Enzyme-Free Ligation of 5'-Phosphorylated Oligodeoxynucleotides in a DNA Nanostructure.

Multicomponent reactions are difficult synthetic transformations. For DNA, there is a special opportunity to align multiple strands in a folded nanostructure, so that they are preorganized to give a specific sequence. Multistrand reactions in DNA origami structures have previously been performed using photochemical crosslinking, 1,3-diploar cycloadditions or phosphoramidate-forming reactions. Here we report carbodiimide-driven phosphodiester formation in a small origami sheet that produces DNA strands up to 600 nucleotides in length in a single step. The method uses otherwise unmodified oligodeoxynucleotides with a 5'-terminal phosphate as starting materials. Compared to an enzymatic multistrand ligation involving linear duplexes, the carbodiimide-driven ligation gave fewer side products, as detected by gel electrophoresis. The full-length 600mer product was successfully amplified by polymerase chain reaction.