Human Norovirus Aptamer Exhibits High Degree of Target Conformation-Dependent Binding Similar to That of Receptors and Discriminates Particle Functionality

Figures

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  FIG 1. Conformation-dependent binding of three different ligands to SYV VLPs treated at selected time-temperature combinations. (a) Signals of aptamer M6-2, HBGA type A, and antibody NS14 binding to SYV VLPs treated at various temperature for 1 min are reported as percentages of signals of binding to untreated VLPs. (b) TEM images of SYV VLPs treated at 68°C for 5, 10, 15, or 25 min. The scale bar represents 100 nm. (c) Signals of aptamer M6-2, HBGA type A, and antibody NS14 binding to SYV VLPs treated at 68°C for various times are reported as percentages of signals of binding to untreated SYV VLPs.

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  FIG 2. Apparent percentages of sequence-dependent binding of three human norovirus ligands. Normalized signal for aptamer M6-2, HBGA type A, and antibody NS14 binding to completely denatured SYV VLPs (treated at 80°C for 5 min) as percentages of signal for binding to untreated SYV VLPs.

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  FIG 3. DLS results for SYV VLPs treated at different temperatures and/or times. (a) Hydrodynamic diameter of SYV VLPs treated at different temperatures for 1 min. Error bars represent the standard error of three measurements taken for each of two samples at each temperature. (b) Kinetics of SYV VLP aggregation for three samples treated at 68°C for various times.

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  FIG 4. Molecular docking of M6-2 to SYV VP1. M6-2 secondary and tertiary structures used in the molecular docking process are shown in panel A with different motifs colored accordingly. The color scheme of the secondary and tertiary structures is consistent. The structural ensemble for the M6-2 ssDNA aptamer is shown in cartoon format with the average structure highlighted in darker colors and the full ensemble used in the molecular docking faded in the background to visually provide a sense of the conformational flexibility of the ssDNA aptamer. Panel B conveys the potential novel interaction surface of M6-2 (orange spheres), HBGA (magenta spheres), and antibody NS14 (cyan spheres). Panels C and D show the HADDOCK-docked M6-2 ssDNA aptamer clusters to SYV VP1. The dimeric partner of SYV VP1 is shown in faded cartoon format to hightlight the dimerization interface. Each individual domain of SYV VP1 is colored differently, i.e., gray, yellow, and red for the S, P1, and P2 domains, respectively.

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  FIG 5. Binding analysis of predicted aptamer M6-2 binding region. Binding ELASAs of native aptamer M6-2 compared to three versions of M6-2 with the suspected binding region sequence mutated (Table 2; see Fig. S3 in the supplemental material) were conducted. Different concentrations of the aptamers were evaluated for binding to SYV VLPs. Ratios of the absorbance of VLP wells to the absorbance of no-VLP wells are reported.