Cell-free systems enable mechanistic characterization of genetically encoded RNA strand exchange circuits for programmable protein expression

Nucleic acid circuits are powerful tools for programming biology, but the design principles for operating these circuits in complex cellular environments remain poorly understood compared to simple in vitro settings. Cell-free expression systems (CFES) are uniquely suited to address this challenge, as they can integrate measurements ideal for either in vitro or in cyto settings . Further, because CFES are open systems, they enable a level of control over component concentration unattainable in cells. Here, we use CFES to characterize genetically encoded RNA circuits that operate via toehold-mediated strand exchange (TMSE). These circuits have been extensively characterized in vitro and were recently deployed as translational riboregulators in E. coli , revealing key differences between the two environments. By systematically modulating the design parameters of these RNA circuits in both purified protein and lysate-based CFES, we elucidate the mechanisms linking TMSE to protein expression. Further, we combine measurements and alterations in CFES composition that are infeasible in cells to investigate interactions between cellular components and RNA circuit components, identifying a potential interaction with ribosomes that informs circuit design. Our results establish a unified set of principles for designing and operating genetically encoded TMSE circuits across in vitro , CFE, and bacterial environments, which should catalyze the widespread adoption of this platform for new applications in molecular programming, synthetic biology, and biotechnology.