Functionalizing DNA Origami by Triplex-Directed Site-Specific Photo- Crosslinking

Here we present a universal method to introduce functionality and improve the structural integrity of DNA origami in a one-pot reaction. Our strategy involves adding nucleotide sequences to adjacent staple strands so that, upon origami assembly, the add-on sequences form short hairpin duplexes targetable by psoralen-labelled triplex-forming oligonucleotides (pso-TFOs) bearing other functionality. Subsequent irradiation with UVA light generates psoralen adducts with one or both hairpin staples leading to site-specific attachment of the pso-TFO to the origami with >80% efficiency. Bis-adduct formation between strands in proximal hairpins further tethers the TFO to the structure and generates ‘super-staples’ that improve the structural integrity of the complex. We also show that crosslinking reduces the sensitivity of the functionalized origami to thermal denaturation and disassembly by T7 RNA polymerase. Our strategy is scalable and cost-effective as it works with existing DNA origami structures, does not require scaffold redesign, and can be achieved with just one psoralen- modified oligonucleotide. It is also non-damaging to the origami scaffold, as well as to introduced fluorescent functionalities.