A comparison of H-pin and hairpin polyamide motifs for the recognition of the minor groove of DNA

In order to compare strategies for covalent linkage of pyrrole-imidazole (Py-Im) polyamide subunits, equilibrium association constants (K-a) were determined for a series of polyamides coupled either C-N with a gamma-aminobutyric acid linker (hairpin motif) or linked across a central Py/Py pair through a tetramethylene spacer (H-pin motif). Compared to the well-characterized hairpin motif, the H-pin motif represents a unique and relatively unexplored approach for increasing the affinity and the specificity of 2:1 polyamide-DNA complexes. Three H-pin polyamides containing six or 10 aromatic amino acid residues were synthesized by solid-phase methods using a Boc-protected bispyrrole monomer combined with bi-directional synthesis. The DNA-binding properties of six-ring and 10-ring H-pin polyamides were analyzed by quantitative DNase I footprint titration on a DNA fragment containing five or seven base pair match and mismatch sequences, respectively. The homodimeric H-Pin (ImPyPy-beta-Dp)(2)C-4 (site of the tetramethylene linker indicated in bold type) binds to the seven base pair match sequence 5'-TGTCA-3' with K-a = 9.3 x 10(6) M-1 and 9.4-fold specificity relative to the single base mismatch sequence 5'-TGT (T) under bar A-3' (K-a = 9.9 x 10(6) M-1). The heterodimeric H-Pin (ImPyPy-beta-Dp)C-4(AcPyPyPy-beta-Dp) binds a 5'-TGTTA-3' match sequence with K-a = 2.0 x 10(6) M-1 and 3.5-fold specificity versus the single base mismatch sequence 5'-TGT (C) under bar A-3' (K-a = 5.7 x 10(5) M-1). The 10-ring H-pin (ImPyPyPyPy-beta-Dp)(2)C-4 binds to the seven base pair match sequence 5'-TGTAACA-3' with K-a = 4.4 x 10(8) M-1 and 28-fold specificity versus the single base pair mismatch sequence 5'-TG (G) under bar AACA-3' (K-a = 1.6 x 10(7) M-1). We find that H-pin polyamides bind with four- to 180-fold enhanced affinity and two- to 10-fold enhanced specificity relative to unlinked analogues, but with 25- to 150-fold reduced affinity and approximately one- to 20-fold reduced specificity compared to the corresponding hairpin polyamides. These results indicate that H-pin polyamides represent a viable motif for the recognition of predetermined sequences in the DNA minor groove; however, DNA-binding properties appear inferior to the corresponding hairpins.