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

Kröll, Sandra ORCID iD icon; Rabe, Kersten S. ORCID iD icon; Niemeyer, Christof M. ORCID iD icon


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.

Verlagsausgabe §
DOI: 10.5445/IR/1000141020
Veröffentlicht am 09.12.2021
DOI: 10.1002/smll.202105095
Zitationen: 8
Web of Science
Zitationen: 8
Zitationen: 9
Cover der Publikation
Zugehörige Institution(en) am KIT Institut für Biologische Grenzflächen (IBG)
Publikationstyp Zeitschriftenaufsatz
Publikationsjahr 2021
Sprache Englisch
Identifikator ISSN: 1613-6810, 1613-6829
KITopen-ID: 1000141020
HGF-Programm 43.33.11 (POF IV, LK 01) Adaptive and Bioinstructive Materials Systems
Erschienen in Small
Verlag John Wiley and Sons
Band 17
Heft 51
Seiten Art.Nr. 2105095
Vorab online veröffentlicht am 25.11.2021
Nachgewiesen in Scopus
Web of Science
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