Selective placement of an aliphatic beta-alanine (beta) 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 gamma-aminobutyric acid (gamma) "turn" and beta-alanine "spring" aliphatic amino acid residues. The binding affinities and specificities of these polyamides are regulated by the placement of paired beta/beta, Py/beta, and Im/beta residues. Quantitative footprint titrations demonstrate that replacing two Py/Py pairings in a 12-ring hairpin (6-gamma-6) with two Py/beta pairings affords 10-fold enhanced affinity and similar sequence specificity for an 8-bp target sequence. The 6-gamma-6 hairpin ImPyImPyPyPy-gamma-ImPyPyPyPyPy-beta-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-beta-ImPyPyPy-gamma-ImPyPyPy-beta-Py-beta-Dp binds to the 8-bp match sequence 5＇-TGTGAACA-3＇ with an equilibrium association constant of K-a, = 2.4 x 10(10) M-1 and greater than or equal to 48-fold specificity versus the 5＇-TGTTAACA-3＇ single-base-pair mismatch site. Modeling indicates that the p-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-P-Im polyamide subunit. This observation provided the basis for design of a hairpin polyamide, Im-beta-ImPy-gamma-Im-beta-ImPy-beta-Dp, which incorporates Im/beta pairings to recognize a "problematic" 5＇-GCGC-3＇ sequence at subnanomolar concentrations. These results identify Im/beta and beta/Im pairings that respectively discriminate G.C and C.G from A.T/T.A as well as Py/beta and beta/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.