(66g) Multi-Enzyme Co-Localization On Nanoparticles Facilitated By DNA Hybridization

Multi-enzyme complexes (MECs) in Nature exhibit highly efficient catalytic mechanism(s) in reaction cascades. Researchers have developed several strategies to co-localize multiple enzymes on nano-carriers to improve multi-enzyme catalytic efficiency by mimicking the structure and function of MECs. Numerous studies have indicated that the spatial arrangement and orientation of multiple enzymes in confined space are of paramount importance in facilitating cooperative enzymatic activity in multi-enzyme co-localization. Biomolecule scaffolds based on DNA hybridization are very attractive for multi-enzyme co-localization because of their ability to enable unique effective control of the relative positions of different enzymes. In this work, glucose oxidase (GOX) and horseradish peroxidase (HRP) were co-localized onto polystyrene nanoparticles via DNA hybridization. The rationale behind selecting the particulate geometry in contrast to a planar surface is that the particulate geometry is more suitable for co-localization of multiple layers of enzymes due to lower steric hindrance. Free DNA hybridization and co-localization efficiency were studied using Förster resonance energy transfer (FRET) techniques. Various capture DNA concentrations were compared and optimized. The immobilization of one enzyme was studied initially to investigate the steric hindrance effect. Evidence of co-localization of GOX and HRP was obtained by FRET studies of dye-labeled tag DNAs. Finally, it was demonstrated that co-localizing GOX and HRP via DNA hybridization significantly improved the overall reaction efficiency in comparison to a mixture of immobilized single enzymes. In summary, DNA-directed co-localization of multiple enzymes provides excellent control of the relative positions of the enzymes, thereby mimicking MECs, and leading to high reaction efficiency.