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

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.