Spatial control of protein binding with DNA nanostructures

<p dir="ltr">The physical and chemical properties of DNA, including its structure predictability thanks to Watson-Crick base pairing, make it into an obvious polymer of choice to use as a biomaterial for the fabrication of complex three dimensional nanostructures. These nanostructures are produced by the technique of DNA origami, where a long single stranded circular DNA, called scaffold, is folded into a pre-designed shape by the hybridization of partially complementary oligonucleotides, the staples. The programmability of DNA origami, i.e. the specific control, by design, of the location of every DNA sequence within the structure, can be harnessed for the positioning of molecules with nanoscale precision. In the present thesis, we have explored different branches of the DNA origami technology, from its functionalization with proteins, its structural characterization and its application for method development, targeted treatment and study of molecular processes taking place at the nanoscale.</p><p dir="ltr">In Paper I, we develop an approach to quantify the incorporation of proteins in DNA origami using DNA-PAINT, a multiplexed super-resolution imaging method that allows the characterization of structures with single-molecule resolution and high reliability. Even as protein-decorated DNA nanostructures become increasingly important tools for biomedical sciences, the existing strategies to study and maximize protein incorporation provide limited insight into functionalization. With DNA-PAINT, we were able to explore factors influencing incorporation rate such as oligonucleotide quality, protein size, or purification method, rank their impact, and model their combined effects. Thus, we were able to offer a comprehensive view of functionalization efficiency and precise parameters for yield optimization, which can have significant implications for the application of DNA origami in fields beyond academia.</p><p dir="ltr">In Paper II, we introduce the new method PLASTIQ for assessing DNA origami structural integrity in vivo with a detection sensitivity of 0.01 femtomolar. Despite the potential of DNA origami in therapeutics, the lack of structural assessment methods for in vivo use hampers its clinical initiation. Sampling only 1 ul of blood, PLASTIQ allowed us to monitor the degradation patterns of nanostructures over time. We examined the protective effects of PEGylation and obtained the pharmacokinetic profiles of different origamis. Additionally, we were able to observe differential degradation of structural regions depending on how exposed they were to the environment. Altogether, PLASTIQ is an accurate tool for assessing structural stability, offering valuable insights for advancing DNA origami-based drug development.</p><p dir="ltr">In Paper III, we present PANMAP, a method based on antigen patterning on DNA nanostructures to measure antibody affinity while taking multivalency into account. Multivalency is fundamental in many biological systems, especially in antibody-antigen interactions where multiple binding sites improve affinity and specificity. However, conventional affinity assays such as ELISA provide only a limited perspective on binding characterization and do not address multivalency. With PANMAP overcoming these limitations, we found that antibody binding equilibrium is influenced by antigen spacing, leading to competitive exclusion at close distances, optimal bivalent binding at intermediate distances, and a monovalent regime at longer distances. Thus, PANMAP enabled a complete profile of multivalency and the binding states that constitute it, potentially providing useful insights into biological processes and engineering applications.</p><p dir="ltr">In Paper IV, we apply DNA origami displaying Jag1 ligands to stimulate neuroepithelial stem-like cells to study the molecular mechanism leading to Notch receptor activation. The Notch pathway is a highly evolutionarily conserved signaling system that plays a key role in embryonic and nervous system development. However, it is unclear how the activation unravels, with the leading hypothesis being force-driven conformational changes. Here, we demonstrate that Notch triggering can occur without pulling forces and that it instead proceeds upon extended binding. These findings suggest an alternative molecular mechanism for receptor activation, suggesting potential for the design of soluble agonists.</p><p dir="ltr">In Paper V, we engineer a pH-responsive DNA robotic switch that selectively displays death receptor ligands only in acidic tumor microenvironments to induce apoptosis of cancer cells. As these receptors are responsible for initiating cell death but are ubiquitously expressed on the membranes of most cells, a targeted approach for their use in tumor therapies is desired. The DNA robotic switch hides the ligands, arranged in a hexagonal pattern inside a cavity while at neutral pH, until encountering pH 6.5 where the ligands are revealed, leading to clustering of DR and triggering apoptosis of breast cancer cells. Our results probe the functionality of the nanodevice in vitro and shows the significant tumor volume reduction in mice with human breast cancer xenografts. Overall, this work highlights the potential for targeted cancer treatment using DNA origami.</p><h3>List of scientific papers</h3><p dir="ltr">1. <b>Iris Rocamonde-Lago</b>, Ferenc Fördös, Cagla Sahin, Ian T. Hoffecker & Björn Högberg. Exploring DNA origami protein functionalization using super resolution imaging. [Manuscript]</p><p dir="ltr">II. Yang Wang*, <b>Iris Rocamonde-Lago</b>*, Janine Waldvogel, Shuya Zang, Igor Baars, Alexander Kloosterman, Boxuan Shen, Ian T. Hoffecker, Qin He & Björn Högberg. DNA origami structural integrity tracked in vivo using proximity ligation. [Manuscript]</p><p dir="ltr">III. <b>Iris Rocamonde-Lago</b>, Ieva Berzina, Ian T. Hoffecker & Björn Högberg. Profiling of multivalent binding with DNA origami reveals spatial determinants of antigen-antibody interactions. [Manuscript]</p><p dir="ltr">IV. Ioanna Smyrlaki, Ferenc Fördös, <b>Iris Rocamonde-Lago</b>, Yang Wang, Boxuan Shen, Antonio Lentini, Vincent C. Luca, Björn Reinius, Ana I. Teixeira & Björn Högberg. Soluble and multivalent Jag1 DNA origami nanopatterns activate Notch without pulling force. Nature Communications, 15(1), 465. <a href="https://doi.org/10.1038/s41467-023-44059-4" rel="noreferrer" target="_blank">https://doi.org/10.1038/s41467-023-44059-4</a></p><p dir="ltr">V. Yang Wang, Igor Baars, Ieva Berzina, <b>Iris Rocamonde-Lago</b>, Boxuan Shen, Yunshi Yang, Marco Lolaico, Janine Waldvogel, Ioanna Smyrlaki, Keying Zhu, Robert A. Harris & Björn Högberg. A DNA robotic switch with regulated autonomous display of cytotoxic ligand nanopatterns. Nature Nanotechnology, 19(9), 1366-1374. <a href="https://doi.org/10.1038/s41565-024-01676-4" rel="noreferrer" target="_blank">https://doi.org/10.1038/s41565-024-01676-4</a></p><p dir="ltr">*Shared first authorship</p>