter of 18.7 nm. The products of nanoparticle-templated growth tend to be more stable upon storage than empty CLPs. Similar to the case of the brome mosaic plant virus, maximum efficiency correlates with capsid-like particle increased stability and diameters that are close to the wt CLP diameter. Charge neutralization is necessary but not sufficient to drive Au−CLP assembly, unlike the case of previously studied nonenveloped plant viruses. The main specific difference for alphavirus CLPs is the requirement for the existence of encapsidation signal DNA free in solution simultaneously with adsorbed DNA on the artificial cores.

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