AbstractCells execute complex transcriptional programs by deploying distinct protein regulatory assemblies that interact with cis-regulatory elements throughout the genome. Using concepts from DNA nanotechnology, we synthetically recapitulated this feature in cell-free gene networks actuated by T7 RNA polymerase (RNAP). Our approach involves engineering nucleic-acid hybridization interactions between a T7 RNAP site-specifically functionalized with single-stranded DNA (ssDNA), templates

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... g cis-regulatory ssDNA domains, and auxiliary nucleic-acid assemblies acting as artificial transcription factors (TFs). By relying on nucleic-acid hybridization, de novo regulatory assemblies can be computationally designed to emulate features of protein-based TFs, such as cooperativity and combinatorial binding, while offering unique advantages such as programmability, chemical stability, and scalability. We illustrate the use of nucleic-acid TFs to implement transcriptional logic, cascading, feedback, and multiplexing. This framework will enable rapid prototyping of increasingly complex in vitro genetic devices for applications such as portable diagnostics, bio-analysis, and the design of adaptive materials.