Aliphatic/Aromatic Amino Acid Pairings for Polyamide Recognition in the Minor Groove of DNA

Abstract

Selective placement of an aliphatic β-alanine (β) residue paired side-by-side with either a pyrrole (Py) or imidazole (Im) aromatic amino acid is found to compensate for sequence composition effects for recognition of the minor groove of DNA by hairpin pyrrole−imidazole polyamides. A series of polyamides were prepared which contain pyrrole and imidazole aromatic amino acids, as well as γ-aminobutyric acid (γ) “turn” and β-alanine “spring” aliphatic amino acid residues. The binding affinities and specificities of these polyamides are regulated by the placement of paired β/β, Py/β, and Im/β residues. Quantitative footprint titrations demonstrate that replacing two Py/Py pairings in a 12-ring hairpin (6-γ-6) with two Py/β pairings affords 10-fold enhanced affinity and similar sequence specificity for an 8-bp target sequence. The 6-γ-6 hairpin ImPyImPyPyPy-γ-ImPyPyPyPyPy-β-Dp, which contains six consecutive amino acid pairings, is unable to discriminate a single-base-pair mismatch site 5‘-TGTTAACA-3‘ from a 5‘-TGTGAACA-3‘ match site. The hairpin polyamide Im-β-ImPyPyPy-γ-ImPyPyPy-β-Py-β-Dp binds to the 8-bp match sequence 5‘-TGTGAACA-3‘ with an equilibrium association constant of Ka = 2.4 × 1010 M-1 and ≥48-fold specificity versus the 5‘-TGTTAACA-3‘ single-base-pair mismatch site. Modeling indicates that the β-alanine residue relaxes ligand curvature, providing for optimal hydrogen bond formation between the floor of the minor groove and both Im residues within the Im-β-Im polyamide subunit. This observation provided the basis for design of a hairpin polyamide, Im-β-ImPy-γ-Im-β-ImPy-β-Dp, which incorporates Im/β pairings to recognize a “problematic” 5‘-GCGC-3‘ sequence at subnanomolar concentrations. These results identify Im/β and β/Im pairings that respectively discriminate G·C and C·G from A·T/T·A as well as Py/β and β/Py pairings that discriminate A·T/T·A from G·C/C·G. These aliphatic/aromatic amino acid pairings will facilitate the design of hairpin polyamides which recognize both a larger binding site size as well as a more diverse sequence repertoire.