Probing the Cooperativity of Binding Networks with High-Throughput Thermophoresis

The formation of supramolecular complexes is found in many natural systems and is the basis for cooperative behavior. Here, we report on the development of a high-throughput platform to measure the complex binding behavior in 500 nL volumes and 1 536-well plates. The platform enabled us to elucidate the thermodynamic properties of a heterotrimeric DNA complex that portrays the structure of a biological relevant three-way junction. In a complementing set of cooperative networks, binding constants from ∼0.1 nM to ∼10 μM were measured by sampling a high-dimensional concentration space. Each intermediate binding state was probed simultaneously with only a single fluorescent label. Through systematic base pair variations, we observed the influence of the cooperative effect on single base pair mismatches. We further found coupled binding between seemingly independent binding sites through the complex structure of the three-way junction that could not have been observed without the measurement of the entire network. These results promote automated high-throughput thermophoresis to characterize arbitrary binding networks.