Discussion
We have established and refined mRNA and cDNA display methods utilizing the cell-free translation system (PURE system) for efficient and stable mRNA and mRNA/cDNA-peptide conjugate synthesis and downstream selection (Figure 1). One of the highlights of our method is the capability of preventing carry over components through two separate electroelution steps to ensure the purity of mRNA and mRNA/cDNA-peptide conjugates. This could be useful if one intends to target the molecules present in the PURE system such as nucleotides and translation related RNA and proteins. Also in comparison to affinity beads purification, polypeptides do not require peptide affinity tags. Performance of the two display methods was validated by conducting an epitope search from approximately 1012 peptide sequences against the well-known anti-FLAG M2 antibody (Roosild et al., 2006). A rapid enrichment of the canonical FLAG epitope: DYKDDDxx after three rounds of selection and further, was identified with high-throughput sequencing. This led to new insights into the variable residues, resulting in the discovery of the consensus FLAG motif sequences; DYK(DLN)(DFLNY)D. This motif appeared in both of the sites that were able to accommodate the motif within the restrictions imposed by the library design. Only three sites in the original library had degenerate codon sequences that can code for tyrosine, and of those three sites, one is too close to the C-terminal end of the random region to be part of the FLAG motif.
Two display methods resulted in overall similar performance, showing consistency of our method but with slightly higher enrichment for mRNA display over cDNA display. The mRNA display FLAG motif sequences were slower to elute from the beads than that of cDNA display, possibly because the RNA-conjugated peptides smaller size allows it access to more binding sites on the beads than the larger mRNA/cDNA-peptide conjugates. In contrast, the cDNA display library appears to have more non-specific binding on the remaining beads.
High-throughput sequencing has become a powerful approach for variousin vitro selection and evolution experiments. We show that round-by-round sequencing and stepwise competitive elution with the FLAG peptide revealed consensus FLAG motif over other sequence variants by the 4th and final round. We also observed additional enrichment of several amino acids adjacent to the FLAG motif in a library dependent manner (FLAG-random vs 10aa-random) suggesting high-order epistasis between residues may exist to account for the optimal binding conformation. The resulting large sequence database also contains important information regarding the fitness of individual amino acid residues. By comparing the enrichment pattern of sequences with a single residue difference, we were able to highlight the most influential residues important for anti-FLAG M2 antibody recognition. Such patterns would be useful to further explore the high-affinity epitope candidates, along with the recent advancement of array based high-throughput and quantitative protein assay able to discover high-avidity epitope variants (Layton, McMahon, & Greenleaf, 2019). Hence, we expect this method can be applied to broad range of molecular targets to allow rapid detection of consensus binding motifs.