From blueprint to biodistribution : harnessing DNA nanotechnology for biomedical applications

<p dir="ltr">DNA nanotechnology has enabled the use of DNA as a versatile tool for various biomedical applications, including mapping of biological environments and the formation of precise nanoscale DNA structures. DNA origami nanostructures (DONs) are self-assembled architectures that enable the precise and tunable immobilization of bioactive molecules with nanometer-scale spatial control. DONs have been applied in a range of biomedi- cal contexts, from targeted drug delivery to the modulation of cellular communication. However, translating these advances into functional biomedical tools requires a deeper understanding of how design parameters influence biological interactions and in vivo behavior. To this end, this thesis explores strategies for characterizing, optimizing, and applying DNA nanostructures for targeted and biologically relevant applications, ranging from the characterization of blueprint functionalization to studies of their in vivo biodis- tribution.</p><p dir="ltr">To address the blueprint of DON functionalization, in <b>Paper I</b> we developed NanoSiTE (Nanostructure Site-occupancy characterization through Tag Extension), a sequencing- based platform for high-throughput characterization and quantification of DON func- tionalization. By encoding structure-, position-, and identity-barcodes, NanoSiTE en- ables high-resolution mapping of the spatial organization and molecular identity of DON- bound components across hundreds of thousands of nanostructures in a single assay. When applied to sheet-like DONs, NanoSiTE detected an unintentional error in the func- tionalization, which was further validated using DNA-PAINT super-resolution microscopy. Moreover, NanoSiTE provided site-specific information about the error that could not be resolved through DNA-PAINT analysis alone. This approach offers an accessible and design-agnostic strategy for the characterization of functionalized DONs, facilitating the broader application of DNA nanotechnology in nanomedicine.</p><p dir="ltr">The precise immobilization of bioactive ligands on DONs poses a promising platform for the development of multivalent therapies. In <b>Paper II</b>, we investigated the multivalent activation of insulin receptors (IRs) by arranging insulin ligands with varying spatial or- ganization and valency on DONs. Our results demonstrated that IRs form clusters at the cell membrane, and that multivalent activation of these clusters led to increased IR phosphorylation. Moreover, multivalent DONs significantly reduced free glucose levels in an in vivo zebrafish model. The multivalent insulin-DONs also elicited transcriptional responses comparable to those of monovalent insulin, but achieved these effects at sub- stantially lower ligand concentrations caused by a saturation of transcriptional signaling. These findings indicate that both the spatial organization and valency of insulin ligands strongly influence IR-mediated signaling, establishing these parameters as design vari- ables for fine-tuning receptor activation. This work provides insight into the multivalent applications of DONs and highlights the potential of exploiting multivalency as a strategy for targeting insulin resistance.</p><p dir="ltr">To translate the applications of DONs from in vitro performance to biomedical impact, it is essential to understand their in vivo properties. In <b>Paper III</b>, we employed zebrafish embryos as an in vivo model to investigate the effects of a stabilizing coating on DON biodistribution. We found that the coating not only influenced the biodistribution and cellular interactions of the DONs, but also mitigated their clearance by scavenger en- dothelial cells and macrophages. This work provides valuable insights into the in vivo behavior of DONs and establishes protocols for the use of zebrafish embryos as an in- termediate screening model for in vivo DON studies. Furthermore, employing zebrafish embryos as an intermediate model prior to experiments in higher-order animals aligns with the 3Rs (reduce, refine, replace) principles, offering ethical and practical advantages for in vivo investigation.</p><p dir="ltr">Finally, in <b>Paper IV</b>, we repurposed a click-chemistry-based approach, previously de- veloped for detecting drug targets in vivo, to decouple nanostructure pre-labelling from in situ detection. This method was applied to visualize the biodistribution of DONs in cleared and fixed tissues across multiple tissue types and spatial scales. Future work will focus on extending these analyses to whole-organ imaging. By eliminating the need for pre-incorporated fluorophores, which are susceptible to quenching during tissue pro- cessing and may induce undesired biological interactions, this approach enables high- resolution detection of DONs with single-cell precision in mouse models, while maintain- ing fluorescence integrity throughout extensive tissue processing.</p><p dir="ltr">In summary, the work presented in this thesis collectively advance the use of DNA nan- otechnology and DONs as versatile tools for biomedical applications. By addressing the complete trajectory from the blueprint functionalization to in vivo biodistribution, this work establishes new strategies for the characterization, optimization, and application of DONs. Together, these findings provide a foundation for the rational design of DNA- based nanomaterials and their future translation into precise and effective nanomedical platforms.</p><h3>List of scientific papers</h3><p dir="ltr">I. <b>Enya Engström</b>, Georges Kiriako, Tade Idowu, David F. Bonet, Joel Spratt, Yang Wang, Elena Ambrosetti, Ian T. Hoffecker, and Ana I. Teixeira. NanoSiTE: Sequencing-Based Characterization of Site-Occupancy in Functionalized Nanostructures through Combinatorial Barcoding [Manuscript]</p><p dir="ltr">II. Joel Spratt, José M. Dias, Christina Kolonelou, Georges Kiriako, <b>Enya Engström</b>, Ekaterina Petrova, Christos Karampelias, Igor Cervenka, Natali Papanicolaou, Anto- nio Lentini, Björn Reinius, Olov Andersson, Elena Ambrosetti, Jorge L. Ruas & Ana I. Teixeira. Multivalent Insulin Receptor Activation using Insulin-DNA Origami Nanostructures Nat. Nanotechnol. 19, 237-245 (2024) <a href="https://doi.org/10.1038/s41565-023-01507-y" rel="noreferrer" target="_blank">https://doi.org/10.1038/s41565-023-01507-y</a></p><p dir="ltr">III. Christina Kolonelou, <b>Enya Engström</b>, Lars Bräutigam, Steven Edwards, José M. Dias, Joel Spratt, Christos Karampelias, Iris Rocamonde-Lago, Björn Högberg, Stefan Wennmalm, Hjalmar Brismar, Olov Andersson, Ana I. Teixeira. Effects of oligolysine-Polyethylene Glycol Coating on the Biodistribution of Wireframe DNA origami Nanosheets Zebrafish Embryos ACS Nano 19 (36), 32145-32157 (2025) <a href="https://doi.org/10.1021/acsnano.5c05801" rel="noreferrer" target="_blank">https://doi.org/10.1021/acsnano.5c05801</a></p><p dir="ltr">IV. <b>Enya Engström</b>, Tade Idowu, Verina Leung, Cailynn Wang, Alexandra Selke, Christo- pher Glynn, Iris Rocamonde-Lago, Chayenne V. Tillack, Tess Edman, Jose M. Dias, Zhengyuan Pang, Björn Högberg, Li Ye and Ana I. Teixeira. In Situ Fluorescent Click-labelling of DNA Nanostructures for Subcellular Resolution Imaging In Vivo [Manuscript]</p>