An Orthogonal Covalent Connector System for the Efficient Assembly of Enzyme Cascades on DNA Nanostructures.
Small
; 17(51): e2105095, 2021 12.
Article
em En
| MEDLINE
| ID: mdl-34825457
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.
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1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Nanoestruturas
Idioma:
En
Revista:
Small
Assunto da revista:
ENGENHARIA BIOMEDICA
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Alemanha