Strategies for the site-specific decoration of DNA origami nanostructures with functionally intact proteins

Abstract

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 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 3 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 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.

Stochastic modeling of antibody binding predicts programmable migration on antigen patterns

“…This is due to the design of the DNA origami nanostructure substrates in which hapten antigens are tethered by short, flexible spacers with rotational freedom. Future studies could explore rotational spatial tolerance as well as degrees of freedom in the Z direction by including additional terms in equation (1) calibrated with the PSPR data that systematically modulate relative epitope orientations or employ structures that incorporate pathogenic protein antigens [33][34][35][36] for more realistic structural complexity and physiological relevance.…”

DNA Framework-Programmed Ligand Positioning to Modulate the Targeting Performance

“…32−35 Based on the unique properties, DNA nanostructures could be applied to be optionally decorated with functional units to realize the manipulation of their self-structures and precise surface functionalization for the exploration of biological applications at the nanoscale, 36−38 such as the simulation of virus particles 39 and their surface modification regulation of functional molecules. 40,41 Inspired by the precise and programmable DNA frameworks, which offer high potential for spatial control of molecules in a quantitative manner, herein we report a DNA frameworkprogrammed approach to construct nanovectors with homogeneous ligand-modification patterns. A DNA tetrahedron (TDN) was used as a representative template for site-specific modification of model ligand folic acid (FA), an extensively applied tumor-targeting ligand, to obtain series FA-modification patterns.…”