Durapore filter plates

Equilibrium dissociation constants (K_D) of synthetic 50-mer modified aptamers (40 nucleotides from the SELEX library modified region and five nucleotides from the 5′ and 3′ primer regions for 40N libraries) and 40-mer modified aptamers (30 nucleotides from the SELEX library modified region and five nucleotides from the 5′ and 3′ primer regions for 30N libraries) were determined by filter binding assay. K_D values of modified aptamers were measured in SB18T buffer. Modified aptamers were 5′ end labeled using T4 polynucleotide kinase (New England Biolabs) and γ-32PATP (PerkinElmer). Radiolabeled aptamers (∼20,000 CPM) were mixed with spike proteins at concentrations ranging from 10⁻⁷ to 10⁻¹² M and incubated at 37°C for 40 min. Following incubation, an equal volume of 10 mM dextran sulfate was added, and bound complexes were partitioned on His-tag Dynabeads at 37°C shaking at 1,850 RPM for 5 min and then captured on Durapore filter plates (EMD Millipore). The fraction of bound aptamer was quantified with a phosphorimager (Typhoon, GE Healthcare, Piscataway, NJ, USA), and data were analyzed in ImageQuant (GE Healthcare). To determine binding affinity, data were fit using the equation $\mathrm{y}=(\max -\min )\left(\mathrm{p}\mathrm{r}\mathrm{o}\mathrm{t}\mathrm{e}\mathrm{i}\mathrm{n}\right)/({\mathrm{K}}_{\mathrm{D}}+\mathrm{p}\mathrm{r}\mathrm{o}\mathrm{t}\mathrm{e}\mathrm{i}\mathrm{n})+\min$ and plotted using GraphPad Prism v.7.00.