KRED-Origami: Synthesis and Characterization of Ketoreductase Cascades Assembled on DNA Origami Nanostructures

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This project aims to harness concepts of DNA-nanotechnology for the production, characterization and understanding of functional, spatially organized biomolecular networks. We will synthesize and characterize supramolecular DNA origami constructs bearing distinctive arrangements of recombinant ketoreductases (KRED) and NAD(P)H regenerating enzymes (NRE). A chromatographic assay based on the stereoselective conversion of a CS-symmetric nitrodiketone will be used as read-out to generate quantitative data on the overall stereoselectivity of the supramolecular constructs as well as on the activity of the individual KREDs built into these constructs. Specific tasks include the molecular cloning of bioconjugatable KREDs and NREs, the establishment of site-selective DNA-ligation methods, the assembly and analysis of origami-based NRE-KRED cascades, and the study of cascades bearing both (S)- and (R)-selective KREDs to control the overall stereoselectivity of the constructs. Supported by theoretical work based on reaction-diffusion models, we plan to identify construction rules for effective systems which aid in the understanding of the fundamental mechanisms of spatially organized, diffusion-based, multi-center biocatalysts and open the door to future applications.