Design and Assembly of a Cargo-Agnostic Hollow Two-Lidded DNA Origami Box

DNA origami, a method of folding DNA into precise nanostructures, has emerged as a powerful tool for the design of complex nanoscale shapes. It has great potential as a technology to encapsulate and release cargos spanning small molecules through large proteins, while remaining stable in a variety of <i>ex vivo</i> processing conditions and <i>in vivo</i> environments. While DNA origami has been utilized for drug delivery applications, the vast majority of these structures have been flexible, flat 2D or solid 3D nanostructures. There is a crucial need for a hollow and completely enclosed design capable of holding and eventually releasing a variety of cargos. In this paper, we present the design and assembly of a hollow DNA origami box with two lids. We characterize the isothermal conditions for structural assembly within minutes. We demonstrate that passive loading of small molecules is charge dependent. We also outline an approach to design staple extensions pointing into the cavity or outside of the hollow DNA origami, allowing for the active loading of protein or the potential for decoration with passivating or targeting molecules.