Reaction enhancement via product scavenging and phase separation-driven pathway canalization

Abstract Targeted biomolecular modification requires recognizing and binding to extended sequence domains, which can limit product dissociation rates and, therefore, catalytic efficiency. Product removal by downstream enzymes or condensates, common in biological catalysis, pushes catalytic reactions toward product formation, but cannot overcome intrinsically low product off-rates. Here, we demonstrate that designed affinity reagents, termed scavengers, can actively displace a product from an enzyme. Designed scavengers for three nucleic acid enzymes increase catalytic turnover up to 39-fold. Additionally, co-localizing enzymes and substrates, but not scavengers, within a biomolecular condensate further enhances >6-fold in catalytic rate by biasing productive substrate-enzyme interaction over inhibitory substrate-scavenger complexation. Scavenging and colocalization can be combined into a single generalizable and programmable framework, multiphase-controlled product scavenging (MCPS). By enabling rapid and exquisitely specific biomolecular modification, MCPS could lead to the development of potent nucleic acid enzyme therapeutics or efficient, controllable pathways for biomolecular modification, cleavage, and synthesis.