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Enhanced Catalysis from Multienzyme Cascades Assembled on a DNA Origami Triangle

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  • William P. Klein, National Research Council (IBFM-CNR)
  • ,
  • Rasmus P. Thomsen
  • ,
  • Kendrick B. Turner, National Research Council (IBFM-CNR)
  • ,
  • Scott A. Walper, National Research Council (IBFM-CNR)
  • ,
  • James Vranish, Ave Maria Univ
  • ,
  • Jorgen Kjems
  • Mario G. Ancona, US Naval Res Lab, United States Department of Defense, United States Navy, Naval Research Laboratory, Elect Sci & Technol Div Code 6800
  • ,
  • Igor L. Medintz, National Research Council (IBFM-CNR)

Developing reliable methods of constructing cell-free multienzyme biocatalytic systems is a milestone goal of synthetic biology. It would enable overcoming the limitations of current cell-based systems, which suffer from the presence of competing pathways, toxicity, and inefficient access to extracellular reactants and removal of products. DNA nanostructures have been suggested as ideal scaffolds for assembling sequential enzymatic cascades in close enough proximity to potentially allow for exploiting of channeling effects; however, initial demonstrations have provided somewhat contradictory results toward confirming this phenomenon. In this work, a three-enzyme sequential cascade was realized by site-specifically immobilizing DNA-conjugated amylase, maltase, and glucokinase on a self-assembled DNA origami triangle. The kinetics of seven different enzyme configurations were evaluated experimentally and compared to simulations of optimized activity. A 30-fold increase in the pathway's kinetic activity was observed for enzymes assembled to the DNA. Detailed kinetic analysis suggests that this catalytic enhancement originated from increased enzyme stability and a localized DNA surface affinity or hydration layer effect and not from a directed enzyme-to-enzyme channeling mechanism. Nevertheless, the approach used to construct this pathway still shows promise toward improving other more elaborate multienzymatic cascades and could potentially allow for the custom synthesis of complex (bio)molecules that cannot be realized with conventional organic chemistry approaches.

TidsskriftACS Nano
Sider (fra-til)13677-13689
Antal sider13
StatusUdgivet - dec. 2019

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