Assembly and Purification of Enzyme-Functionalized DNA Origami Structures

The positioning of enzymes on DNA nanostructures for the study of spatial effects in interacting biomolecular assemblies requires chemically mild immobilization procedures as well as efficient means for separating unbound proteins from the assembled constructs. We herein report the exploitation of free-flow electrophoresis (FFE) for the purification of DNA origami structures decorated with biotechnologically relevant recombinant enzymes: the S-selective NADP(+)/NADPH-dependent oxidoreductase Gre2 from S.Cerevisiae and the reductase domain of the monooxygenase P450BM3 from B.megaterium. The enzymes were fused with orthogonal tags to facilitate site-selective immobilization. FFE purification yielded enzyme-origami constructs whose specific activity was quantitatively analyzed. All origami-tethered enzymes were significantly more active than the free enzymes, thereby suggesting a protective influence of the large, highly charged DNA nanostructure on the stability of the proteins.