Tailoring the Nanoscale Environment of Enzymatic Cascades on 3D DNA Scaffolds

DNA nanotechnology has increasingly been used as a platform to scaffold materials based on its unmatched ability to create structure in a designed, desired format. Our group has focused on the organization of materials using 3D DNA scaffolds, ranging from individual DNA origami to mesoscale lattices. A particular source of interest across multiple disciplines has been the organization of enzymatic cascades, whereby enzymes can be arranged according to their associated substrates and intermediate exchange pathways. Yet while the concept of a nanoscale factory has been appealing, colocalization of enzymes on charged scaffolds introduces complex considerations arising from scaffold effects on the local reaction environment. The contribution of different effects makes it difficult to distill leading causes of activity changes associated with scaffolded enzymatic cascades. In working towards functional assemblies that present higher activity and guided reaction pathways, we can also shed light on questions of mechanistic causes for higher enzymatic activity on DNA scaffolds. Traditionally, this work has utilized 2D DNA scaffolds, but we utilize 3D wireframe DNA origami as a basis for our designs. Our group has organized enzymatic systems on multiple organizational scales in three dimensions, and has gained insight into the contribution of enzyme spacing, arrangement, and location on DNA scaffolds. We show that rather than being strongly dependent on specific spacing and DNA sequence makeup, enzyme activity is more strongly dependent on the nanoscale environment, providing experimental support that mesoscale organizations can yield significant enhancement in activity without direct substrate channeling between enzymes.