Engineered retrons generate genome-independent protein-binding DNA for cellular control

DNA-protein interactions are core components of myriad natural and synthetic gene networks. Despite the potential new design space, DNA-protein interactions remain underexploited in vivo due to challenges in controlling specific DNA segments, including protein-binding sequences, independently of the genome. Here we engineer retrons, prokaryotic retroelements, to intracellularly generate genome-independent programmable small DNA for sequence-specific protein-binding. Using reprogrammed retron-derived DNA for allosteric transcription factor, we demonstrated dynamic regulation of synthetic gene networks and construction of automated feedback circuits for signal amplification, adaptation, and memory. Furthermore, we developed a new class of stimuli-responsive molecular "bait and prey" that enable modular, rapid, and post-translational control of protein subcellular localization. This work substantially expands possible application area of DNA-protein interactions, laying the foundation for technical advances in synthetic biology. One-Sentence SummaryWe demonstrate new ways to control, design, and exploit DNA-protein interactions in living cells using engineered retron-generated small DNA.