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Nanostructured molecular decoders for the quantitative, multiplexed, layer-by-layer detection of disease-associated proteins

Periodic Reporting for period 2 - Immuno-NanoDecoder (Nanostructured molecular decoders for the quantitative, multiplexed, layer-by-layer detection of disease-associated proteins)

Periodo di rendicontazione: 2017-04-01 al 2019-03-31

Immunofluorescence and in situ hybridization imaging are established techniques for detecting tissue-specific, cumulative changes in protein or nucleic acid profiles associated with cancer development and progression. However, the current use of fluorochromes (typically one per biomarker) does not permit the co-localization of more than four different biomarkers in the same histological section, due to the paucity of fluorochromes with non- overlapping absorption/emission spectra. In addition, fluorochromes have qualitatively distinct fluorescence properties, which limits the determination of relative amounts of biomarkers to semiquantitative visual scoring.

The proposed method intends to overcome the aforementioned limits of fluorescence-based imaging to achieve definitive, multiplexed biomarker detection and quantification in situ. The value of the approach undertaken in this project is based on the premise that the quantification of only one or several markers is imprecise, but that the analysis of a larger set of defined biomarkers would permit accurate prognostic classification of the disease phenotype.

This project primarily aims to develop a prototypical, functional nanodevice and to demonstrate its potential as an innovative tool for detecting multiple disease-associated proteins belonging to two widespread, and etiologically different diseases: melanoma and glycogenosis type II, the latter being a rare genetic lysosomal storage disorder. In particular, we will focus our immuno-nanodecoders to (i) the analysis of Melanoma Antigen GEnes (MAGE) for an increased capability for disease classification, and (ii) the detection acid alpha-glucosidase (GAA) and selected proteins involved in the autophagic process to in vitro assess the efficacy of small molecules to revert the cellular pathologic phenotype of glycogenosis type II. Due to the complex molecular profiles involved in such disease (see below), their study will provide an ideal testing workbench for our immuno-nanodecoders.
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.
This project has provided a new approach to solve the problem of localizing biomolecules, including protein and nucleic acids, in biological samples in a quantitative and multiplexed fashion, and using standard fluorescence microscopy platforms, which are available in basically any biomedical and clinical laboratory.

The impact of the Research, Training and Collaborative activities of this project can be summarized as follows:
- It has led to a promising approach for the diagnosis and treatment of specific tissue-related diseases, to help refine the identification of novel biomarkers indicative of disease progression and the response to novel therapeutic approaches.
- It has led to a potential breakthrough technology that can open up new, potentially transformative approaches to clinical research, and with great potential for commercialization. In particular, this breakthrough technology may open up a new and potentially transformative approach to the characterization and treatment of high-impact as well as rare diseases, as well as the practice of molecular pathology.
- It has strengthened and internationalized the research network of the partners, leading to several new research initiatives between Italy, UK, Argentina and USA that address technological progress towards new approaches to biosensing and cell manipulation.
- It has trained a new generation of nanotechnologists, biochemists, biologists, and clinicians in the field of multiplexed and quantitative protein imaging and has prepared several European junior researchers to become the future leaders of this new field.