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Assembly and purification of enzyme-functionalized DNA origami structures
Christopher Timm1, Christof M Niemeyer2
1Karlsruhe Institute of Technology (KIT), Institute for Biological Interfaces (IBG 1), Hermann-von-Helmholtz-Platz, 76344 Eggenstein-Leopoldshafen (Germany).
Angewandte Chemie (International Ed. in English)
|April 29, 2015
Summary
Free-flow electrophoresis purifies enzyme-DNA origami nanostructures. Tethered enzymes showed increased activity, suggesting DNA nanostructures protect protein stability.
Area of Science:
- Biochemistry
- Nanotechnology
- Molecular Biology
Background:
- Enzyme immobilization on DNA nanostructures requires mild conditions and efficient purification.
- Understanding spatial effects in biomolecular assemblies necessitates controlled enzyme positioning.
Purpose of the Study:
- To utilize free-flow electrophoresis (FFE) for purifying enzyme-decorated DNA origami nanostructures.
- To analyze the specific activity of immobilized enzymes and assess the influence of DNA nanostructures on protein stability.
Main Methods:
- Site-selective immobilization of recombinant enzymes (Gre2 and P450 BM3 reductase domain) onto DNA origami using orthogonal tags.
- Purification of enzyme-origami constructs via free-flow electrophoresis (FFE).
- Quantitative analysis of specific enzyme activity for both free and immobilized enzymes.
Main Results:
- Successful purification of enzyme-DNA origami constructs using FFE.
- Quantitatively analyzed specific activity revealed significantly higher activity in origami-tethered enzymes compared to free enzymes.
- The DNA nanostructure appears to confer a protective effect on enzyme stability.
Conclusions:
- Free-flow electrophoresis is an effective method for purifying enzyme-DNA nanostructures.
- DNA nanostructures can enhance the stability and activity of immobilized enzymes.
- This approach facilitates the study of spatial effects in biomolecular assemblies.
