Adler, N. A.; Antelo, G. T.; Villarruel Dujovne, M.; Rondon, J. J.; Le, M. T.; Giedroc, D. P.; Peinetti, A. S.; Capdevila, D. A.

In vitro transcription (IVT) systems regulated by allosteric transcription factors (aTFs) are central to emerging cell-free biosensing and synthetic biology platforms, yet their performance is often limited by suboptimal protein DNA interactions and the need for well-characterized regulatory elements. Here, we report an in vitro evolution strategy to engineer DNA operator sequences that enables tunable and multivalent aTF DNA interactions without requiring prior detailed knowledge of the native operator or regulatory mechanism. Using a SELEX-based approach with integrated positive and counter-selection steps, we evolved non-natural operators for the sulfane sulfur responsive transcriptional repressor SqrR. The selected sequences preserve ligand-responsive allostery while enhancing binding affinity and reducing transcriptional leakage. Notably, we identify operator architectures that promote multivalent protein DNA interactions, resulting in improved repression efficiency and dynamic range compared to the native operator. Incorporation of these evolved operators into IVT circuits enables the development of the first ROSALIND based sensor for sulfane sulfur species, achieving sensitive and selective detection in a fully cell free format. More broadly, this work establishes programmable operator evolution as a generalizable strategy to optimize transcription factor DNA interactions and expand the applicability of transcription-based biosensors to less-characterized systems.