DNA tools for imaging and manipulation of biological samples

Spatial organization within biological systems is important for understanding mechanisms underlying health and disease. This thesis investigates the role of spatial distribution at cellular and molecular levels, utilizing DNA-based tools to elucidate its impact on receptor activation and creating methods for studying spatial organization of cells. The research of this thesis is structured into three studies, two focusing on clustering receptors with the aim of inducing apoptosis, one focusing on imaging and mapping cells with their transcriptomic contents.

In Paper I, we investigate the spatial clustering of Death Receptor 5 (DR5) on cellular surfaces, examining its significance in the induction of apoptosis. The study demonstrates that a hexagonal arrangement of DR5-binding peptides on DNA origami structures can induce apoptosis. We identify that a 6 nm spacing in this hexagonal shape is the optimal peptide spacing for apoptosis induction. This finding highlights the significance of spatial organization in receptor signaling and introduces DNA origami as a potential platform for cancer therapy.

Paper II expands on the foundation laid in Paper I with the development of a pH- sensitive DNA origami structure capable of selective DR5 activation in acidic tumor environments by using Triplex Forming Oligos (TFOs). This structure dynamically presents DR5-binding peptides in response to pH variations, thereby concentrating apoptotic activity within tumor sites where pH is lower than that of the normal physiological value. We demonstrate that the TFO forms a triplex at pH 6.5, but not pH 7.4, and can do so repeatedly. Furthermore, we demonstrate that this structure can induce apoptosis in cancer cells at pH 6.5, but not at pH 7.4, and does not induce apoptosis in other cell types at pH 7.4 either. Finally, we also show that injection of this structure into mice with growing tumors creates a reduction in tumor growth. The efficacy of this approach in inducing targeted apoptosis underscores the therapeutic potential of responsive DNA origami drug delivery systems.

In paper III, we propose a novel method for spatial transcriptomics that enables the mapping of DNA spatial organization without the use of optics. Through PCR and DNA sequencing, this technique allows us to generate a spatial map of biological samples, overlayed with transcriptomic data to illustrate the spatial distribution of gene expression. We demonstrate the capacity of this method to create pure sequencing libraries. Furthermore, we show that this technique can reconstruct spatial location of artificially placed targets, preserving their relative spatial relations to each other. This method offers a potential new way for examining spatial dimensions of cellular function and gene expression.

This thesis presents DNA-based methods in which the spatial organization of biological samples can be investigated or manipulated. The studies contribute to the advancement of targeted therapeutic interventions and spatial transcriptomics, offering insights that could inform future research and applications in biotechnology and medicine.

List of scientific papers

I. Yang Wang, Igor Baars, Ferenc Fördös, Björn Högberg. Clustering of Death Receptor for Apoptosis Using Nanoscale Patterns of Peptides. ACS Nano. 2021, 15(6), 9614−9626.
https://doi.org/10.1021/acsnano.0c10104

II. Yang Wang, Igor Baars, Iris Rocamonde Lago, Yunshi Yang, Ieva Berzina, Keying Zhu, Marco Lolaico, Boxuan Shen, Robert A Harris, Björn Högberg. Peptide-pattern display from DNA origami driven by cancer acidity for selective apoptosis. Nature Nanotechnology. [Accepted]

III. Igor Baars, Alexander Kloosterman, Marco Lolaico, Jakub Horváth, Anna Andersson, and Björn Högberg. Spatial reconstruction of a patterned sample using in situ PCR and DNA sequencing. [Manuscript]