High-Throughput Screening of Cell-Free Riboswitches By Droplet Sorting

A riboswitch is a regulatory element present in untranslated regions of some messenger RNAs that up- or down- regulates gene expression in response to the interaction of its aptamer domain with specific chemical signals. The possibility of developing aptamers for desired ligands by in vitro selection makes riboswitches attractive gene regulatory devices for synthetic biology applications. Although a large number of RNA aptamers have been discovered by in vitro selection, many of them cannot be developed into riboswitches in vivo due to various reasons (biocompatibility, bioavailability, different in vitro and intracellular conditions, etc.); however, such aptamers may still be useful for riboswitches in cell-free systems. Cell-free systems allow more precise control over experimental conditions with fewer biological constraints than in vivo systems. Consequently, they offer unique opportunities to study riboswitches in a controlled environment that can lead to fundamental insights of their mechanisms and design principles. Cell-free riboswitches are also attractive devices for applications such as cell-free metabolic engineering and artificial cells. Previous efforts to engineer cell-free riboswitches have mostly been based on low-throughput trial-and-error processes, or transferring riboswitches optimized in bacteria by high-throughput screening and genetic selection. The latter approach often results in compromised riboswitch performance in cell-free systems, and it cannot be used for chemical signals that are not compatible with living cells due to low bioavailability or toxicity. In this work, we report our ongoing efforts to develop a high-throughput strategy for screening riboswitches directly in a cell-free environment. We used a microfluidic device to generate a water-in-oil emulsion of ~20 μm diameter droplets containing a riboswitch-controlled reporter gene and a reconstituted transcription-translation system. Using control constructs in ON- and OFF-state, we demonstrated ~70-fold enrichment of desired droplets using a droplet sorter. We plan to apply this screening platform to engineer cell-free riboswitches with superior characteristics.