A modular DNA origami nanocompartment for engineering a cell-free, protein unfolding and degradation pathway.

Within the cell, chemical reactions are often confined and organized through a modular architecture. This facilitates the targeted localization of molecular species and their efficient translocation to subsequent sites. Here we present a cell-free nanoscale model that exploits compartmentalization strategies to carry out regulated protein unfolding and degradation. Our synthetic model comprises two connected DNA origami nanocompartments (each measuring 25 nm × 41 nm × 53 nm): one containing the protein unfolding machine, p97, and the other housing the protease chymotrypsin. We achieve the unidirectional immobilization of p97 within the first compartment, establishing a gateway mechanism that controls substrate recruitment, translocation and processing within the second compartment. Our data show that, whereas spatial confinement increases the rate of the individual reactions by up to tenfold, the physical connection of the compartmentalized enzymes into a chimera efficiently couples the two reactions and reduces off-target proteolysis by almost sixfold. Hence, our modular approach may serve as a blueprint for engineering artificial nanofactories with reshaped catalytic performance and functionalities beyond those observed in natural systems.