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.