# 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
SELEX aims at selecting aptamers based on target affinity and selectivity but not on their competence to exert an effective switching activity. The inclusion of an in vivo screening of sensor candidates after SELEX could contribute to overcoming this limitation. [PERSON] group developed a sensor implementation pipeline that led to the generation of a set of antibiotic sensors in eukaryotic cells. The procedure consists of a few rounds of SELEX, cloning of the resulting library in a reporter gene expression platform, and an in vivo screening for ligand-dependent expression of the reporter system [47; 97; 98]. On the one hand, the SELEX step enriches the pool in target binders. On the other hand, it reduces the diversity of the initial pool to a library size that allows yeast transformation with high coverage. With a subsequent in vivo screening step, it is possible to fish out only those aptamer candidates that efficiently switch reporter gene expression in response to ligand binding. The recent implementation of Capture-SELEX has tremendously increased the efficiency of this pipeline and represents a key improvement in RNA-based sensor development. Here, the RNA pool is immobilized to a resin containing a sequence tag that hybridizes with a constant region present in all aptamer candidates. An elution step with the free ligand will release only structure-switching aptamers [99]. Alternatively, sensing and actuator domains can be joined by a rational design inspired by the ligand-induced conformational changes of the aptamer. [PERSON] et al. used SHAPE to examine conformational changes undergone by FBP aptamer upon ligand binding. The aptamer is a stem-loop where FBP binding alters the tertiary structure of the loop without affecting the stem. Thus, the absence of a secondary structure switching activity prompted to design a sensor candidate library based on the HHRz aptazyme framework, which allows engineering RNA devices with ligand-responsive ribozyme tertiary interactions (Figure 1B) [62]. Finally, issues related to the sensor readout have also been addressed in the last few years. For instance, light-up sensors yielded low fluorescence due to poor folding and reduced stability of fluorogenic aptamers, especially in mammalian cells. To improve the stability and folding of the sensor construct, [PERSON] et al. embedded it within the three-way junction RNA motif (3 WJ), a common fold frequently found in bacterial riboswitches [75]. In specific, the 3 WJ motif was used as a scaffold to integrate the sensing and fluorogenic aptamers such that interhelical interactions are only stabilized as a result of ligand binding (Figure 1E) [65]. [PERSON] et al. designed a self-circularizing RNA device framework named _Tornado_. Here, a SAM light-up sensor is inserted between the sequences of two small Twister ribozymes. Upon transcription, Tornado is rapidly self-cleaved in a way that allows a subsequent RNA circularization by a pervasive cellular RNA ligase. The circular RNA sensor is much more stable, achieving a cellular abundance necessary for the accurate detection of metabolite dynamics (Figure 1F) [66]. Another important issue is the robustness of the signal. Natural fluctuations in cellular components and metabolic activity contribute to cell-to-cell heterogeneity (or _extrinsic noise_) within isogenic populations [100]. This noise affects the resolution of sensor readout, such that small signal changes resulting from biologically-relevant metabolite oscillations might become blurred. An additional fluorescence source that does not depend on ligand concentration but is sensitive to extrinsic noise serves to normalize the sensor signal. Thus, the normalization of the fluorescence intensity of the sensor by an unregulated fluorescence source reduces the variability of sensor readout among individual cells [101]. For aptazyme-based sensors of FBP, xanthine, and c-di-GMP in eukaryotic cells, an unregulated fluorescent protein was used to normalize the sensor signal, generating a ratiometric readout (Table 1) [60; 62].