Systematic Evaluation of Different Ribonucleoprotein Complexes as Posttranscriptional Biosensors in Cell-Free TX-TL...

Synthetic biology has enabled the development of sophisticated cell-free biosensors capable of complex, sensitive signal detection with promising clinical and environmental applications due to their ease of use, fast readout development, portability, and built-in biosafety. Nevertheless, the potential of ribonucleoprotein (RNP) complexes as posttranscriptional biosensors in cell-free transcription-translation (TX-TL) systems remains largely unexplored. In this study, we test the performance of three different posttranscriptional circuits (RISC:miRNA, MS2-CNOT7:ms2L, and L7Ae:k-turn) in different TX-TL cell-free platforms (eukaryotic, prokaryotic, and recombinant) and evaluate the robustness of their reconstitution in vitro. We find that miRNA sensors encoded in in vitro-transcribed (IVT) luciferase RNAs work effectively, yet the difficulty in reliably quantifying the basal expression levels of the reporter gene represents a major bottleneck of IVT RNA-based miRNA sensors. Although the MS2-CNOT7:ms2L RNP circuit was robustly reconstituted in eukaryotic lysates, we found that lysate-dependent constraints hamper its use as an in vitro biosensor, reducing its programmability potential in the cell-free context. On the other hand, L7Ae:k-turn was successfully reconstituted in all cell-free platforms tested following thorough characterization of plasmid-specific and protocol-dependent effects on reporter expression and its conditional repression by L7Ae. Finally, we successfully introduced an additional control layer by implementing protease-responsive L7Ae-mediated conditional repression in the PURE system, indicating the potential of the L7Ae:k-turn RBP circuit as a promising cell-free biosensor for protease-based viral detection in vitro.