Exploiting conformational changes in a caffeine aptamer to engineer synthetic RNA devices

RNA devices, including riboswitches, aptazymes, and RNA-based biosensors, have become essential components in synthetic and molecular biology. These systems make use of RNA's modularity and structural diversity to build programmable tools for sensing, regulation, and cellular computation. To drive these systems, RNA aptamers usually need to undergo a conformational change upon ligand binding. However, the limited availability of such aptamers has restricted the development and application of RNA devices, particularly in mammalian systems. Traditional selection methods often prioritize high-affinity binding, resulting in a scarcity of conformation-switching aptamers. Here, we addressed this limitation by selecting a caffeine-binding aptamer with RNA Capture-SELEX and in vivo screenings. This aptamer functioned as a modular regulator of riboswitches and ribozymes in Saccharomyces cerevisiae and mammalian cells. Through new optofluidic screenings, we overcame throughput and sequence-function challenges inherent in ribozyme screening for mammalia. Additionally, a straightforward grafting method transformed the aptamer into a fluorogenic iSpinach aptasensor. This work demonstrates the successful selection of a modular and communicating aptamer for a challenging target like caffeine and establishes robust strategies for their modular integration into diverse RNA platforms. These strategies pave the way for broadening the repertoire of aptamers and expanding the potential of RNA-based synthetic biology.