Hydroxybenzamide/Pyrrole Pair Distinguishes T·A from A·T Base Pairs in the Minor Groove of DNA

Abstract

A new aromatic pair, 2-hydroxy-6-methoxybenzamide/1-methylpyrrole at the terminal position of hairpin polyamides has been designed for distinguishing T·A from A·T base pairs and both from G·C/C·G in the minor groove of DNA. Four eight-ring hairpin polyamides with benzamide (Bz), 2-hydroxybenzamide (Hb-1), 2-hydroxy-6-methylbenzamide (Hb-2), and 2-hydroxy-6-methoxybenzamide (Hb-3) at the N-terminal position were synthesized. The equilibrium association constants (K_a) were determined at four DNA sites which differ at a single common position, 5‘-TNTACA-3‘ (N = T, A, G, C). Quantitative DNase I footprint titration experiments reveal that (Hb-3)PyPyPy-(R)^H2^Nγ-ImPyPyPy-β-Dp (4) bound the sequences 5‘-TTTACA-3‘ and 5‘-TATACA-3‘ with high affinity; K_a = 2.6 × 10^(10) M^(-1) and K_a = 8.4 × 10^9 M^(-1), respectively, a 3-fold specificity for T vs A was found. Importantly, the sequences 5‘-TGTACA-3‘ and 5‘-TCTACA-3‘ are bound with 50 and 200 times lower affinity, revealing an overall specificity of Hb-3/Py of T > A ≫ G > C. These results expand the repertoire of sequences targetable by hairpin polyamides.