The widespread natural ability of RNA to sense small molecules and regulate genes has become an important tool for synthetic biology in applications as diverse as environmental sensing and metabolic engineering. Previous work in RNA synthetic biology has engineered RNA mechanisms that independently regulate multiple targets and integrate regulatory signals. However, intracellular regulatory networks built with these systems have required proteins to propagate regulatory signals. In this work, we remove this requirement and expand the RNA synthetic biology toolkit by engineering three unique features of the plasmid pT181 antisense-RNA-mediated transcription attenuation mechanism. First, because the antisense RNA mechanism relies on RNA-RNA interactions, we show how the specificity of the natural system can be engineered to create variants that independently regulate multiple targets in the same cell. Second, because the pT181 mechanism controls transcription, we show how independently acting variants can be configured in tandem to integrate regulatory signals and perform genetic logic. Finally, because both the input and output of the attenuator is RNA, we show how these variants can be configured to directly propagate RNA regulatory signals by constructing an RNA-meditated transcriptional cascade. The combination of these three features within a single RNA-based regulatory mechanism has the potential to simplify the design and construction of genetic networks by directly propagating signals as RNA molecules.gene networks | regulatory systems | orthogonal regulators N oncoding RNA has been found to play a central role in regulating gene expression in both prokaryotes and eukaryotes. Recently, the diverse roles of RNA-mediated regulation have become important tools for synthetic biology applications ranging from detecting metabolic state (1), balancing metabolic pathway expression (2), tightly regulating toxin genes (3), and detecting environmentally harmful chemicals (4). In particular, RNA-based genetic parts have been engineered that regulate transcription through RNA-mediated transcription factor recruitment (5, 6), transcript stability through small-molecule-mediated ribozyme cleavage (1, 7) and siRNA targeted degradation (8), and translation through cis-acting mRNA conformational changes (9) and trans-acting antisense RNA-mRNA interactions (10,11).This wide array of RNA function is beginning to be used to engineer programmable genetic circuitry required for the next level of synthetic biology applications (12). By interfacing with protein-based transcription factors and repressors, hybrid RNA∕ protein cascades have been made that perform sophisticated logic evaluation (8) and even count extracellular events (13). Much like previous work on protein-based cascades (14), protein regulators propagate the signal between different levels of the hybrid cascades. This makes the inner workings of these cascades complicated by the many interconversions between mRNA and protein that must take place. This not only incre...

New regulatory roles continue to emerge for both natural and engineered noncoding RNAs, many of which have specific secondary and tertiary structures essential to their function. Thus there is a growing need to develop technologies that enable rapid characterization of structural features within complex RNA populations. We have developed a high-throughput technique, SHAPE-Seq, that can simultaneously measure quantitative, single nucleotide-resolution secondary and tertiary structural information for hundreds of RNA molecules of arbitrary sequence. SHAPE-Seq combines selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry with multiplexed paired-end deep sequencing of primer extension products. This generates millions of sequencing reads, which are then analyzed using a fully automated data analysis pipeline, based on a rigorous maximum likelihood model of the SHAPE-Seq experiment. We demonstrate the ability of SHAPESeq to accurately infer secondary and tertiary structural information, detect subtle conformational changes due to single nucleotide point mutations, and simultaneously measure the structures of a complex pool of different RNA molecules. SHAPE-Seq thus represents a powerful step toward making the study of RNA secondary and tertiary structures high throughput and accessible to a wide array of scientific pursuits, from fundamental biological investigations to engineering RNA for synthetic biological systems.chemical probing | RNA sequencing | RNA folding | genomics O ver the past several years, there has been an explosion in the discovery of noncoding, but functional RNAs that play central roles in maintaining, regulating, and defending the genome (1). At the same time, RNA-based mechanisms have emerged as powerful tools for engineering synthetic biological systems (2). Many of these natural and synthetic RNAs have specific secondary and tertiary structures essential to their function, and there is a growing need to develop technologies that enable rapid characterization of structural features within complex RNA populations. Such a high-throughput structure characterization assay would allow rapid assessment of the impact of sequence on structure and function and enable RNA engineers to design libraries of RNA molecules with desired structural properties.Two techniques for high-throughput RNA structure characterization have recently been reported: parallel analysis of RNA structures (PARS) (3) and fragmentation sequencing (FragSeq) (4). Both techniques couple classic in vitro nuclease probing techniques that are traditionally performed one RNA at a time, with deep sequencing of RNA fragments to simultaneously probe a complex mixture of RNAs sampled from transcriptomes. Although important first steps, these techniques provide only low-resolution secondary structure information due to the limitations inherent in nuclease probing (5).We have developed a high-throughput technique, SHAPESeq, that can simultaneously measure quantitative, single nucleotide-resolution secondary and tertia...

RNAs can begin to fold immediately after emerging from RNA polymerase during transcription. Interactions between nascent RNAs and ligands during cotranscriptional folding can direct the formation of alternative RNA structures, a feature exploited by non-coding RNAs called riboswitches to make gene regulatory decisions. Despite their importance, cotranscriptional folding pathways have yet to be uncovered with sufficient resolution to reveal how cotranscriptional folding governs RNA structure and function. To access cotranscriptional folding at nucleotide resolution, we extend selective 2’-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) to measure structural information of nascent RNAs during transcription. With cotranscriptional SHAPE-Seq, we determine how the B. cereus crcB fluoride riboswitch cotranscriptional folding pathway undergoes a ligand-dependent bifurcation that delays or promotes terminator formation via a series of coordinated structural transitions. Our results directly link cotranscriptional RNA folding to a genetic decision and establish a framework for cotranscriptional analysis of RNA structure at nucleotide resolution.