IN VITRO TRANSCRIPTIONAL REGULATORY NETWORKS FOR AUTONOMOUS CONTROL OF BIOINSPIRED MATERIALS

Cells use genetic regulatory networks (GRNs) composed of interconnected genes that regulate one another to orchestrate complex behaviors such as differentiation, stress response, and self-healing. Many of the key dynamics identified in cellular GRNs have been recapitulated using synthetic chemistries in vitro, with emerging applications in autonomous control and dynamic regulation of materials. In this context, in vitro GRN analogs could imbue materials with the aforementioned capabilities of living systems. In vitro transcriptional circuits, composed of short synthetic genelets and a few inexpensive enzymes, have emerged as a simple, yet potentially powerful tool for assembling synthetic GRNs. However, only small genelet modules that exhibit a single function have been developed. Given cellular GRNs build complexity by integrating many functional modules together, identifying design rules that allow reliable integration of multiple genelet modules into larger networks is a critical step in developing sophisticated multifunctional GRN analogs. Here we report an updated genelet toolbox that enables the construction of large multifunctional regulatory networks. We develop the toolbox by identifying sources of undesired interactions between network components and designing strategies to mitigate these effects. Using these design principles, we assemble multi-module genelet networks that exhibit key functionalities inspired by cellular decision making and differentiation pathways. These results introduce a new class of mesoscale synthetic networks that can orchestrate increasingly complex regulatory processes by design. The genelet regulatory networks we construct can be programmed to autonomously control diverse nucleic acid-responsive materials such as nanostructures, hydrogels, and nanoparticle assemblies. Thus, in parallel to in vitro transcriptional circuit construction, we develop dynamic and reconfigurable DNA nanostructure architectures, inspired by the cellular cytoskeleton, that could be controlled by the RNA signals of our regulatory networks. A key challenge in developing autonomously controlled materials is coupling the materials with the regulating reaction networks, where compatibility, modularity, and crosstalk between components are critical considerations. Thus, we also highlight important design rules necessary for successful coupling of DNA-based materials with T7 RNA polymerase transcriptional systems and other nucleic acid-based reaction networks