In vitro evolution of DNA operators enables multivalency protein−DNA interactions: towards programmable transcription factor regulation

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 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, with some sequences exhibiting enhanced binding affinity and reducing transcriptional leakage. Notably, we identify operator with binding behaviors consistent with cooperative recruitment of multiple SqrR dimers, suggesting that sequence architecture can modulate aTF–DNA interactions beyond affinity alone. Incorporation of these operators into IVT circuits improves transcriptional control and dynamic range, enabling the development of ROSALIND-based sensors for sulfane sulfur species, achieving sensitive and selective detection in a fully cell-free format. More broadly, this work establishes operator evolution as a programmable strategy to optimize transcription factor–DNA interactions and expand the design space of transcription-based biosensors, including for systems lacking well-characterized genetic components.