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Leveraging Microfluidics for High‐Throughput Studies of Transcription Factor/DNA Binding

Gene expression is regulated primarily by transcription factors (TFs) that bind specific regulatory DNA sequences within the genome, thereby recruiting or repressing the transcriptional machinery to activate or repress transcription, respectively. However, despite decades of research, we remain largely unable to predict TF binding and/or downstream gene expression from regulatory sequence alone. Here, we leverage a microfluidic platform to yield quantitative, high‐throughput measurements of TF‐DNA binding affinities and dissociation rates for hundreds of basic helix‐loop‐helix TF variants interacting with varying DNA sequences. These data establish that sequences outside of known consensus sites can have surprisingly large effects on equilibrium binding and quantitatively map interactions across the TF‐DNA interface. We anticipate that these results will prove broadly useful for computational efforts to predict functional effects of rare mutations in precision medicine and promoter design in synthetic biology.