Minutes-timescale in situ assembly of DNA origamis with living cells

Synthetic DNA strand is a highly accessible and biocompatible building block that can be synthesized in virtually any length and sequence, chemically modified, and combined with high specificity via hybridization. Applied within biological environments, in situ hybridization is a powerful biotechnological tool but limited to the use of small numbers of DNA strands. Ex vivo, this principle is the basis of structural DNA nanotechnology, in which hundreds of synthetic DNA strands are designed to self-assemble into highly programmable and versatile platforms, with applications ranging from drug delivery to immunobiology. However, the formation of such structures requires conditions such as temperature and ionic environments that are not only incompatible with living cells but also very long, imposing an ex situ fabrication step before any application with living matter. Here, we report that complex cocktails of DNA strands, coding for so-called DNA origamis, can faithfully self-assemble in situ at 37 °C, in a variety of common cell culture media and directly in the presence of living cells, in just a few minutes. We apply this approach for the in situ assembly of two- and three-dimensional DNA origamis in the presence of human cell lines in 2D culture as well as with 3D assemblies of human induced pluripotent stem cells successfully evolving into cerebral organoids. This life-compatible and highly programmable self-assembly method, requiring only a few minutes of incubation at 37 °C, drastically simplifies the preparation of user-defined complex DNA nanostructures and enables the creation of in situ self-assembling nanomachines for direct and adaptive interactions with living cells.