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Characteristics of DNA-AuNP networks on cell membranes and real-time movies for viral infection

This data article provides complementary data for the article entitled “DNA-AuNP networks on cell membranes as a protective barrier to inhibit viral attachment, entry and budding” Li et al. (2016) [1]. The experimental methods for the preparation and characterization of DNA-conjugated nanoparticle networks on cell membranes were described. Confocal fluorescence images, agarose gel electrophoresis images and hydrodynamic diameter of DNA-conjugated gold nanoparticle (DNA-AuNP) networks were presented. In addition, we have prepared QDs-labeled RSV (QDs-RSV) to real-time monitor the RSV infection on HEp-2 cells in the absence and presence of DNA-AuNP networks. Finally, the cell viability of HEp-2 cells coated by six types of DNA-nanoparticle networks was determined after RSV infection.


Specifications table
The data is useful for developing movies to real-time monitor the inhibition of viral infection. It provides multiform ways to investigate the stability and antiviral ability of DNA-AuNP networks. This data may provide a pathway for the development of broad-spectrum antiviral agents.

Data
This paper presents data related to the research article entitled "DNA-AuNP networks on cell membranes as a protective barrier to inhibit viral attachment, entry and budding" [1]. The data here include the characterization of DNA-conjugated nanoparticle networks on cell membranes, the stability test of DNA-AuNP networks against enzymatic cleavage, the immunofluorescence images of HEp-2 cells coated by DNA-magnetic nanoparticle (DNA-MNP) networks, the real-time movies for RSV infection, and cell viability of HEp-2 cells fabricated by six types of DNA-nanoparticle networks.

Chemicals and materials
Streptavidin (SA) was purchased from Beijing Biosynthesis Biotechnology Co. Ltd. (Beijing, China) and bovine serum albumin (BSA) was purchased from Beijing Dingguo Changsheng Biotechnology Co. Ltd. (Beijing, China). Streptavidin-coated iron oxide nanoparticles (SA-MNPs, Ocean Nanotech) of 10 nm and 30 nm diameter were resuspended at 0.1 mg/mL in 100 mM phosphate-buffered saline (PBS). Citrate coated gold nanoparticles (AuNPs) of 13 nm, 30 nm, and 50 nm diameters were prepared by altering the quantity of HAuCl 4 and reducing agents according to a previously published method [2]. Citrate coated silver nanoparticles (AgNPs) of 30 nm diameter were obtained by reducing AgNO 3 with trisodium citrate according to the modified Lee-Meisel method [3].

Preparation and characterization of MNP-P1-P2 and AuNP-SA
MNP-P1, MNP-P2, or MNP-P1-P2 were obtained by incubating SA-MNPs of 30 nm (34 nM, 500 μL) with P1, P2, or P1/P2 (100 μM, 100 μL) in PBS buffer (pH 7.4) at 25°C for 1 h with gentle shaking at 120 r/min. Then the solution was ultracentrifuged at 10000 r/min for 5 min three times to remove the free DNA. At last, MNP-P1, MNP-P2, or MNP-P1-P2 were redispersed in PBS buffer (pH 7.4) and stored at 4°C, respectively. MNP-P1-P2 was further hybridized with linker DNA by mixing MNP-P1-P2 with linker DNA in PBS buffer for 1 h at room temperature. Transmission electron microscope (TEM) images were used to demonstrate the aggregation of MNP-P1-P2 by DNA hybridization with a Hitachi S-4800 scanning electron microscope (Tokyo, Japan) at 20.0 kV. SA-AuNPs was prepared by incubating the as-prepared AuNPs of 13 nm (920 μL) with SA (1 mg/mL, 80 μL) for about 30 min at room temperature, followed by the addition of BSA (50 mg/mL, 100 μL) to react another 30 min in order to block the excessive binding sites on AuNPs surface. AuNPs modified with BSA only was used as a control (BSA-AuNPs). SA-AuNPs was further conjugated with P1 or P2 to obtain AuNP-P1 or AuNP-P2 following the above protocol for the preparation of MNP-P1or MNP-P2. UV-vis absorption spectra of AuNPs, BSA-AuNPs and SA-AuNPs were measured by a U-3010 spectrophotometer (Hitachi, Tokyo, Japan) ( Fig. 2A). The hydrodynamic diameters and zeta potential of nanoparticles were determined by Zetasizer Nano-ZS System (Malvern Inc) (Fig. 2B).

Characterization of DNA-nanoparticle networks on cell membranes
Scanning electron microscope (SEM) images of HEp-2 cells anchored by DNA-AuNP networks were obtained using a gradient ethanol dehydration method. Accordingly, HEp-2 cells were seeded and cultured on 24-well plates with cover glass slides for about 24 h to reach 50% confluence. After fabrication of DNA-AuNP networks on cell membranes, cells were first fixed by 500 mL of 2.5% glutaraldehyde at room temperature for 2 h, followed by a gradient series of ethanol dehydration from 30%, 50%, 75%, 80%, 95%, to 100%. Each step was maintained for 15 min to ensure complete ethanol saturation throughout the cells. The samples were then sputter-coated with gold for 30 s, and observed using a Hitachi S-4800 scanning electron microscope (Tokyo, Japan) at an operating voltage of 20.0 kV.
Dark-field light scattering imaging of HEp-2 cells was used to demonstrate the formation of DNA-AuNP networks on cell membranes. HEp-2 cells were seeded and cultured on 24-well plates with cover glass slides for about 24 h to reach 50% confluence. After PBS washing, cells were anchored by SA-AuNPs, BSA-AuNPs, AuNP-P1-P2 and DNA-AuNP networks on cell membranes, respectively. Then cells were fixed with 4% paraformaldehyde, sealed with glycerin, and then transferred for dark-field light scattering imaging under an Olympus BX-51 System microscope (Tokyo, Japan) with an Olympus E-510 digital camera (Tokyo, Japan) [2].
Confocal fluorescence images of HEp-2 cells anchored by DNA-AuNP networks were carried out. The fluorescent dye of Cy3 was modified at the 5 0 end of oligonucleotide P1. Briefly, HEp-2 cells (1.0 Â 10 5 cells mL À 1 ) were cultured in the 35 mm glass-bottom cell culture dishes (NEST. Corp) over 24 h. After fabrication of DNA-AuNP networks on cell membranes, cells were further incubated at 37°C for 0 h, 16 h, 24 h and 48 h, respectively (Fig. 3). After washing by PBS, cells were fixed with 4% paraformaldehyde for 20 min. Fluorescent images were acquired using an Olympus IX-81 inverted microscope equipped with an Olympus IX2-DSU confocal scanning system and a Rolera-MGi EMCCD. Colocalization analysis was performed with Image-Pro Plus software. Fluorescent dye of Cy3 was excited at 530-550 nm and detected with a barrier filter BA575-625 nm.

Stability of DNA-AuNP networks against enzymatic cleavage
Agarose gel electrophoresis images were first carried out. At first, DNA sequence P1, the hybrid of P1 þP2 þ linker DNA, and DNA-AuNP networks were incubated with DNaseI at 37°C for 20 min, respectively. Then 10 μL of reaction products were mixed with 2 μL 6 Â loading buffer, and then dropped into 2% agarose gel. The gel electrophoresis was run at 120 V for about 50 min in TBE buffer, followed by the Sybr Gold staining (1:2000) at room temperature for 40 min. Finally, gel electrophoresis was photographed with a digital camera under the irradiation of visible light and the UV light, respectively [4] (Fig. 4A and B). In addition, hydrodynamic diameter measurement was used to  characterize the size change of the DNA-AuNP networks with or without incubation with DNaseI (Zetasizer Nano-ZS System, Malvern Inc) (Fig. 4C).

Optical microscopic images of biotinylated HEp-2 cells with cytopathogenic effect (CPE)
To visualize the presence of CPE after infection by RSV, optical microscopic images of biotinylated HEp-2 cells infected by RSV in the absence and presence of DNA-AuNP networks were taken after 2 days (Fig. 5). CPE was indicated by the arrows.
2.6. Characterization on the formation of DNA-magnetic nanoparticle (DNA-MNP) networks and the inhibition ability on viral attachment The formation of DNA-MNP networks in solution was characterized by TEM (Fig. 6A ab) and hydrodynamic diameter measurements (Fig. 7A). Then the fabrication of DNA-MNP networks on cell  membranes was characterized by SEM using a gradient ethanol dehydration method (Fig. 6A cd). The inhibition ability of DNA-MNP networks on viral attachment was investigated by confocal immunofluorescence images taken after RSV infection at 4°C for 30 min (Fig. 6B). At last, the in vitro cytotoxicity of DNA-MNP networks to biotinylated HEp-2 cells was tested using the CCK-8 cell viability assay. (Fig. 7B).

Preparation of QDs-labeled virus and real-time monitoring of RSV infection
QDs labelling viruses involves three steps. Firstly, the viruses were concentrated by ultracentrifugation (Beckman Type 70 Ti) at 110,000 Â g for 40 min at 4°C. After this spin, a virus pellet was clearly visible and the viruses were resuspended in PBS buffer. Subsequently, the concentrated viruses were biotinylated by incubation with 1 mg/mL biotinylation reagent (Sulfo-NHS-LC-LC-Biotin, Thermo Scientific) at 4°C for 30 min. Unreacted biotinylation reagent was removed with a desalting NAP-5 column. At last, the biotinylated RSV were specifically labeled with QDs through the tight interaction between biotin and streptavidin by incubation with QDs-SA (605 nm, Wuhan Jiayuan Quantum Dots Co.., Ltd.) at 4°C for 30 min. To remove the free QDs-SA, the reaction solution was loaded onto sucrose cushion (30% sucrose in 0.1 M sodium chloride, 0.01 M Tris-HCl, 0.001 M EDTA, 1 M urea, pH ¼ 7.5) and ultracentrifuged (Beckman Type 70 Ti) at 110,000 Â g for 40 min at 4°C. Purified QDs-labeled RSV (QDs-RSV) were resuspended in PBS buffer and stored at À 80°C for further experiments.
The blocking of QDs-RSV attachment to HEp-2 cell membrane by DNA-AuNP networks was realtime monitored by Supplementary movie 1. The inhibition of the entrance and internalization of QDs-RSV into HEp-2 cells by DNA-AuNP networks was subsequently recorded by Supplementary movie 2.