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

Abstract

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.

Atmospheric Pressure Cold Plasma: A Friendly Environment for Dry Enzymes

“…The machinery of life is based on chains of several enzymes that catalyse in cascade a series of selective reactions. To work properly, the different enzymes must be present at the optimal ratios and often in a given spatial arrangement . In principle, such an approach could be implemented in many analytical, industrial, and biotechnological applications .…”

DNA nanotweezers for stabilizing and dynamically lighting up a lipid raft on living cell membranes and the activation of T cells

“…2 Recently, DNA nanostructures with lipidic anchors were demonstrated to be promising membrane protein mimics that enable one to monitor molecular encounter events on living cell membranes. [33][34][35][36][37][38][39][40][41] In the present study, we demonstrate that DNA nanotweezers could stabilize and dynamically light up lipid ras on living cell membranes. The proposed DNA nanotweezers create an exogenous cholesterol-rich region on living cell membranes, which results in the recruitment of raassociated lipids and proteins, and possibly also endogenous cholesterol.…”

A Thermodynamic Limit on the Role of Self-Propulsion in Enhanced Enzyme Diffusion

“…Alternative technologies for measuring D t , such as NMR diffusion spectroscopy [47,48], may be informative. In addition, direct tracking of single enzyme molecules [49] could be useful both to confirm the phenomenon and also to provide details that might bear on mechanism.…”