Having confirmed that PADI4_3_H4(2) was a covalent binder of PADI4, we synthesised three model mRNA templates for use in a test selection. The first template encoded for the wildtype PADI4_3 sequence. The second encoded a sequence where His4 in the PADI4_3 sequence was substituted for a Met codon that could be reprogrammed to FAO, PADI4_3_H4M. The third template encoded a control sequence where an arginine residue in the sequence, Arg2, was replaced by a Met codon, PADI4_3_R2M, which we anticipated would not be correctly positioned to covalently react with PADI4 (Figure , Figure ). We performed a single cycle of RaPID screening (clone assay), with each of the individual mRNA templates, to assess peptide binding. In each case, translated RaPID peptide was incubated with PADI4 at room temperature for 1 hour to allow time for covalent reaction, before affinity panning was performed both with and without denaturing guanidinium chloride washes. qPCR was used to quantify DNA recovery for each peptide. In the absence of guanidinium chloride washes, all three peptides bound to PADI4, whilst only PADI4_3_H4M was retained after guanidinium chloride washes (Figure ). This confirmed that the translated warhead was competent to react with cysteine residues in the target protein, that the guanidinium chloride washes were effective at retaining only covalently bound peptides and that we were not observing high levels of non-specific peptide reaction. Having confirmed that our encoded electrophile RaPID setup could identify covalent cyclic peptides, we set out to perform a de novo peptide screen for covalent binders of PADI4. For this we used an mRNA-displayed library encoding peptides with between six and ten randomised positions, flanked by an initiator codon and a CGSGSGS C-terminal linker. Peptide cyclisation was enabled by flexizyme-mediated reprogramming of the initiator codon to Nchloroacetyl-D-tyrosine which would spontaneously cyclise with the cysteine in the C-terminal linker. All internal Met codons were reprogrammed to 2. Following translation and reverse transcription, this library was used in a covalent RaPID selection. In each round, the library was preincubated with PADI4 at room temperature prior to denaturing affinity panning against PADI4. We saw low recovery when the library was incubated with biotinylated streptavidin beads (negative selection) and increasing positive library recovery for the first 4 rounds with PADI4-bound beads (Figure ). Following 5 rounds of selection, next-generation sequencing was performed on the DNA libraries recovered after each selection round (Supplementary Data 1). Sequencing results suggested that our RaPID setup rapidly enriched for covalent peptides, because from round 2 onwards 99% of sequences had an internal Met codon, indicating warhead presence (Figure ). Interestingly, however, the sequencing data from later rounds did not resemble typical successful RaPID selections. Although our total library recovery increased through the rounds, rather than finding a smaller number of highly enriched peptide sequences, contributing substantially to the total recovered libraries, we saw many different individual sequences each with relatively low abundance (Figure ). This suggested that reaction with 2 was permissible within a wide range of peptide sequence contexts. Despite this, multiple sequence alignments indicated the enrichment of certain families and a clear increase in the abundance of certain sequences within these families round by round, indicative of target binding (Figure ). We selected 6 of these sequences for synthesis by SPPS and further characterisation.