To identify cancers and viral infections, T cell receptors (TCRs) scan the surface of cells in search of abnormal peptides presented by MHC molecules (pMHC). This TCR–pMHC interaction is largely responsible for activation of the T cell response. Recent evidence suggests that mechanical forces at TCR–pMHC bonds have a crucial role in downstream signalling. However, the exact mechanisms by which TCR-transmitted forces regulate immune responses remain unclear. To study the mechanical forces experienced by individual cells or molecules, researchers rely on tension sensors, which are molecular tools that are designed to detect and report on mechanical forces within a biological system. However, the existing probes are immobilized on hard substrates, which inhibits lateral motion and prevents TCR clustering, in addition to potentially overestimating force magnitudes. Such limitations make these techniques unsuitable for studying fluid TCR–pMHC interactions in the physiological context of cell membranes. To address this gap, we developed a new tool: DNA origami tension sensors (DOTS). DOTS enable the measurement of mechanical tension at dynamic intermembrane junctions.
DOTS are composed of a DNA origami base (a nanostructure of folded DNA), a force-sensitive DNA hairpin component, a variable number of DNA ‘legs’ for anchoring the structure to a lipid membrane, and a fluorescent reporter for identifying and tracking the nanostructure. The origami base/scaffold of DOTS is a 40 nm by 80 nm rectangular sheet, which is linked on one side to the DNA hairpin structure. This hairpin is labelled with a fluorophore–quencher pair and presents a pMHC ligand for TCR binding. When mechanical force is applied, the hairpin unfolds, which separates the fluorophore and the quencher to produce a measurable fluorescence signal. On the opposite side of the origami base are the single-stranded DNA legs, which allow the structure to embed into and move within the fluid lipid bilayers via complementary cholesterol-modified DNA strands. DOTS are also labelled by a second fluorophore that is used for tracking, density reporting and stoichiometric measurements. This reporter fluorophore must be spectrally separate from the fluorophore that indicates force and they must be spaced at a minimum of 20 nm apart to prevent unwanted interactions between the fluorophores. At the immune synapse, TCRs engage antigen-loaded DOTS, applying forces that pull the DNA hairpins open. Unlike rigid substrates, DOTS move laterally within the membrane, which enables the measurement of forces in a dynamic, fluid context. This mobility mimics physiological conditions, allowing the study of transient TCR forces and molecule clustering behaviour at the immune synapse.
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