Temperature effects on the stability and mobility of DNA origami platforms and his-tag pMHC on supported liipid bilayers

Abstract

Supported lipid bilayers (SLBs) provide a modular, laterally fluid biointerface for presenting membrane-tethered ligands in reconstituted T cell activation assays. In this thesis, the temporal stability of SLB-based ligand presentation was assessed for SLB-anchored rectangular DNA origami platforms as well as peptide-MHC (pMHC) complexes. Experiments were performed at 26 →C and 37 →C. Temporal stability was analyzed over observation windows of approximately 120–200 minutes following washing of SLB-wells, with typically three to four measurements acquired per SLB during this time period. Time-resolved TIRF microscopy was used to quantify (i) lateral diffusion via single-particle tracking and (ii) normalized bulk fluorescence intensity as a proxy for surface coverage. In addition, single-molecule brightness served as a control metric for tracking quality and potential aggregation effects. Across conditions, diffusion coefficients remained temporally stable within the observation window, indicating that ligand mobility is preserved over time and that no progressive immobilization or loss of membrane fluidity occurs on experimentally relevant timescales. Bulk fluorescence intensities were either stable or showed only gradual, well-resolved changes, consistent with overall robust surface presentation rather than rapid ligand depletion. Importantly, the absence of pronounced time-dependent brightness increases argues against substantial aggregation-driven artifacts, supporting the reliability of the mobility measurements. Together, these results demonstrate that the investigated SLB-based membrane systems provide sufficiently stable and laterally mobile ligand presentation over the timescales relevant for downstream experiments in tested conditions. This temporal robustness supports their suitability as standardized platforms for T cell activation assays, where controlled surface densities and sustained ligand mobility are essential to probe activation thresholds and immunological synapse formation under physiologically relevant conditions.