Extended Data Fig. 2: Gaiting and selection strategies for in vitro evolution of the SARS-CoV-2 RBD domain.

(A, B) Gating strategy for FACS sorting. In the first step, yeast cells were isolated by their FSC-A and SSC-A properties (A). In the second step (B), single cells were isolated by their FSC properties (area and height) on the diagonal plot. The Green area represents the gated region. (C) Selection strategy for affinity maturation. The library was titrated with a range of ACE2 concentrations to select the concentration with limited signal (inset 1). Under such conditions, the tighter binding clones gain the highest advantage over the parental population. Using less stringent selection (insets 2 – 4) reduces the advantage of the tighter binders. Using sub-optimal concentrations of ACE2 protein will also result in loss of selectivity. (D) Affinity maturation library after 3 sorts, where the separation between parental and tighter binding population is well defined. The top 0.1 – 0.3 % of cells were sorted – green region. (E) Fast-association selection strategy. The library was incubated with a constant concentration (30 pM) of ACE2 for different times. The time with minimal signal was determined and used for the selection of clones with faster association (library B6(FA)). The same shape of the sorting region as in (D) was applied.