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

Abstract

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)γ] residue form hairpin polyamide structures with enhanced DNA binding properties. In hairpin polyamides, 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 cyclo-(γ-ImPyPyPy-(R)γ-ImHpHpPy-) (1) provides a 20-fold increase in affinity for the 5′-tAGAACTt-3′ site (K_a=7.5×10^7 M^(−1)) relative to ImPyPyPy-(R)γ-ImHpHpPy-C3-OH (2). A cyclic polyamide of sequence composition cyclo-(γ-ImHpPyPy-(R)γ-ImHpPyPy-) (3) binds a 5′-tAGTACTt-3′ site with an equilibrium association constant K_a=3.2×10^9 M^(−1), representing a fivefold increase relative to the hairpin analogue ImHpPyPy-(R)γ-ImHpPyPy-C3-OH (4). Arrangement of Hp/Py pairs in a 3′-stagger regulates specificity of cyclo-(γ-ImPyHpPy-(R)γ-ImPyHpPy-) (5) for the 5′-tAGATCTt-3′ site (K_a=7.5×10^7 M^(−1)), threefold increase in affinity relative to the hairpin analogue ImPyHpPy-(R)γ-ImPyHpPy-C3-OH (6), respectively. This study identifies cyclic polyamides as a viable motif for restoring recognition properties of polyamides containing Hp/Py pairs.

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