# Real-Time Assessment of Intracellular Metabolites in Single Cells through RNA-Based Sensors
## 5 Development of RNA-Based Sensors for Intracellular Metabolites: A Challenging Endeavor
functional riboswitches is a mechanism of actuation evolved as a gene-regulatory module. Third, publicly available crystal structures of riboswitches, and especially those in different switched states, serve to identify the mechanism of switching and the sequence elements involved [68; 84]. This way, ligand binding, and the derived switching activity are already optimized by nature to work within cells. On the other hand, knowing the mechanism of switching smoothens the rational design of the sensor. However, the potential of generating sensors with novel specificities is limited by the availability of identified aptamers in natural riboswitches. It is possible to expand the catalog of available aptamers through computational searches for novel riboswitches. In this regard, comparative genomics and metagenomics in bacteria and archaea aimed to identify novel structured RNA motifs in intergenic regions unveiled a number of new riboswitch classes [85; 86], and it has been proposed that most riboswitch classes among the global bacterial population are still to be discovered [31; 87]. Novel riboswitches can also be identified by means of experimental approaches. For instance, the binding of a metabolite ligand triggers an RNA conformational change in naturally occurring aptamers, which can be mapped at the transcriptome-wide level in vitro and in vivo [88; 89; 90]. The most relevant feature contributing to the scalability of engineered RNA-based sensors is the faculty to generate custom RNA-sensing moieties by means of the systematic evolution of ligands by exponential enrichment (SELEX). SELEX is a general iterative approach that allows for aptamer selection from an RNA combinatorial library (or _pool_) based on the capacity to bind a specific metabolite in vitro [91; 92]. Despite the enormous potential of applying SELEX to generate aptamers de novo for RNA-based sensors, to my knowledge, only five sensors for intracellular metabolites have been constructed thus far with in vitro selected aptamers: hypoxanthine, FBP, ADP/ATP, 5-hydroxytryptophan (5-HT) and 3,4-dihydroxyphenylalanine (L-DOPA) (Table 1) [44; 62; 63; 75; 93; 94]. There is a number of issues related to small molecule-aptamer selection and its integration into RNA device frameworks that could halt the construction of custom sensors [92]. In vitro-selected aptamers may not be functional within cells because the physicochemical conditions used for in vitro selection are different from those found in the intracellular niche. These differences might affect, for instance, the affinity and selectivity of the aptamer for the target metabolite. For FBP aptamer selection, both SELEX and the biochemical characterization (determination of the affinity, selectivity, and ligand-induced structural changes) were performed in a buffer in which the pH, the ionic composition, and the concentration of non-target metabolites and macromolecules were adjusted to resemble the conditions of eukaryotic cytoplasm [62; 93]. In addition, in vitro-selected aptamers might not fold properly within cells and/or might exhibit reduced stability. This is the case of Spinach, for example, which requires being embedded within a folding scaffold to yield an optimal signal [95; 96]. To circumvent folding and stability issues, aptamers could be selected in vitro embedded within a natural RNA scaffold. [PERSON] et al. identified the three-way junction motif as a common RNA motif in bacterial riboswitches and utilized it as a scaffold to generate the pool for SELEX. They selected aptamers for neurotransmitter precursors 5-HT and L-DOPA, which were then engineered to generate RNA-based sensors functional in the cellular context [75]. The idea of using structural scaffolds from functional riboswitches can expedite the implementation of sensors because these motifs have an efficient switching activity in vivo and, thus, could be better suited for coupling to readout domains.