With the aim of improving aqueous solubility, we designed and synthesized five N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides capable of recognizing 9-bp sequences. Their DNA-binding affinities and sequence specificities were evaluated by SPR and Bind-n-Seq analyses. The design of polyamide 1 was based on a conventional model, with three consecutive Py or Im rings separated by a beta-alanine to match the curvature and twist of long DNA helices. Polyamides 2 and 3 contained an 8-amino-3,6-dioxaoctanoic acid (AO) unit, which has previously only been used as a linker within linear Py-Im polyamides or between Py-Im hairpin motifs for tandem hairpin. It is demonstrated herein that AO also functions as a linker element that can extend to 2-bp in hairpin motifs. Notably, although the AO-containing unit can fail to bind the expected sequence, polyamide 4, which has two AO units facing each other in a hairpin form, successfully showed the expected motif and a K-D value of 16 nM was recorded. Polyamide 5, containing a beta-alanine-beta-alanine unit instead of the AO of polyamide 2, was synthesized for comparison. The aqueous solubilities and nuclear localization of three of the polyamides were also examined. The results suggest the possibility of applying the AO unit in the core of Py-Im polyamide compounds. (C) 2016 Elsevier Ltd. All rights reserved.