# Real-Time Assessment of Intracellular Metabolites in Single Cells through RNA-Based Sensors
## 3 Prokaryotic Cells Utilize RNA to Sense Intracellular Metabolites
Consistent with this hypothesis, it is possible that ligand-binding domains in today's riboswitches could have been _recycled_ from a primordial RNA-based catalytic machinery in the RNA world that transformed or synthesized these small molecules in metabolism [32; 33]. Although riboswitches can bind diverse intracellular ligands, the response is generated through a reduced set of molecular mechanisms. In most cases, this regulatory activity is directed at transcription or translation by ligand-dependent stabilization of one of the two mutually exclusive RNA conformations. In the case of transcription, ligand binding favors the formation of a stable double-stranded RNA stem structure followed by a polyuridine tract, which serves as a transcription terminator. The formation of this terminator promotes a premature termination of transcription of the downstream gene. Under low ligand concentrations, the riboswitch folds as an anti-terminator structure, which allows for a transcriptional read-through. In the case of translation, the conformational switch induced by the ligand influences the accessibility of the ribosome-binding site (RBS) and the initiation codon. In the OFF conformation, these sequence elements become occluded within a stem, which halts the initiation of translation [34]. The _glmS_ riboswitch represents a widespread class that uses a singular mechanism. It is a sequence-specific self-cleaving ribozyme whose endonucleolytic activity depends on \(N\)-acetyl-glucosamine-6-phosphate (GlcNAcbF) [35; 36]. Interestingly, structural and biochemical data suggest that GlcN6P does not induce a conformational switch but participates directly in RNA cleavage activity [28; 37]. Additional but less frequent mechanisms that rely on regulatory proteins have also been described [38]. Conserved sensing domains can occur in combination with different types of expression platforms. For instance, thiamin pyrophosphate (TPP)-binding riboswitch (Thi-box) is widely distributed in the three kingdoms of life. In _Escherichia coli_, the binding of TPP prevents translation initiation by sequestering the RBS and the start codon, while in _Bacillus subtilis_, it leads to transcription attenuation [39; 40]. In eukaryotes, the riboswitch occurs within introns or 3\({}^{\prime}\)-UTRs, where TPP binding makes an occluded splice site accessible to the splicing machinery. Isoforms derived from this alternative splicing event either lead to the occurrence of premature stop codons in the mature mRNA or to the destabilization of the transcript, depending on the species [34]. Thus, similar ligand-induced conformational changes in conserved sensing RNA domains can trigger different regulatory activities depending on the adjacent expression platform. This observation indicates that riboswitches are made of functional modules. In fact, this natural modular architecture has been implemented in synthetic RNA devices, where different expression platforms could be coupled to a variety of natural and synthetic aptamers [41; 42]. Overall, riboswitches are naturally-evolved sensors for intracellular metabolites that serve to monitor intracellular metabolism. They show a modular architecture. This means that a defined set of RNA-sensing domains with different specificities or aptamers can be combined with a few basic gene regulatory modules to generate the cellular response. These properties make riboswitches a suitable model to simulate for engineering synthetic RNA-based biosensors for metabolites.