The researchers, led by Veikko Linko and Mauri Kostiainen from Aalto University, use a single-stranded, circular DNA scaffold, which upon assembly forms a rod-shaped DNA origami, measuring 16 nm in diameter and 107 nm in length. It is composed of 24 parallel double helix bundles (24HB origami). For surface functionalization, Seitz et al. exploit the highly negatively charged surface of the DNA origami for electrostatic immobilization of a two-component protein coating comprising a targeting moiety and an inert camouflaging entity. In their approach, the proteins of interest are covalently attached via their cysteine residues to the single N-maleimide group on a synthetic dendron. The latter comprises spermine groups that serve as the positively charged counterparts for electrostatic assembly on the DNA origami. To render the camouflaging protein-dendron complexes photoresponsive, the authors incorporate photolabile o-nitrobenzyl groups that are cleaved in response to ultraviolet light exposure, triggering release of camouflaging protein. “Key features in our system are that the camouflaging agent is serum albumin, the most abundant protein in blood circulation. This enables spatiotemporal control over the protection and targeting of the underlying DNA nanostructure,” says Kostiainen.
To test the feasibility of their two-component approach Seitz et al. develop a fluorescence-based immunoassay to detect breast cancer marker protein human epidermal growth factor receptor 2 (HER2) on a solid phase. For the specific recognition of HER2 as an antigen, the authors used small, genetically engineered antibody fragments called single-chain variable fragments (scFv) derived from anti-HER2 monoclonal antibodies. These anti-HER2 scFvs are conjugated to the non-photoresponsive dendrons and function as the analyte recognition element on the DNA origami. For fluorescence readout, fluorescent dye ATTO488-tagged DNA is integrated into the origami structure by hybridization to staples containing a 3′ overhang. In proof-of-principle photoreversibility experiments, the researchers demonstrate that UV irradiation between 3–5 minutes is sufficient to fully dissociate the camouflaging serum albumin and restore the binding of anti-HER2 scFvs to the breast cancer marker, while the non-photolabile camouflaged controls fail to do so. It is important to note that their light addressable assay remains functional even in the presence of serum proteins up to 10% in cell culture media. Concerning resistance against nucleases, the authors show camouflaging with serum albumin prevents DNA origami degradation by DNAse for up to an hour at physiological temperature, with negligible impact on assay performance.
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