The spatial organization of biochemical reaction systems by molecular nanoscaffolds

Cellular functions rely on the precise regulation of biochemical reactions to transform chemicals and energy, thereby supporting various living activities. The mimicking and replication of such control in artificial systems can offer promising applications in various fields. Spatial organization has been considered a key factor in affecting the efficiency and specificity of multienzyme cascade reactions. However, it remains a challenge to engineer the precise spatial arrangement of biomolecule systems and to investigate the mechanistic kinetics to affect the catalytic functions. In this dissertation, I systematically studied how enzymes were immobilized on nano- and microscale scaffolds with controlled spatial arrangements to modulate their catalytic functions. To achieve this, I first developed methods for chemical conjugation to assemble large protein and ligand complexes onto DNA nanoscaffolds. I also established a sepharose-based size exclusion chromatography method for efficient purification of DNA nanoparticles from unbound biomolecules. I designed and developed DNA nanostructure-based systems to control and study enzyme activity. My work involved using aptamer switches to modulate local substrate concentration and applying bioconjugation strategies, such as click chemistry, to attach enzymes onto DNA. I conducted a comprehensive analysis of enzyme immobilization strategies on microscale scaffolds and quantified their catalytic performance relative to free enzymes in solution. In parallel, I investigated the mechanistic basis for the enhanced catalytic activity of aptamer-linked hGQ, a topic that has long been debated in relation to substrate enrichment. Collectively, these projects aimed to explore how nanoscale organization and biomolecular interfaces influence catalytic performance in enzyme assemblies.These findings and developments have provided new insights into the spatial interaction of natural and biomimetic catalytic systems and will be useful for the development of biomimetic nanoreactors and nanostructures with potential applications in molecular sensing, drug delivery, and biocatalysis.