Nucleic Acid Aptamers for Human Norovirus GII.4 and GII.17 Virus-Like Particles (VLPs) Exhibit Specific Binding and Inhibit VLPs from Entering Cells

Figures

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  Figure 1. Secondary structure prediction and Kd values of candidate aptamers AP4-2 and AP17-4. Secondary structure prediction of (A) AP4-2 and (B) AP17-4, some common motifs (≥ 6 bases in length) occurring in the aptamers for HuNoV GII.4 and GII.17 VLPs are marked by different colored boxes and the number of occurrences is listed. Binding and dissociation curves and Kd values of (C) AP4-2 and (D) AP17-4.

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  Figure 2. Affinity analysis of aptamers for targets. (A) ELASA schematic diagram. T/N of (B) AP4-2 and (C) AP17-4 at different HuNoV GII.4 and GII.17 VLP concentrations (1.5 μg/mL, 3.0 μg/mL and 10 μg/mL) and different aptamer concentrations (0.01 μM, 0.1 μM, 1.0 μM, 1.5 μM and 2.0 μM). The data are reported as means ± SEM of triplicate wells.

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  Figure 3. Specificity analysis of aptamers for targets. Dot blotting results of (A) AP4-2 and (B) AP17-4 for target HuNoV VLPs versus other unrelated pathogens/proteins. Datametric rendering of dot blotting points of (C) AP4-2 and (D) AP17-4 using ImageJ. Comparison of T/N of (E) AP4-2 and (F) AP17-4 to target HuNoV VLPs versus unrelated viruses/proteins using ELASA (****, P < 0.0001). The data are reported as means ± SEM of triplicate wells.

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  Figure 4. The capacity of blocking interaction between HuNoV VLPs and PGM III of (A) AP4-2 and (B) AP17-4 by ELISA. The data are reported as means ± SEM of triplicate wells.

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  Figure 5. Generation of HuNoV HiBiT VLPs, 293T-LgBiT cell, and probing the blocking function of aptamers on VLPs internalised cells. (A) WB of GII.4 VLP, HiBiT GII.4 VLP, GII.17 VLP, HiBiT GII.17 VLP using polyclonal antibodies against HuNoV GII.4 VLP and HuNoV GII.17 VLP. (B) HiBiT blotting of GII.4 VLP, HiBiT GII.4 VLP, GII.17 VLP, HiBiT GII.17 VLP. Transmission electron microscopy of (C) GII.4 VLP (left), HiBiT GII.4 VLP (right) and (D) GII.17 VLP (left), HiBiT GII.17 VLP (right). (E) mRNA expression levels of LgBiT relative to β-actin in 293T and 293T-LgBiT cells were determined by qRT-PCR. (F) LgBiT expression levels in 293T and 293T-LgBiT cells were detected by WB with anti-LgBiT antibody, β-actin served as an internal control. Effects of (G) AP4-2 and (H) AP17-4 on HuNoV GII.4 and GII.17 VLPs cell internalisation. The data in the bar graphs are reported as means ± SEM of triplicate wells (*0.01 ≤ P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

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  Figure 6. Effect of aptamers on cell viability. Effect of treatment of 293T-LgBiT and Caco2 cells with (A) AP4-2 and (B) AP17-4 for one day on viability as determined by cytotoxicity LDH assay. The data are reported as means ± SEM of triplicate wells.