Construction of a CO 2 ‐Fixing Compartment Using a Shape‐Transforming DNA Scaffold

ABSTRACT A shape‐transforming three‐dimensional DNA nanoscaffold was constructed to mimic the confined microenvironment of a carboxysome for carbon dioxide (CO 2 ) fixation. The DNA scaffold, designed as a shallow hexagonal prism (SHP), exhibited a structural transition between open and closed states through DNA linker hybridization, allowing direct comparison of identical enzyme populations under distinct microenvironments. Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) was covalently immobilized onto the SHP through a modular CLIP‐GCN4 adaptor composed of a CLIP‐tag and GCN4 peptide. The quantitative and site‐specific assembly of the adaptor‐fused RuBisCO (CG‐RuBisCO) was confirmed by AFM imaging and electrophoretic mobility assays. The efficient structural transformation from the open to closed state was verified by fluorescence resonance energy transfer analysis. In the closed state, RuBisCO enzymes were brought into self‐contacting proximity, thereby mimicking the natural carboxysomal environment at the molecular level. A quantitative comparison between the CO 2 ‐fixing reaction in the open and closed states revealed that the profiles for both states were comparable. This reconfigurable DNA‐based compartment enables quantitative control over the number of enzymes and their spatial proximity, providing a versatile platform that facilitates the analysis of the effects of nanoscale confinement and spatial organization of enzymes on metabolic efficiency within synthetic microcompartments.