Scaffold-based multi-enzyme assembly strategies have significantly advanced biocatalysis by enhancing reaction efficiency through precise spatial organization of enzymes. While DNA- and protein-based scaffolds have been extensively studied, RNA scaffolds present unique advantages, including structural flexibility, dynamic regulation, and functional diversity. However, their application in vitro has been limited due to challenges related to stability and cost. Here, we developed a programmable RNA scaffold system that leverages catalytically inactive MbpAgo to spatially organize natural protein macromolecules into multi-enzyme complexes for in vitro cascade reactions. This strategy significantly enhances the catalytic efficiency of multi-enzyme systems in vitro. We utilized Förster resonance energy transfer experiments demonstrated tunable protein localization along the scaffold. By designing short guide DNAs (gDNAs) to direct MbpAgo-enzyme assembly onto yeast ribosomal RNA scaffolds, we achieved precise positioning of three enzymes in the ATP biosynthesis pathway, resulting in a 5.5-fold increase in catalytic yield after 3 h compared to scaffold-free multi-enzyme complexes. Additionally, the modular design of the Ago-gDNA-RNA scaffold system allows for dynamic reconfiguration of enzyme arrangements through simple modifications of gDNAs, enabling adaptability to diverse multi-enzyme reactions. This study underscores the potential of Argonaute-mediated RNA scaffolds as a versatile and efficient platform for in vitro multi-enzyme assembly.

• PMID: 40334882
• DOI full text
• PubMed
• PubTator

Reference Type

Journal Article

Authors

Yan G, Li X, Yu X, Zhai C, Li W, Ma L

Primary Lit For

Additional Lit For

Review For