Real-Time Imaging of Single-Molecule Enzyme Cascade Using a DNA Origami Raft

The dynamics of enzymes are directly associated with their functions in various biological processes. Nevertheless, the ability to image motions of single enzymes in a highly parallel fashion remains a challenge. Here, we develop a DNA origami raft-based platform for in-situ real-time imaging of enzyme cascade at the single-molecule level. The motions of enzymes are rationally controlled via different tethering modes on a two-dimensional (2D) supported lipid bilayer (SLB). We construct an enzyme cascade by anchoring catalase on cholesterol-labeled double-stranded (ds) DNA and glucose oxidase on cholesterol-labeled origami rafts. DNA functionalized with cholesterol can be readily incorporated in SLB via the cholesterol-lipid interaction. By using a total internal reflection fluorescence microscope (TIRFM), we record the moving trajectory of fluorophore-labeled single enzymes on the 2D surface: the downstream catalase diffuses freely in SLB, whereas the upstream glucose oxidase is relatively immobile. By analyzing the trajectories of individual enzymes, we find that the lateral motion of enzymes increases in a substrate concentration-dependent manner and that the enhanced diffusion of enzymes can be transmitted via the cascade reaction. We expect that this platform sheds new light on studying dynamic interactions of proteins and even cellular interactions.