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

Abstract

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.

Switchable and dynamic G-quadruplexes and their applications

“…An important advance in DNA nanotechnology has involved the programmed assembly of higher order assemblies, such as Yshaped or cross-over Holliday junction DNA assemblies, 91 twodimensional or three-dimensional DNA origami structures 92 or three-dimensional DNA tetrahedra. 93,94 The engineering of these assemblies allows the programmed positioning of nucleic acids strands on the scaffold, allowing the positioning of protruding tethers that act as anchoring units for binding auxiliary constituents such as enzymes 95 or nanoparticles. 96,97 In addition, the decoration of the edges of these structures with functional nucleic acids allows the introduction of dictated nucleic acid catalysts or stimuli-responsive tethers that control switchable assembly and dissociation of DNA nanostructures.…”

Mechanistic Aspects for the Modulation of Enzyme Reactions on the DNA Scaffold

“…Compared with the 2D DNA scaffolds, the 3D DNA scaffolds provide more ideal space for the spatial arrangement of enzymes. Kahn et al [ 104 ] created a library of 3D DNA wireframe octahedron structures for the spatial organization of GOx and HRP. The contribution of enzyme spacing, arrangement and location on the 3D DNA scaffold to cascade efficiency was investigated ( Figure 13 b).…”

“…( a ) Three types of enzyme, malic dehydrogenase/oxaloacetate decarboxylase/lactate dehydrogenase (LDH/MDH/OAD), co-assembled on a three-point star DNA scaffold to produce lactic acid from malic acid [ 103 ]. ( b ) The spatial arrangement of gOx and HRP on the DNA wireframe octahedrons [ 104 ]. Reprinted with permission from: ( a ) Ref.…”

The enhancement of enzyme cascading via tetrahedral DNA framework modification

“…For example, a recent study utilized a 3D DNA wireframe octahedron to organize glucose oxidase and horseradish peroxidase and deciphered the influence of the spatial organization on the cascade enzymatic activity. 25…”

“…For example, a recent study utilized a 3D DNA wireframe octahedron to organize glucose oxidase and horseradish peroxidase and deciphered the influence of the spatial organization on the cascade enzymatic activity. 25 Despite the remarkable progress that has been made in recent years, there are still limitations to cluster enzymes in vitro. One of the big challenges that remain unsolved in this field is how to accurately immobilize enzymes on the scaffold without influencing their activity.…”