Fig. 3: Increased selection stringency yields more potent daunomycin-binding HFNAPs.
a Sequence convergence for selection 1 and selection 2. b The evolutionary trajectory of the top ten most enriched sequences in selection 2. c MST characterization of the binding affinity for the top ten most enriched sequences from selection 2. Dm-HS-4, 5, and 9 are omitted for clarity, as Dm-HS-1, 4, 5 and Dm-HS-3, 9 are each related by 1 nucleotide. 1 ∆ s.c. denotes the sequence (Dm-HS-1) without side-chains. Sequences were found to bind with Kd = 5–65 nM. Values and error bars reflect the mean and SEM for n = 3 independent replicates. Error bars for some values are too small to extend beyond the data point. d 14 Dm-HS-1 mutants were translated where a single building block is replaced with the corresponding building block lacking any side-chain. MST characterization of the binding affinity for these Dm-HS-1 mutants is shown. CGA ∆ s.c. denotes Dm-HS-1 without a side-chain for the CGA building block. The table shows individual side-chain removals that result in the largest increase in Kd. The activity of the original Dm-HS-1 sequence is shown in black for comparison. Values and error bars reflect the mean and SEM for n = 3 independent replicates.
