Cascaded Enzyme Reactions over a Three-Dimensional, Wireframe DNA Origami Scaffold

[Image: see text] DNA nanotechnology has increasingly been used as a platform to scaffold enzymes based on its unmatched ability to structure enzymes in a desired format. The capability to organize enzymes has taken many forms from more traditional 2D pairings on individual scaffolds to recent works introducing enzyme organizations in 3D lattices. As the ability to define nanoscale structure has grown, it is critical to fully deconstruct the impact of enzyme organization at the single-scaffold level. Here, we present an open, three-dimensional (3D) DNA wireframe octahedron which is used to create a library of spatially arranged organizations of glucose oxidase and horseradish peroxidase. We explore the contribution of enzyme spacing, arrangement, and location on the 3D scaffold to cascade activity. The experiments provide insight into enzyme scaffold design, including the insignificance of scaffold sequence makeup on activity, an increase in activity at small enzyme spacings of <10 nm, and activity changes that arise from discontinuities in scaffold architecture. Most notably, the experiments allow us to determine that enzyme colocalization itself on the DNA scaffold dominates over any specific enzyme arrangement.