Following the immuno-nanodecoder conceptual function, we have identified a promising strategy for its implementation. A proof-of-principle design has been achieved with a combination of activities described in WP1, WP2, and WP3. The core of the Nanodecoder structure consists of a self-assembled DNA tile termed Encoder (E), which is conjugated with the antibody through biotin-streptavidin recognition. E recognizes a fluorescently-labelled, self-assembled hybrid DNA-RNA tile (termed Decoder, D) through hybrid hybridization between two ssDNAs of E and two ssRNAs of D. Therefore, the entire D structure functions as an ON-code (which activate the fluorescence signal), while the OFF-code (which deactivate the fluorescence signal) consists in the action of the enzyme RNase H, which dissociates D from E by the degradation of the RNA probe involved in hybrid DNA-RNA duplex serving as linker between E and D. In this way, the Encoder returns in a completely reversible OFF state (in the sense that several ON-OFF-ON cycles can be implemented). Unlimited amounts of orthogonal Encoders and Decoders can be designed in principle just by varying the DNA-RNA sequence of E-D linker. According to our proposed technology, multiplexed immuno-fluorescence imaging would require cycling encoder-decoder association-followed-by-dissociation reactions, one for each E-conjugated antibody on the biological sample.
In parallel, according to WP4, we tested a prototypical immuno-nanodevice coupled to an antibody via biotin-streptavidin recognition, for the detection of MAGE proteins in cultured cells. A protocol for sample treatment was set up and preliminary data of immuno-nanodecoder functionality in fixed cells have been obtained. In WP5, similar tests were carried out using different cell lines including primary cells obtained from patients with the glycogenosis type II disease or healthy donors with the scope of optimizing developed protocols.
To implement a computational approach to study the biochemical reactions involved in the function of nanodecoder DNA nanostructures, we started from recent experimental results in our group on the action of restriction endonucleases on two-dimensional DNA origami such as the sharp triangle (Stopar et al., NAR 2018). We found that restriction endonuclease reactions in the DNA origami triangle can be tuned in a binary “on/off” manner. Our interpretation is that the intertwining of DNA in self-assembled nanostructures, as stabilized by Watson-Crick base-pairing, can introduce specific structural elements functioning as negative(anti) determinants of restriction endonuclease site reactivity, and could be used to program DNA reactivity. We modeled the system using oxDNA, a model designed to reproduce specific structural and thermodynamic properties of DNA, such as persistence length, bridging and melting temperature, and stochastic molecular dynamics simulations. In turn, we could provide the first theoretical characterization of the mechanical properties of a triangle DNA origami, and their effect on restriction enzyme binding. We found two metastable states for the triangle, with the free energy barrier separating them that can be decreased by the effect of a small defect in the DNA matrix. This leads to increased mechanical flexibility and higher accessibility for the enzyme. Our findings accurately reproduce experimental observations and support the notion that DNA origami show strong allosterism [1]
[1] Suma, A.; Stopar, A.; Nicholson, A. W.; Castronovo, M.; Carnevale, V., Allosteric modulation of local reactivity in DNA origami. bioRxiv 2019, 640847.
