(189o) De Novo Design of DNA Origami Spheres As Biological Tools

Complex geometries like curves and spheres are found both in artificial constructs and in nature. Spheres and globular structures with intricate 3D curvature are inherently advantageous in nature because of their high surface area to volume ratio as well as their ability to carry or deliver cargo loads. Using molecular design and assembly approaches, there are examples of nanospheres built from polymers, lipids, and proteins. However, these nanospheres are often limited by symmetry, lack of size control, and their inability to be precisely modified. Using DNA as a material to build nanostructures can overcome these limitations with the advantages of self-assembly, precise biological functionality, and size control. DNA origami nanotechnology is a technique that relies on the self-assembly of a long-strand of single-stranded DNA (ssDNA) called a “scaffold” and hundreds of ssDNA oligos called “staples”. The staples bind in a piecewise complementary manner to the scaffold to form a predefined geometry. Advances in DNA origami design tools have pushed the capabilities of macromolecular structure production, especially in the 3D space. Previous DNA origami sphere designs were often limited by design heuristics and relied on tricks by skipping bases or automating 2D curvature with precise staple crossovers between helices to incorporate flexibility and curvature. These spheres are not truly closed with structural gaps ranging from 6 to 10nm due to the limitations in radius of curvature of DNA and therefore cannot encapsulate or trap biomolecules effectively without leakage.

Inspired by functional spherical structures coupled with recent developments of de novo DNA origami design in the 3D space using an algorithmic approach, we can now design fully closed DNA origami spheres. We overcome common limitations by designing spheres in a “tennis-ball”-like shape with two half spheres fortified by a “seam”. By using an algorithmic approach, we can map the correct number of double-stranded DNA (dsDNA) helices in each half and the number of helices in the “seam” to respect the inter-helix distance of the dsDNA helices given a chosen radius. Here, we designed a library of monolayer DNA spheres ranging from 6 to 50 nanometers in radius showing size control and geometric complexity. We experimentally validated the designs and imaged well-folded spheres using cryo-electron microscopy. With this molecular design and assembly approach, we can envision using the DNA spheres in a variety of applications such as encapsulating and delivering nucleic acids for gene therapy or drug delivery, creating nano bio-reactors, or for molecular tracking.