New dsDNA binding unnatural oligopeptides with pyrimidine selectivity Original Research Article

Solid phase peptide library screening followed by extension of a lead recognition element for binding to a dsDNA sequence (NF binding site of IL6) using solution phase screening, delivered a new DNA binding peptide, Ac-Arg-Ual-Sar-Chi-Chi-Tal-Arg-CONH2. In the present research, the contribution of the different amino acid side chains to the binding strength of the peptide to dsDNA was investigated using an ethidium bromide displacement test. Based on these results, the lead structure was optimized by deconvolution. Eight new unnatural amino acids were evaluated at two positions of the heptapeptide replacing the Ual-Sar fragment. The strongest dsDNA binding was observed using {[(3-chlorophenyl)methyl]amino}acetic acid (Cbg) and β-cyclohexyl-l-alanine (Cha) respectively, at those two positions. A 10-fold increase in affinity compared to the Ual-Sar sequence was obtained. Further enhancement of dsDNA binding was obtained with hybrid molecules linking the newly developed peptide fragment to an acridine derivative with a flexible spacer. This resulted in ligands with affinities in the μM range for the dsDNA target (Kd of 2.1×10−6 M). DNase I footprinting with the newly developed oligopeptide motifs showed the presence of a pronounced pyrimidine specificity, while conjugation to an intercalator seems to redirect the interaction to mixed sequences. This way, new unnatural oligopeptide motifs and hybrid molecules have been developed endowed with different sequence selectivities. The results demonstrate that the unnatural peptide library approach combined with subsequent modification of selected amino acid positions, is very suited for the discovery of novel sequence-specific dsDNA binding ligands.