Recognition of 5‘-(A,T)GG(A,T)_2-3‘ Sequences in the Minor Groove of DNA by Hairpin Polyamides

A series of four hairpin pyrrole−imidazole polyamides, ImImPy-γ-PyPyPy-β-Dp, PyPyPy-γ-ImImPy-β-Dp, AcImImPy-γ-PyPyPy-β-Dp, and AcPyPyPy-γ-ImImPy-β-Dp (Im = N-methylimidazole-2-carboxamide, Py = N-methylpyrrole-2-carboxamide, Dp = N,N-dimethylaminopropylamide, γ = γ-aminobutyric acid, β = β-alanine, and Ac = acetyl), designed for recognition of 5‘-(A,T)GG(A,T)_2-3‘ sequences in the minor groove of DNA were synthesized using solid phase methodology and analyzed with respect to DNA binding affinity and sequence specificity. Quantitative DNase I footprint titration experiments reveal that the optimal polyamide ImImPy-γ-PyPyPy-β-Dp binds a designated 5‘-TGGTT-3‘ match site with an equilibrium association constant of K_a = 1.0 × 10^8 M^(-1) and the single base pair mismatch sites, 5‘-TGTTA-3‘ and 5‘-GGGTA-3‘, with 50-fold and 100-fold-lower affinity, respectively (10 mM Tris·HCl, 10 mM KCl, 10 mM MgCl_2, and 5 mM CaCl_2, pH 7.0 and 22 °C). Polyamides of sequence composition AcImImPy-γ-PyPyPy-β-Dp and AcPyPyPy-γ-ImImPy-β-Dp, which differ only by the position of the γ-linker, bind with similar affinities and specificities. Recognition of sequences containing contiguous G·C base pairs expands the sequence repertoire available for targeting DNA with pyrrole−imidazole polyamides