Spatial Organization of Enzyme Cascade on a DNA Origami Nanostructure

: Self-assembled DNA nanostructures hold great promise to organize multi-enzyme systems with the precise control of the geometric arrangements. Enzymes modified with single-stranded DNA anchors are assembled onto the DNA origami tiles by hybridizing with the corresponding complementary strands displayed on the surface of the DNA nanostructures. Here, we describe a protocol of assembling a two-enzyme cascade on a discrete, rectangular DNA origami tile, where the distance between enzymes is precisely controlled for investigating the distance-dependent cascade activities.