Discrimination of A/T sequences in the minor groove of DNA within a cyclic polyamide motif

Eight-ring cyclic polyamides containing pyrrole (Py), imidazole (Im), and hydroxypyrrole (Hp) aromatic amino acids recognize predetermined six base pair sites in the minor groove of DNA. Two four-ring polyamide subunits linked by (R)-2,4-diaminobutyric acid [(R)(H2N)gamma] residue form hairpin polyamide structures with enhanced DNA binding properties. In hairpin poly amides, substitution of Hp/Py for Py/Py pairs enhances selectivity for T.A base pairs but compromises binding affinity for specific sequences. In an effort to enhance the binding properties of polyamides containing Hp/Py pairings, four eight ring cyclic polyamides were synthesized and analyzed on a DNA restriction fragment containing three 6-bp sites 5'-tAGNNCTt-3', where NN = AA, TA, or AT. Quantitative footprint titration experiments demonstrate that contiguous placement of Hp/Py pairs in cy clo-(gamma -ImPyPyPy-(R)(H2N)gamma -ImHpHpPy-) (1) provides a 20-fold increase in affinity for the 5'-tAGAACTt-3' site (K-a = 7.5 x 10(7) M-1) relative to ImPyPyPy-(R)(H2N)gamma -ImHpHpPy-C3-OH (2). A cyclic polyamide of sequence composition cyclo-(gamma -ImHpPyPy-(R)(H2N)gamma -ImHpPyPy-) (3) binds a 5'-tAGTACTt-3' site with an equilibrium association constant K-a = 3.2 x 10(9) M-1, representing a fivefold increase relative to the hairpin analogue ImHpPyPy-(R)(H2N)gamma -ImHpPyPy-C3-OH (4). Arrangement of Hp/Py pairs in a 3'stagger regulates specificity of cyclo-(ImPyHpPy-(R)(H2N)gamma -ImPyHpPy-) (5) for the 5'-tAGATCT(t-3') site (K-a=7.5x 10(7) M-1), threefold increase in affinity relative to the hairpin analogue ImPyHpPy-(R)(H2N)gamma -ImPyHpPy-C3-OH (6), respectively. This study identifies cyclic polyamides as a viable motif for restoring recognition properties of polyamides containing Hp/Py pairs.