Construction of synthetic protein-binding non-genetic DNA systems in living cells

Life's development and adaption to fluctuating environments are underpinned by DNA-protein interactions, which have also spurred the development of artificial systems ranging from genetic circuits to nanoarchitectures. Nonetheless, in cellulo there remains an untapped design space for DNA-protein systems that are incompatible with the role of DNA as genetic material. Here we engineer retrons to intracellularly express non-genetic small DNAs embedding specific protein-binding sequences. These DNA species are products of genes and therefore their quantitative, spatial and functional control is decoupled from genetic stability, enabling alternative architectures of DNA-protein systems with unique functions. Using synthetic networks of proteins and the engineered retron-DNA, we demonstrated precise, multiplexed gene regulation and construction of feedback circuits for dynamic responses. Further, we developed DNA-based molecular scaffolds and bridges that enable modular, post-translational and spatial control of multiple proteins within cells. Finally, we transformed an allosteric transcription factor into inducible post-translational switches. Our work suggests that the non-genetic DNA-protein systems represent a promising control layer for creating synthetic cellular behaviours.