Expanding the recognition of the minor groove of DNA by incorporation of β-alanine in hairpin polyamides

Abstract

In order to expand the recognition code by hairpin polyamides to include DNA sequences of the type 5′-CWWC-3′ two polyamides, PyPyPyPy-(R)^(H_2^N)γ-ImPyPyIm-β-Dp (1) and PyPyPyPy-(R)^(H_2^N)γ-ImPy-β-Im-β-Dp (2) were synthesized which have in common an Py/Im pair in the terminal position for targeting C·G but differ with respect to internal placement of a β-alanine residue. 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 the eight-ring hairpin PyPyPyPy-(R)^(H_2^N)γ-ImPyPyIm-β-Dp (1) binds the four binding sites with similar affinities, K_a=1.3–1.9×10^(10) M^(−1) indicating that there is no preference for the position N. In contrast, a redesigned polyamide PyPyPyPy-(R)^(H_2^N)γ-ImPy-β-Im-β-Dp (2) that places an internal flexible aliphatic β-alanine to the 5′-side of a key imidazole group bound the match site 5′-TCTACA-3′ with high affinity and good sequence discrimination (K_a(match)=4.9×10^(10) M^(−1) and the single base pair mismatch sites with 5- to 25-fold lower affinity). These results expand the repertoire of sequences targetable by hairpins and emphasize the importance of β-alanine as a key element for minor groove recognition.