Pairing niles have been developed to predict the sequence specificity of minor groove binding polyamides containing pyrrole (Py) and imidazole (Im) amino acids. An Im/Py pair distinguishes G . C from C . G and both of these from A . T/T . A base pairs. A Py/Py pair appears not to distinguish A . T from T . A base pairs. To test the extent of this degeneracy, the affinity and binding orientation of the hairpin polyamide ImPyPy-gamma-PyPyPy-beta-Dp were measured for eight possible five base pair 5'-TG(A,T)(3)-3' match sites. Affinity cleavage experiments using a polyamide with an EDTA . Fe(II) moiety at the carboxy terminus, ImPyPy-gamma-PyPyPy-beta-Dp-EDTA . Fe(II), are consistent with formation of an oriented 1:1 hairpin polyamide complex al all eight 5'-TG(A,T)(3)-3' binding sites [20 mM HEPES, 200 mM NaCl, 50 mg/ml glycogen, pH 7.0, 22 degrees C, 5 mM DTT, 1 mM Fe(II)]. Quantitative DNase I footprint titration experiments reveal that ImPyPy-gamma-PyPyPy-beta-Dp binds all eight 5'-TG(A,T)(3)-3' target sites with only a 12-fold difference in the equilibrium association constants between the strongest site, 5'-TGTTT-3' (K-a = 2.1 x 10(8) M(-1)), and the weakest site, 5'-TGAAT-3' (K-a = 1.8 x 10(7) M(-1)) (10 mM Tris . HCl, 10 mM KCI, 10 mM MgCl2, 5 mM CaCl2, pH 7.0, 22 degrees C). This relatively small range indicates that the Py/Py pair is approximately degenerate for recognition of A,T base pairs, affording generality with regard to targeting sequences of mixed A . T/T . A composition.
