title: Evolution of a split RNA polymerase as a versatile biosensor platform.
abstract: Biosensors that transduce target chemical and biochemical inputs into genetic outputs are essential for bioengineering and synthetic biology. Current biosensor design strategies are often limited by a low signal-to-noise ratio, the extensive optimization required for each new input, and poor performance in mammalian cells. Here we report the development of a proximity-dependent split RNA polymerase (RNAP) as a general platform for biosensor engineering. After discovering that interactions between fused proteins modulate the assembly of a split T7 RNAP, we optimized the split RNAP components for protein-protein interaction detection by phage-assisted continuous evolution (PACE). We then applied the resulting activity-responsive RNAP (AR) system to create biosensors that can be activated by light and small molecules, demonstrating the 'plug-and-play' nature of the platform. Finally, we validated that ARs can interrogate multidimensional protein-protein interactions and trigger RNA nanostructure production, protein synthesis, and gene knockdown in mammalian systems, illustrating the versatility of ARs in synthetic biology applications.
authors.affiliation: Department of Chemistry, The University of Chicago, Chicago, Illinois, USA.
authors.forename: Jinyue
authors.initials: J
authors.lastname: Pu
authors.affiliation: Department of Chemistry, The University of Chicago, Chicago, Illinois, USA.
authors.forename: Julia
authors.initials: J
authors.lastname: Zinkus-Boltz
authors.affiliation: Department of Chemistry, The University of Chicago, Chicago, Illinois, USA.
authors.forename: Bryan C
authors.initials: BC
authors.lastname: Dickinson
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D002478:Cells, Cultured; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D025941:Protein Interaction Mapping / Q000379:methods
chemical_list: D012321:DNA-Directed RNA Polymerases