Optimizing a DNA origami-based model system to assess T- cell activation

Abstract

The role of microclustering of T-cell receptors (TCRs) in T-cell activation, and whether pre-clustered ligands can promote this clustering and thus facilitate activation, remains a subject of ongoing investigation. A convenient approach to investigate this question is to use model systems, as they allow researchers to dissect biological processes at the molecular level. In this thesis, an existing protocol for the creation of such a model system based on DNA origami and supported lipid bilayers (SLBs) was optimized with the aim of producing a reliable system that could be used for the assessment of T-cell activation. To this end, the thermal folding process of DNA origami, the purification of folded platforms, the functionalization of DNA origami platforms, and the anchoring of platforms to SLBs were analyzed and refined. An optimized thermal folding protocol was developed, with a focus on reliability as well as time efficiency. Ultra-filtration was identified as a more efficient purification method than agarose gel electrophoresis due to its reduced labor intensity and time requirements. Platform anchoring was found to be more stable when mediating cholesterol anchoring with MgCl2 , and platform binding efficiency of pMHC was found to be clearly higher when functionalizing platforms on the day of the experiment. In conclusion, the work provided insight into the essential steps in the creation of a DNA origami-based model system, helping to create a reliable model system for the assessment of T-cell activation and enabling researchers to make informed decisions for the creation of a protocol of their own.