DNA three-way junction (3WJ) structures with three amino acid side chains in the core have been synthesized via post-synthetic DNA modification. Amide condensation reactions of oligonucleotides containing 2'-aminouridine with activated esters yielded DNA strands modified with His, Cys and Asp side chains to form modified 3WJs. Even a 3WJ with three negatively charged Asp side chains formed stably at room temperature. Furthermore, DNA hybridization alone placed two (His and Asp) and three (His, Cys, and Asp) side chains within the 3WJs, indicating that the DNA 3WJs are a useful platform for spatial arrangement of amino acid side chains.