Figure 1. RaPID selection of ^K48Ub_n binding cyclic peptides.

a) Chemical synthesis of biotin-K48-linked di/tetra-Ub chain. b) Flexizyme, an artificial tRNA aminoacylation ribozyme, can load nonproteinogenic amino acids onto tRNA (black), for use by the ribosome in in vitro translation. nonproteinogenic amino acids with an N-terminal ClAc group will spontaneously react with intramolecular cysteine and can be used to form macrocyclic peptides. c) Large libraries (~10¹³) of DNA-tagged cyclic peptides can be generated in the RaPID system, from which sequences can be isolated to tightly bind a protein target. d) Using ^K48Ub₂ as a target, RaPID DNA libraries became enriched in certain peptide sequences (grey lines), with Ub2i and Ub2ii (black) as the dominant peptides (selection repeat shown as dashed lines). Using ^K48Ub₄ as a target, Ub4i and Ub4ix (black) emerged amongst the enriched peptides. e) (top to bottom) Synthesized peptides Ub2i, Ub2ii, Ub4i, Ub4ix and their binding to ^K48Ub₂ (left) and ^K48Ub₄ (right) detected using SPR. Highest and lowest peptide SPR concentrations are shown in blue and grey respectively.