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

ABSTRACT 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.