Pyrrole-imidazole (Py-Im) polyamides containing stereospecifically alpha-amino-or alpha-hydroxylsubstituted gamma-aminobutyric acid as a 5'-TG-3' recognition element were synthesized by machine- assisted Fmoc solid-phase synthesis. Their binding properties to predetermined DNA sequences containing a core binding site of 5'-TGCNCA-3'/3'-ACGN'GT-5' (N,N'= A center dot T, T center dot A, G center dot C, and C center dot G) were then systematically studied by surface plasmon resonance (SPR). SPR results revealed that the pairing of stereospecifically alpha-amino-/alpha-hydroxyl-substituted gamma-aminobutyric acids, (R or S)-alpha, gamma-diaminobutyric acid (gamma RN or gamma SN) and (R or S)-R-hydroxyl-alpha-aminobutyric acid (gamma RO or gamma SO), side-by-side with beta-alanine (beta) in such polyamides significantly influenced the DNA binding affinity and recognition specificity of hairpin polyamides in the DNA minor groove compared with beta/beta, and beta/gamma, pairings. More importantly, the polyamide Ac-Im-gamma SO-ImPy-gamma-ImPy beta Py-Dp(beta/gamma SO) favorably binds to a hairpin DNA containing a core binding site of 5'-TGCNCA-3'/3'-ACGN'GT-5' (N center dot N' = A center dot T with dissociation equilibrium constant (K-D) of 1.9 x 10(-7) M over N center dot N' = T center dot A with K-D = 3.7 x 10(-6) M, with a 19-fold specificity. By contrast, Ac-Im-gamma-SN-ImPy-gamma-Im beta Py-Py-beta-Dp (beta/gamma SN) binds to the above sequence with N center dot N' = A center dot T with K-D = 8.7 x 10(-7) M over N center dot N' = T center dot A with K-D 8.4 x 10(-6) M, with a 9.6-fold specificity. The results also show that the stereochemistry of the alpha-substituent, as well as the alpha-substituent itself may greatly alter binding affinity and recognition selectivity of hairpin polyamides to different DNA sequences. Further, we carried out molecular modeling studies on the binding by an energy minimization method, suggesting that alpha-hydroxyl is very close to N-3 of the 3'-terminal G to induce the formation of hydrogen bonding between hydroxyl and N3 in the recognition event of the polyamide Ac-Im-gamma SO-ImPy-gamma-ImPy beta Py-beta-Dp (beta/gamma SO) to 5'-TGCNCA-3'/3'-ACGN'GT-5' (N center dot N' = A center dot T). Therefore, SPR assays and molecular modeling studies collectively suggest that the (S)-alpha-hydroxyl-gamma-aminobutyric acid (gamma SO) may act as a 5'-TG-3' recognition unit.