(A) Structure of novel coronavirus spike RBD complexed with its receptor, ACE2 (PDB ID code 6LZG), highlighting the amino acid positions of 24 RBD natural variants selected from the GISAID repository in June 2020, with the subsequent inclusion of S477N, and their proximity to the RBD-ACE2 interface. The structural illustration was generated using PyMol. Variants with a significantly higher affinity to ACE2 as determined in our multiplex assay are highlighted in blue. (B) Cumulative frequency of variants ranked from most frequently isolated to less frequent included in the multiplex array. Frequency of genomes is based on high-quality 2019-nCov genome sequences reported by the GISAID database as of May 25, 2021. Variants with a significantly higher affinity to ACE2 as determined in our multiplex assay are highlighted in blue. (C) Relative EC₅₀ values (μg/mL) obtained for each of the variants. Values are color-coded blue for EC₅₀ less than 10 μg/mL and red for EC₅₀ values more than 10 μg/mL. (D) Mean fold difference for each RBD variant EC₅₀ value relative to WT RBD. Data are presented as dot plots of (n = 4) independent experiments, with the mean ± SEM. Bars for variants with more than a 2-fold difference in affinity to the WT RBD are colored blue. Bars for variants with a slightly increased affinity (< 2-fold) are colored white. Variants with a weaker EC₅₀ value relative to WT are colored red. (E) Bio-layer interferometry sensograms of immobilized ACE2 with 2-fold 6–100 nM serial dilutions of SARS-CoV-2 RBD variants in solution. Binding curves representative of 2 independent experiments for each variant are plotted (solid-colored lines), globally fitted to a 1:1 binding model (black line).