TY - JOUR
T1 - Modular Imaging Scaffold for Single-Particle Electron Microscopy
AU - Aissaoui, Nesrine
AU - Lai-Kee-Him, Josephine
AU - Mills, Allan
AU - Declerck, Nathalie
AU - Morichaud, Zakia
AU - Brodolin, Konstantin
AU - Baconnais, Sonia
AU - Le Cam, Eric
AU - Charbonnier, Jean Baptiste
AU - Sounier, Rémy
AU - Granier, Sébastien
AU - Ropars, Virginie
AU - Bron, Patrick
AU - Bellot, Gaetan
N1 - Publisher Copyright:
©
PY - 2021/3/23
Y1 - 2021/3/23
N2 - Technological breakthroughs in electron microscopy (EM) have made it possible to solve structures of biological macromolecular complexes and to raise novel challenges, specifically related to sample preparation and heterogeneous macromolecular assemblies such as DNA-protein, protein-protein, and membrane protein assemblies. Here, we built a V-shaped DNA origami as a scaffolding molecular system to template proteins at user-defined positions in space. This template positions macromolecular assemblies of various sizes, juxtaposes combinations of biomolecules into complex arrangements, isolates biomolecules in their active state, and stabilizes membrane proteins in solution. In addition, the design can be engineered to tune DNA mechanical properties by exerting a controlled piconewton (pN) force on the molecular system and thus adapted to characterize mechanosensitive proteins. The binding site can also be specifically customized to accommodate the protein of interest, either interacting spontaneously with DNA or through directed chemical conjugation, increasing the range of potential targets for single-particle EM investigation. We assessed the applicability for five different proteins. Finally, as a proof of principle, we used RNAP protein to validate the approach and to explore the compatibility of the template with cryo-EM sample preparation.
AB - Technological breakthroughs in electron microscopy (EM) have made it possible to solve structures of biological macromolecular complexes and to raise novel challenges, specifically related to sample preparation and heterogeneous macromolecular assemblies such as DNA-protein, protein-protein, and membrane protein assemblies. Here, we built a V-shaped DNA origami as a scaffolding molecular system to template proteins at user-defined positions in space. This template positions macromolecular assemblies of various sizes, juxtaposes combinations of biomolecules into complex arrangements, isolates biomolecules in their active state, and stabilizes membrane proteins in solution. In addition, the design can be engineered to tune DNA mechanical properties by exerting a controlled piconewton (pN) force on the molecular system and thus adapted to characterize mechanosensitive proteins. The binding site can also be specifically customized to accommodate the protein of interest, either interacting spontaneously with DNA or through directed chemical conjugation, increasing the range of potential targets for single-particle EM investigation. We assessed the applicability for five different proteins. Finally, as a proof of principle, we used RNAP protein to validate the approach and to explore the compatibility of the template with cryo-EM sample preparation.
KW - DNA origami
KW - electron microscopy
KW - molecular template
KW - nanotechnology
KW - single-particle imaging
UR - http://www.scopus.com/inward/record.url?scp=85101578231&partnerID=8YFLogxK
U2 - 10.1021/acsnano.0c05113
DO - 10.1021/acsnano.0c05113
M3 - Article
C2 - 33586425
AN - SCOPUS:85101578231
SN - 1936-0851
VL - 15
SP - 4186
EP - 4196
JO - ACS Nano
JF - ACS Nano
IS - 3
ER -