Three-ring polyamides containing pyrrole (Py) and imidazole (Im) amino acids covalently coupled by gamma-aminobutyric acid (gamma) form six-ring hairpins that recognize five-base-pair sequences in the minor groove of DNA. Selective chiral substitution of the "gamma-turn" enhances the properties of polyamide hairpins with regard to DNA affinity and sequence specificity. Polyamides of core sequence composition ImPyPy-gamma-PyPyPy which differ by selective stereochemical substitution of the prochiral alpha-position in the gamma-turn were prepared. The DNA binding properties of two enantiomeric polyamides were analyzed by footprinting and affinity cleavage on a DNA fragment containing two match sites (5'-TGTTA-3' and 5'-ACATT-3') and one 5'-TGTCA-3' mismatch site. Quantitative footprint titrations demonstrate that replacement of gamma-aminobutyric acid by (R)-2,4-diaminobutyric acid enhances DNA binding affinity for the 5'-TGTTA-3' match site 13-fold (K-a = 3.8 x 10(9) M-1). The enhanced affinity is achieved without a compromise in sequence selectivity, which in fact increases and is found to be 100-fold higher relative to binding at a single base pair mismatch sequence, 5'-TGTCA-3'. An (S)-2,4-diaminobutyric acid linked hairpin binds with 170-fold reduced affinity relative to the R-enantiomer and only 5-fold sequence specificity versus a 5'-ACATT-3' reversed orientation site. These effects are modulated by acetylation of the chiral amine substituents. This study identifies structural elements which should facilitate the design of new hairpin polyamides with improved DNA binding affinity, sequence specificity, and orientational selectivity.