Effect of single pyrrole replacement with β-alanine on DNA binding affinity and sequence specificity of hairpin pyrrole/imidazole polyamides targeting 5′-GCGC-3′

N-Methylpyrrole (Py)-N-methylimidazole (Im) polyamides are small organic molecules that can recognize predetermined DNA sequences with high sequence specificity. As many eukaryotic promoter regions contain highly GC-rich sequences, it is valuable to synthesize and characterize Py-Im polyamides that recognize GC-rich motifs. In this study, we synthesized four hairpin Py-Im polyamides 1-4, which recognize 5′-GCGC-3′ and investigated their binding behavior with surface plasmon resonance assay. Py-Im polyamides 2-4 contain two, one, and one β-alanine units, replacing the Py units of 1, respectively. The binding affinities of 2-4 to the target DNA increased 430, 390, and 610-fold, respectively, over that of 1. The association and dissociation rates of 2 to the target DNA were improved by 11 and 37-fold, respectively, compared with those of 1. Interestingly, the association and dissociation rates of 3 and 4 were higher than those of 2, even though the binding affinities of 2, 3, and 4 to the target DNA were comparable to each other. The binding affinity of 2 to DNA with a 2 bp mismatch was reduced by 29-fold, compared with that to the matched DNA. Moreover, the binding affinities of 3 and 4 to the same mismatched DNA were reduced by 270 and 110-fold, respectively, indicating that 3 and 4 have greater specificities than 2 and are suitable as DNA-binding modules for engineered epigenetic regulation.