Ultra‐Fast Isothermal Formation of DNA Nanostructures in Culture Media: Application to In Situ Assembly of DNA Origami With Living Cells

Synthetic DNA strands are programmable and biocompatible building blocks that can be combined through hybridization to form user-defined nanostructures, but their assembly traditionally requires cell-incompatible conditions, imposing a lengthy ex situ fabrication step before any application with living matter. Here we demonstrate for the first time that 2D and 3D DNA origami structures can isothermally self-assemble at 37°C within minutes, directly in cell culture media, both in the absence and in the presence of living cells. Scaffold-free structures of extended dimensions, such as micrometer-long DNA nanotubes, can also self-assemble when the system is given more time to evolve. With human cell lines, 2D and 3D origami structures in situ self-assemble in 5 to 15 min, and remain stable for about 24 h and up to 3 days when actin monomers are added. Similar self-assembly performance is observed in the presence of more complex tissue-like systems, such as human induced pluripotent stem cells evolving into cerebral organoids. This ultra-fast, life-compatible self-assembly method drastically simplifies the fabrication of complex DNA nanostructures and enables the creation of in situ self-assembling nanomachines for direct and adaptive interactions with living cells.