The molecular structures of enzyme mimics may be modified to optimize their catalytic properties. In this study, to generate artificial enzyme mimics, Watson-Crick base paired DNA duplexes were designed as scaffolds which were assembled by nucleotides modified with specific functional groups. This process allowed various functional groups to be precisely assembled at different sites on the duplexes. By using this strategy, the 5-[2-(1H-imidazolyl-4)-(E)-ethylene]-2'-deoxythymidine (1) analog with the 5-substituted imidazolyl group was incorporated into single strands of DNA. Upon DNA duplex formation, several combinations of the imidazolyl group were formed. Using p-nitrophenyl acetate as the substrate of the catalytic reaction, we evaluated the hydrolysis capabilities of the imidazolyl assemblies. The catalytic ability was closely related to the distribution of imidazolyl groups in the DNA duplex. The most effective catalytic center was that of the duplex 05-06 construct with three imidazolyl groups. This construct displayed bell-shaped pH-dependent and Mg2+-independent kinetic curves, which are typical characteristics of imidazolyl-mediated catalytic reactions. (C) 2013 Elsevier Inc. All rights reserved.