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J. Hellmeier, R. Platzer, V. Mühlgrabner, M. Schneider, E. Kurz, G. Schütz, J. Huppa, E. Sevcsik:
"Strategies for the Site-Specific Decoration of DNA Origami Nanostructures with Functionally Intact Proteins";
ACS Nano, 15 (2021), 15057 - 15068.

DNA origami structures provide flexible scaffolds for the
organization of single biomolecules with nanometer precision. While
they find increasing use for a variety of biological applications, the
functionalization with proteins at defined stoichiometry, high yield,
and under preservation of protein function remains challenging. In
this study, we applied single molecule fluorescence microscopy in
combination with a cell biological functional assay to systematically
evaluate different strategies for the site-specific decoration of DNA
origami structures, focusing on efficiency, stoichiometry, and protein
functionality. Using an activating ligand of the T-cell receptor (TCR)
as the protein of interest, we found that two commonly used
methodologies underperformed with regard to stoichiometry and
protein functionality. While strategies employing tetravalent wildtype
streptavidin for coupling of a biotinylated TCR-ligand yielded mixed populations of DNA origami structures featuring up to
three proteins, the use of divalent (dSAv) or DNA-conjugated monovalent streptavidin (mSAv) allowed for site-specific
attachment of a single biotinylated TCR-ligand. The most straightforward decoration strategy, via covalent DNA conjugation,
resulted in a 3-fold decrease in ligand potency, likely due to charge-mediated impairment of protein function. Replacing DNA
with charge-neutral peptide nucleic acid (PNA) in a ligand conjugate emerged as the coupling strategy with the best overall
performance in our study, as it produced the highest yield with no multivalent DNA origami structures and fully retained
protein functionality. With our study we aim to provide guidelines for the stoichiometrically defined, site-specific
functionalization of DNA origami structures with proteins of choice serving a wide range of biological applications.

DNA origami, DNA nanostructures, protein conjugation, functionalization, single molecule fluorescence microscopy, T-cell activation

http://dx.doi.org/10.1021/acsnano.1c05411

https://publik.tuwien.ac.at/files/publik_297527.pdf