Recognition of the sequences 5'-NGCACA-3' (N = T, A, C, G) by pyrrole/imidazole polyamides with (R/S)-alpha-hydroxyl/alpha-amino-substituted gamma-aminobutyric acid as a gamma-turn was investigated. Four novel polyamides, 2, 3, 4, and 5, including (R)-alpha-hydroxyl-gamma-aminobutyric acid (gamma RO), (S)-alpha-hydroxyl-gamma-aminobutyric acid (gamma SO), (R)-alpha, gamma-diaminobutyric acid (gamma RN), and (S)-alpha, gamma-diaminobutyric acid (gamma SN) residues, respectively, were synthesized, and their binding affinity to T.A, A.T, G.C, and C.G base pairs at turn position was studied by the surface plasmon resonance (SPR) technique. SPR data revealed that polyamide 3, Aclm beta lmPy-gamma SO-lmPy beta Py-beta-Dp, with a gamma SO turn, possesses a marked binding preference for T.A over A.T with a 25-fold increase in specificity, despite low binding affinity relative to 2, with a gamma RO turn. Similarly, Aclm beta lmPy-gamma SN-lmPy beta Py-beta-Dp (5), with a gamma SN-turn, gives rise to a 8.7-fold increase in specificity for T.A over A.T. Computer-assisted molecular modeling suggests that 3 binds more deeply in the minor groove of the T.A base pair relative to the A.T base pair, allowing hydrogen bonding to O2 of the thymine at the turn position, which explains the SPR results. These results suggest that gamma SO and gamma SN may function as T-recognition units at the turn position, as well as a gamma-turn in the discrimination of polyamides.