Fig. 2.

IFCE SELEX analysis. A) Binding affinity and specificity of the different aptamer pools partitioned by IFCE-SELEX. Slot blot binding assays were made by incubating purified recombinant SARS-CoV-2 S protein (200 nM) with 10 pM of either the radiolabeled M2 randomized pool or the partitioned pools 2R and 3R. Bovine serum albumin (BSA) (200 nM) was used as negative binding control. The residual radioactivity on the nitrocellulose (NC) and nylon (NY) membranes was used to determine the aptamer fraction bound to the proteins. Error bars represent one standard deviation from triplicate analyses. The results were analyzed using one-way ANOVA with post hoc Tukey's multiple comparisons test (99.9% CI). Asterisks indicate statistical significance (n = 3, p < 0.0001). B) NGS variability analysis. The percentage of unique sequences in each dataset was calculated after sequencing each IFCE-SELEX cycle. The number of unique sequences decreased through the IFCE partition. C) NGS enrichment analysis. R1 data set was matched against 2R. Each black dot represents one sequence obtained from the NGS data. Sequence frequency was plotted as a function of its enrichment-fold from R1 to R2. It was observed that some of the most enriched sequences were also frequent. FASTAptamer toolkit was used for the variability and enrichment analysis. D) Unrooted radial phylogenetic tree of the most enriched sequences in R2 containing the ten selected aptamer sequences. E) Unrooted radial phylogenetic tree of the most enriched sequences against NC membrane. The trees were constructed using the 100 most enriched sequences and analyzed by Molecular Phylogenetic Analysis by Maximum Likelihood method based on the Kimura 2-parameter model conducted in MEGA7. Scale bar indicates 0.5 substitution per site.