Abstract
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.
Highlights
Design of SNAP-Tag VariantsThe efficient application of the SNAP-tag as a connector for DNA nanostructures requires a high affinity toward the extremely dense negative charges on the DNA scaffold surface
Introduction orybbR,[6] they still do not always result in good occupancy densities of enzymes on the DNA origami nanostructures (DON).[7]
Since these self-assembling structures can be and the negatively charged nucleic acid nanostructures. To this used as frameworks for arranging proteins with nanometer end, by rational engineering of the binding interface of the precision, a variety of applications are emerging in the field Halo tag domain, five amino acids located around the entry of sensing, biocatalysis, or as tools for studying biological pro- channel for the chlorohexyl ligand (CH) of the Halo-tag processes.[2]
Summary
The efficient application of the SNAP-tag as a connector for DNA nanostructures requires a high affinity toward the extremely dense negative charges on the DNA scaffold surface. We chose to investigate the above described amino acids regarding their influence on DNA affinity and their use as an optimized immobilization tag, generating four different variants, which were compared to the conventional SNAP-tag (Figure 1B). The AGT from Sulfolobus solfataricus (SsOGT_wt)[18] as well as an engineered variant SsOGT_H5[19] were described as tags for extremophilic organisms, as they possess a high thermostability Since they have a specific binding ability to BG substrates comparable to the conventional SNAP-tag, we wanted to test whether they could be used for protein immobilization in DNA-based applications
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