Here we present a novel strategy based on triplex-directed photo-crosslinking that can be used to introduce functionality and improve the structural integrity of DNA origami in a single one-pot reaction (Fig ). Triplex-forming oligonucleotides (TFOs) are sequence-specific DNA recognition agents that bind within the major groove of polypurine-polypyrimidine duplex sequences. TFOs have been used as a means to attach functional groups to DNA by the conjugation of non-nucleic acid components to the 5′ or 3′ ends of the oligonucleotide (e.g. fluorescent dyes, reactive groups, proteins, etc.). The site-specific incorporation of the attached functional group is achieved by targeting the TFO to appropriate polypurinepolypyrimidine sites embedded within the DNA sequence. TFOs have also been used to direct site-specific crosslinking reactions within DNA by attaching the photo-crosslinking agent 4,5,8-trimethylpsoralen (psoralen) to the 5′ end of the oligonucleotide. TFO binding directs psoralen intercalation at a TpA step flanking the 5′-end of the triplex-duplex junction.