Applications of DNA origami encoded nanoscale patterns
Sammanfattning: It was almost four decades ago when the recognition of DNA’s potential use as a programmable, self-assembling building material for nanostructures led to the birth and rapid expansion of the field of DNA nanotechnology, but it was two decades later when the development of the DNA origami technique initiated the widespread use of DNA based nanoconstructs through the simplification of the design process and the reduction of the required control over the quality and stoichiometry of the assembly components by using a single-stranded “scaffold” DNA and a set of “staple” oligonucleotides that “fold” the mentioned scaffold DNA into a predesigned shape by binding different regions of the scaffold strand together. This robust approach not only permitted the construction of intricate two- and three-dimensional structures, but it also allowed the design and fabrication of molecular patterns with unprecedented accuracy as each functionalizible component’s relative position in the DNA origami structure is known to nanometer precision. In this thesis we utilize the DNA origami technology’s before mentioned patterning capability to create research tools for a diverse set of biomedical and biophysical applications. In paper I we studied the effect of different receptor ligand distributions in the ephrin/Eph signaling pathway by following the receptor activation in cancer cells stimulated with DNA origami probes displaying different, rationally designed Eph receptor ligand patterns. We found that incubation of cells with receptor ligands at shorter distance relative to each other led to significantly higher receptor activation and lower invasiveness of these cells. In paper II we used DNA origami to create reference samples for measuring the imaging accuracy of two of the most commonly used super resolution techniques, STED and STORM. We demonstrated that accuracy is a less biased metric for imaging faithfulness than precision and that DNA origami can be used to create a highly conserved and uniform pattern of fluorophores to measure and compare this metric for STED and STORM. In paper III we developed a DNA origami platform to study the photophysical behavior of two reversibly switchable fluorescent protein (rsFP) tags commonly used in microscopy in a quantitative, controlled fashion. With this system we were able to show that rsFPs at low numbers exhibit similar behavior to what was seen for them in bulk measurements, we could optimize imaging parameters more precisely and we could measure the achievable resolution using these samples. We were also able to show that some of the measured parameters scaled linearly with the amount of rsFPs making this DNA origami system a valuable calibration tool for quantitative imaging. In paper IV we developed a DNA origami-based optical tagging system detectable by next generation sequencing and super resolution microscopy to be used for introducing high resolution spatial information into RNA sequencing data. Using a combinatorial enzymatic approach, we were able to create a highly complex barcode library with which we successfully tagged cells and which we made compatible with one of the commonly used single cell RNA sequencing sample preparation techniques.
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