A DNA origami platform for single-pair Förster Resonance Energy Transfer investigation of DNA-DNA interactions and ligation

DNA double-strand breaks (DSBs) pose an everyday threat to the conservation of genetic information and therefore life itself. Several pathways have evolved to repair these cytotoxic lesions by re-joining broken ends, among them the non-homologous end-joining (NHEJ) mechanism that utilizes a DNA ligase. Here, we use a custom-designed DNA origami nanostructure as a model system to specifically mimic a DNA DSB, enabling us to study the end-joining of two fluorescently-labeled DNA double-strands with the T4 DNA ligase on the single-molecule level. The ligation reaction is monitored by Forster Resonance Energy Transfer (FRET) experiments both in solution and on surface-anchored origamis. Due to the modularity of DNA nanotechnology, DNA double-strands with different complementary overhang lengths can be studied using the same DNA origami design. We show that the T4 DNA ligase repairs sticky ends more efficiently than blunt ends and that the ligation efficiency is both influenced by DNA sequence and the incubati...