Our study began with the incorporation of the Bpa moiety into our mRNA displayed cyclic peptide library through including pBpa in the FIT system. We synthesised the photoactivatable amino acid protected with a cyanomethyl ester (CME) group using previously reported procedures. The CME leaving group facilitated efficient charging onto a model tRNA (microhelix RNA, FAM-MiHx_23b) through eFx mediated aminoacylation (Supplementary Figure ). Following charging onto an elongator tRNA (tRNA Asn CAU) to genetically reprogramme the methionine codon, pBpa was successfully incorporated into a model cyclic peptide by in vitro translation using a Met-free FIT system (PURExpress TM , Δaa, ΔtRNA, NEB) (Supplementary Figure ).