Comparative Analysis of DNA-Binding Selectivity of Hairpin and Cyclic Pyrrole-Imidazole Polyamides Based on Next-Generation Sequencing

Many long pyrrole-imidazole polyamides (PIPs) have been synthesized in the search for higher specificity, with the aim of realizing the great potential of such compounds in biological and clinical areas. Among several types of PIPs, we designed and synthesized hairpin and cyclic PIPs targeting identical sequences. Bind-n-Seq analysis revealed that both bound to the intended sequences. However, adenines in the data analyzed by the previously reported Bind-n-Seq method appeared to be significantly higher in the motif ratio than thymines, even though the PIPs were not expected to distinguish A from T. We therefore examined the experimental protocol and analysis pipeline in detail and developed a new method based on Bind-n-Seq motif identification with a reference sequence (Bind-n-Seq-MR). High-throughput sequence analysis of the PIP-enriched DNA data by Bind-n-Seq-MR presented A and T comparably. Surface plasmon resonance assays were performed to validate the new method.