Fig. 3: DP-DEL thrombin selections.

A HTDS of concatenated triplicate selections against thrombin. Building blocks 1, 2 and 3 correspond to each respective axis. The heat bar represents the DNA sequence counts. The Plot is constrained to combinations from version 2 of DP-DEL possessing BB4-position 2, due to the attainment of highest enrichment values from that version of the library (see Supplementary Information, section 5.4.2.). Arrows indicate the three most enriched library members from the selection. Cut-off: 100 counts. B On-LNA experimental validation of highest enriched library members 1-3 displayed on 8-mer locked nucleic acid (LNA) heteroduplexes with a fluorescein label by fluorescence polarization (FP). Conjugates were tested in open form approximating version 1 of DP-DEL and clicked form approximates version 2 of DP-DEL. Affinity values are given as a mean of triplicate experiments (n = 3), with the exception of 1-o, which was measured as a duplicate (n = 2). C Off-DNA inhibitory measurements of selected compound 4 (R = Ahx-(H-Lys-NH₂)-5-carboxyfluorescein) against human alpha-thrombin, synthesized using a selection of linkers between the two cysteine scaffolds to test preferred peptide flexibility. L1-L5 allow the formation of variably sized macrocyclic peptides, while L0 yields a linear peptide. A heat bar shows the relation of IC₅₀ obtained and peptide steric constraint due to L0-L5 of binder. IC₅₀ values are obtained from experimental triplicates (n = 3) and given as a mean. Error bars indicate standard deviation of the replicates.