Modular DNA origami compartments for the engineering of a protein unfolding and degradation pathway

Abstract 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 this compartmentalization principle to carry out regulated protein unfolding and degradation. Our model is composed of two connected DNA origami nanocompartments, one containing the protein unfolding machine, p97, and the other housing the protease chymotrypsin. We achieve the unidirectional immobilization of p97, establishing a ‘gateway’ mechanism that controls compartment accessibility and directionality of substrate processing. Our data show that, whereas spatial confinement increases the reaction rate of each individual enzyme, their physical connection into a chimera further improves their performance, minimizing off-target proteolysis. We anticipate that our modular approach may serve as a blueprint for reshaping biocatalytic pathways and stimulating the creation of nanofactories with capabilities beyond those observed in natural systems.