An Orthogonal Covalent Connector System for the Efficient Assembly of Enzyme Cascades on DNA Nanostructures

Combining structural DNA nanotechnology with the virtually unlimited variety of enzymes offers unique opportunities for generating novel biocatalytic devices. However, the immobilization of enzymes is still restricted by a lack of efficient covalent coupling techniques. The rational re‐engineering of the genetically fusible SNAP‐tag linker is reported here. By replacing five amino acids that alter the electrostatic properties of the SNAP_R5 variant, up to 11‐fold increased coupling efficiency with benzylguanine‐modified oligonucleotides and DNA origami nanostructures (DON) was achieved, resulting in typical occupancy densities of 75%. The novel SNAP_R5 linker can be combined with the equally efficient Halo‐based oligonucleotide binding tag (HOB). Since both linkers exhibit neither cross‐reactivity nor non‐specific binding, they allowed orthogonal assembly of an enzyme cascade consisting of the stereoselective ketoreductase Gre2p and the cofactor‐regenerating isocitrate dehydrogenase on DON. The cascade showed approximately 1.6‐fold higher activity in a stereoselective cascade reaction than the corresponding free solubilized enzymes. The connector system presented here and the methods used to validate it represent important tools for further development of DON‐based multienzyme systems to investigate mechanistic effects of substrate channeling and compartmentalization relevant for exploitation in biosensing and catalysis. Through rational protein engineering, an effective, genetically fusible connector for site‐selective immobilization of enzymes on DNA nanostructures is developed. Due to the greater than tenfold increase in coupling efficiency, a biocatalytic cascade reaction for the stereoselective reduction of carbonyl compounds is established by combining it with an equally efficient orthogonal connector.