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Mapping gene regulatory networks is a significant challenge in systems biology, yet only a few methods are currently capable of systems-level identification of transcription factors (TFs) that bind a specific regulatory element. We developed a microfluidic method for integrated systems-level interaction mapping of TF-DNA interactions, generating and interrogating an array of 423 full-length Drosophila TFs. With integrated systems-level interaction mapping, it is now possible to rapidly and quantitatively map gene regulatory networks of higher eukaryotes.

Characterizing libraries of mutant proteins is a challenging task, but can lead to detailed functional insights on a specific protein, and general insights for families of proteins such as transcription factors. Challenges in mutant protein screening consist in synthesizing the necessary expression-ready DNA constructs and transforming them into a suitable host for protein expression. Protein purification and characterization are also non-trivial tasks that are not easily scalable to hundreds or thousands of protein variants. Here we describe a method based on a high-throughput microfluidic platform to screen and characterize the binding profile of hundreds of transcription factor variants. DNA constructs are synthesized by a rapid two-step PCR approach without the need of cloning or transformation steps. All transcription factor mutants are expressed on-chip followed by characterization of their binding specificities against 64 different DNA target sequences. The current microfluidic platform can synthesize and characterize up to 2,400 protein-DNA pairs in parallel. The platform method is also generally applicable, allowing high-throughput functional studies of proteins.

Gene regulatory networks (GRNs) consist of transcription factors (TFs) that determine the level of gene expression by binding to specific DNA sequences. Mapping all TF-DNA interactions and elucidating their dynamics is a major goal to generate comprehensive models of GRNs. Measuring quantitative binding affinities of large sets of TF-DNA interactions requires the application of novel tools and methods. These tools need to cope with the difficulties related to the facts that TFs tend to be expressed at low levels in vivo, and often form only transient interactions with both DNA and their protein partners. Our approach describes a high-throughput microfluidic platform with a novel detection principle based on the mechanically induced trapping of molecular interactions (MITOMI). MITOMI allows the detection of transient and low-affinity TF-DNA interactions in high-throughput.