Peptides
that recognize artificial materials including synthetic
polymers and small molecules are drawing attention in the fields of
biotechnology and chemical biology. In particular, reversible peptide
aptamers that associate with the target molecules only under specific
conditions are interesting. In this work, peptide aptamers that recognize
a phenolphthalein derivative (PhP: a pH-sensitive organic dye) immobilized
on a solid surface in a pH-dependent manner were selected using an
in vitro display method (cDNA display). Considering the hydrophobic
and aromatic nature of PhP, we prepared a biased DNA library (3A library)
that encodes more aromatic amino acids than the standard random codon
and performed seven rounds of selection from >10
10
peptide
species. The selected peptides including LVFLIWWM (LV59) associated
with PhP-modified solid support (sepharose resin and magnetic beads)
in neutral buffer but readily dissociated under basic conditions where
PhP undergoes large structural change from lactone to quinoid, which
is accompanied by increase of hydrophilicity and anionic charge. Control
experiments suggested that LV59 recognized both phenol and lactone
moieties, and the association under neutral pH is mainly driven by
π-stacking and hydrophobic interaction between the peptide and
PhP. Notably, however, total hydrophobicity and number of aromatic
rings did not completely explain the affinity, and sequence specificity
was observed to some extent. After further optimization, this interaction
pair would be practically useful for protein purification.
Peptide-based
drugs are an attractive new modality of therapeutics,
and in vitro selection from a large-scale library
is a powerful way to identify new lead sequences. In conventional
screenings, peptide specificity and stability in physiological heterogenous
environments are not evaluated, which sometimes makes subsequent optimization
difficult. Here we show that selection using a cDNA display system
can be performed in a high percentage of serum and that this might
be an option to select molecules with high potency and stability in
a biological context. Specifically, we chose interleukin-17A as a
target protein and performed in vitro selection of
cyclic peptide aptamers from a library of approximately 1012 members in the presence of serum. The selected molecules had nanomolar
affinity to the target and were stable in serum. Interestingly, we
found that a component of the DNA linker that connected the peptide
and cDNA may play a pivotal role in target binding.
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