Systematic analysis of biomolecular binding reactions using microfluidic systems

Transcription factor binding to a single binding site on DNA and its functional consequence in a promoter context are beginning to be relatively well understood. However, binding to clusters of sites has yet to be characterized in depth, and the functional relevance of binding site clusters remains uncertain. We employed a high-throughput biochemical method to characterize transcription factor binding to clusters varying across a range of affinities and configurations. We found that transcription factors can bind concurrently to overlapping sites, challenging the notion of binding exclusivity. Furthermore, compared to an individual high-affinity binding site, small clusters with binding sites an order of magnitude lower in affinity give rise to higher mean occupancies at physiologically-relevant transcription factor concentrations in vitro. To assess whether the observed in vitro occupancies translate to transcriptional activation in vivo, we tested low-affinity binding site clusters by inserting them into a synthetic minimal CYC1 and the native PHO5 S. cerevisiae promoter. In the minCYC1 promoter, clusters of low-affinity binding sites can generate transcriptional output comparable to a promoter containing three consensus binding sites. In the PHO5 promoter, replacing the native Pho4 binding sites with clusters of low-affinity binding sites recovered activation of these promoters as well. This systematic characterization demonstrates that clusters of low-affinity binding sites achieve substantial occupancies, and that this occupancy can drive expression in eukaryotic promoters. In the antibody discovery phase, before advancing a particular antibody sequence to clinical trials, a large number of candidates are characterized for properties of therapeutic interest. Above all, this includes their ability to bind to the target antigen. Standard processes currently in use to characterize antibody binding are either only semi-quantitative (ELISA), or low-throughput (SPR). We developed an experimental process to first retrieve DNA encoding promising synthetic antibody variants directly from the output pools of phage and ribosome display methods, followed by expressing these variants on a microfluidic chip for characterization in a fluorescence-based binding assay. We applied our method to characterize the binding of retrieved Sybody variants against the Spike trimer and RBD domain of the SARS-CoV-2 variant. After an initial proof-of-concept, we improved our method by removing bottlenecks, reducing costs, and incorporating a large degree of process automation to enable measuring binding affinities at scale.