# Real-Time Assessment of Intracellular Metabolites in Single Cells through RNA-Based Sensors
## 3 Prokaryotic Cells Utilize RNA to Sense Intracellular Metabolites  
An important endeavor in synthetic biology is to develop nature-inspired systems with programmable architecture and function, such as engineered biosensors. These biosensors are molecular devices that respond to small molecules generating a quantifiable signal or specific functionality. Ideally, they should display a modular architecture, with at least a _sensing_ moiety that binds the small molecules and a _reporter_ moiety that triggers the specific response. This way, different modules could be combined to build sensors with different specificities using the same basic design or sensors that generate different readouts in response to the same small molecule [26]. In the previous section, I have described examples where intracellular metabolites (or second messengers) bind protein sensors. The conformational changes induced upon binding modulate protein activity, which eventually results in a cellular effect. However, the molecular mechanisms that transduce metabolite-binding into a cellular effect through protein sensors are diverse. Thus, the design of natural systems involving protein sensors cannot be easily replicated and systematized to build engineered sensors. Bacteria have a broadly-represented molecular sensing mechanism based on RNA [27]. These natural prokaryotic sensors named riboswitches are RNA segments that occur in two conformations, the transition between which is favored by binding to a metabolite. Riboswitches are mostly found in the untranslated regions at the 5' (5'-UTR) of mRNAs, and each of the two conformations has a different impact on downstream gene expression: the \(ON\) conformation allows gene expression while the \(OFF\) conformation halts it. Riboswitches are made of two structurally and functionally differentiated modules: a sensing domain (or _apatmer_) that binds the metabolite, and an _expression platform_, which modulates gene expression as a result of the structural changes induced upon ligand binding [28]. The riboswitch-regulated downstream gene is functionally associated with the metabolite ligand. For example, in the cyanobacteria _Synechocystis_, glutamine binds a riboswitch that activates the expression of an inhibitory factor of the glutamine synthase (GS) [29]. This riboswitch belongs to a broader class, named \(glnA\) riboswitch, frequently found in the 5'-UTR of genes involved in nitrogen metabolism in cyanobacteria and marine metagenomic sequences, including the GS, glutamate synthase, and ammonium transporters. This functional association indicates that glutamine riboswitch is a central gene regulatory element of nitrogen assimilation that respond to intracellular glutamine levels in cyanobacteria [30]. More than 55 classes of riboswitches, with different ligand binding specificities and aptamer structures, have been experimentally validated thus far. They respond to a broad spectrum of intracellular ligands, including enzyme cofactors (e.g., derivatives of vitamin B, S-adenosylmethionine, FMN, NAD), nucleotide derivatives bases (e.g., guanine, adenine, xanthine), nucleotide-based signaling molecules (e.g., cyclic di-GMP, cyclic di-AMP, guanosine tetraphosphate ppGpp, ZTP), amino acids (e.g., lysine, glycine, glutamine), elemental atoms (e.g., Mn2+, Mg2+, Li+, Na+) and other small molecules [31]. The diversity of intracellular molecules sensed by riboswitches, as well as the fact these ligands carry out crucial functions in fundamental metabolic processes in organisms across all kingdoms of life, endorse the success of riboswitches as natural sensors of metabolism. Interestingly, the pervasiveness in modern bacteria of nucleotide derivatives as riboswitch-binding and signaling molecules might represent a vestige of an ancient RNA world.