Polyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) amino acids can be combined in antiparallel side-by-side dimeric complexes for sequence-specific recognition in the minor groove of DNA. Covalently linking polyamide subunits has led to designed ligands with both increased affinity and specificity. Simple aliphatic amino acid linkers serve as internal guide residues for turn vs extended binding in a head-to-tail-linked polyamide motif. Polyamides of sequence composition ImPyPy-X-PyPyPy containing linkers of incremental length (X = 3-aminopropionic acid (beta), 4-aminobutyric acid (gamma), or 5-aminovaleric acid (delta)) in complex with an undecamer DNA duplex containing a 5'-(A,T)G(A,T)(3)-3' target site were structurally characterized using NMR spectroscopy. Previous quantitative DNase I footprinting studies identified gamma as the highest affinity of these ''turn'' linkers. NMR titrations and 2D NOESY data combined with restrained molecular modeling reveal that polyamides with beta, gamma, and delta linkers all may adopt a hairpin structure. Modeling supports the idea that the linkers in the beta and delta complexes adopt an energetically less favorable turn geometry than the gamma linker and confirms that the three-carbon gamma linker is sufficient and optimal for the hairpin conformation.