# Real-Time Assessment of Intracellular Metabolites in Single Cells through RNA-Based Sensors
## 4 Synthetic RNA-Based Sensors for Intracellular Metabolites
(**C**) [63]. (**D**) [64]. (**E**) [65]. (**F**) [66]. RNA devices based on the allosteric HHRz framework have been implemented as metabolite sensors for the vitamin B derivative TPP, the glycolytic metabolite fructose-1,6-bisphosphate (FBP), the product of catabolism of purines hypoxanthine, and the bacterial second messenger cyclic diguanylate monophosphate (c-di-GMP) (Table 1). TPP biosensor was built by inserting the metabolite-sensing domain of TPP riboswitch into the 5\({}^{\prime}\)-UTR of a GFP expression cassette in _E. coli_, such that TPP-dependent activation of HHRz activity controls the blockade of the RBS [67]. In contrast, in the FBP, hypoxantine, and c-di-GMP biosensors, the aptazyme is located in the 3\({}^{\prime}\)-UTR of a reporter gene and exhibits metabolite-responsive HHRz tertiary structure interactions. Thus, intracellular metabolite concentration-dependent cleavage leads to mRNA destabilization and a concurrent decrease in the abundance of a fluorescent protein (Figure 1B) [60; 62]. In addition to HHRz aptazyme sensors, fluorogenic or light-up RNA-based sensors represent a modular, scalable, and portable framework for engineering genetically-encoded biosensors for intracellular metabolites [54; 68]. Light-up sensors rely on fluorogenic RNA aptamers, such as _Spinach_[63]. _Spinach_ interacts specifically with a non-fluorescent small molecule (3,5-difluoro-4-hydroxybenzylidene imidazolinone, DFHBI) similar to GFP fluorophore, which upon binding to the aptamer, becomes fluorescent [69]. Additional fluorogenic RNA aptamers (with their respective target fluorophores) with different biophysical properties have been developed and implemented into RNA sensors, such as _Broccoli_ (DFHBI-1T) [70] and _Corn_ (DFHO) [71]. In light-up RNA-based sensors, folding of the fluorogenic aptamer (actuator domain) requires binding of the ligand to a metabolite-binding aptamer (sensor domain), such that ligand-induced stabilization of a structural element allows the recruitment of the fluorophore and fluorescence emission (Figure 1C-F). Light-up RNA sensors for intracellular metabolites were initially constructed by joining the sensing aptamer to Spinach through a weak transducer stem that stabilized upon ligand binding (Figure 1C) [63]. The efficacy of the RNA secondary structure changes to transduce a different functional state of the actuator domain (i.e., the _switching activity_) was later improved in a second generation of light-up sensors named _Spinach riboswitches_. In this sensor framework, the actuator domain of natural riboswitches is replaced by Spinach (Figure 1D). This strategy streamlines sensor development because it repurposes a naturally evolved and in vivo functional switching activity to actuate a synthetic device [64]. In addition, a third generation of light-up sensors exhibits much higher sensitivity thanks to improved RNA folding and stability in eukaryotic cells (Figure 1E,F) [65; 66; 72]. An interesting feature of light-up sensors compared to aptazymes is reversibility. Ribozymes catalyze an irreversible endonucleolytic cleavage of reporter mRNA upon a change in ligand concentration, while light-up sensors can be switched on and off over time. These intrinsic properties make aptazyme-based sensors more sensitive to low target concentrations. In contrast, light-up sensors are more suitable for dynamic measurements, where they can serve to monitor oscillations in the concentration of an intracellular metabolite [66; 72]. In addition, while light-up sensors can generate solely fluorescence as the output signal, aptazymes can control the expression of any gene. Thus, aptazyme sensors can transduce metabolite concentration into different cell functionalities, such as a selective advantage or a deleterious protein [73]. In any case, both sensor types are compatible and could be combined depending on the application's needs. Furthermore, the modular architecture of RNA devices allows for combining an aptazyme and a fluorogenic aptamer actuator domains in an individual sensor.