Foot-and-mouth disease virus: DNA aptamer selection for the 3ABC protein

Figures

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  Fig. 1. Representative CE electropherogram of CE-SELEX separation step. The A region refers to oligonucleotides that did not bind to the target. The peak in the B corresponds to the 3ABC-aptamer complexes that were collected. The collection window was 7.2 to 8.5 min. Five runs, which were assigned by distinct color lines, were performed to accumulate enough DNA for PCR amplification. These runs were superimposed in the graph. AU = absorbance.

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  Fig. 2. (A) FMDV1 aptamer sequence with the conserved regions in bold. (B) Secondary structure of the FMDV1 aptamer predicted by Mfold (∆G = −10.42 kcal/mol).

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  Fig. 3. Evaluation of the increase in affinity to the 3ABC protein obtained with the aptamer selection process. The 3ABC protein (0.67 µg) was incubated separately with 25 nM of the FMDV1 aptamer and 25 nM of the oligonucleotide library used as start material for selection. Protein-bound DNA was quantified by qPCR. Statistical analyzes were performed using the GraphPad Prism 5 software (**p < 0.01).

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  Fig. 4. Dissociation constant (Kd) determination of FMDV1 aptamer. The saturation curve was obtained by plotting the aptamer concentration (nM) versus the total ssDNA (ng) bound to 3ABC protein. Protein-bound aptamer was quantified by qPCR. The Kd was calculated by non-linear regression analysis (One-Site Specific Binding with Hill slope software GraphPad Prism 5), resulting in Kd = 22.69 ± 1.79 nM.

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  Fig. 5. Specificity assay. Binding of FMDV1 aptamer to 3ABC and BSA proteins was performed by qPCR. The graph shows the ssDNA amount (ng) recovered after incubation of 25 nM of FMDV1 aptamer with 0.67 µg of each protein. Statistical analyzes were performed using the GraphPad Prism 5 software (**p < 0.01).