# Real-Time Assessment of Intracellular Metabolites in Single Cells through RNA-Based Sensors
## 4 Synthetic RNA-Based Sensors for Intracellular Metabolites  
A synthetic RNA-based sensor is a genetically-encoded RNA molecule consisting of a metabolite-binding aptamer (_sensor_ domain) and a transducer region that converts the interaction of the metabolite into a quantifiable response (the _actuator_ domain) (Figure 1A). The first demonstration of a synthetic RNA-based sensor was an allosteric ribozyme developed by [PERSON] and [PERSON] in 1997 [43]. By using a rational design, an ATP-binding aptamer (sensor domain) [44] was joined to a hammerhead ribozyme (actuator domain) through different linker sequences. The generated constructs were then screened for an ATP-driven inhibition of the self-cleaving activity of the ribozyme in vitro [43]. Testing different linker regions is a necessary step to identify the construct that more efficiently transduces metabolite-binding into the output signal [45]. Although this primordial RNA device was not genetically encoded, it was the first proof-of-principle of an engineered genetic device de novo whose activity could be controlled by a small molecule. Since then, a plethora of RNA devices that control gene expression in a ligand-dependent fashion have been developed. Such systems utilized a reduced set of sensingaptamers to prove valid different mechanisms of actuation. These synthetic RNA-device prototypes usually included the aptamer of theophylline [46], tetracyclin [47], or thiamin pyrophosphate (TPP) [39] coupled to an actuation domain that allowed for allosteric control of the functionality of translational machinery [48; 49], translation initiation [47; 50; 51; 52], mRNA stability [53; 54; 55], splicing [56] and microRNA (miRNA) maturation [57]. Some devices are exclusively functional in either bacteria or eukaryotes because the functionality of the actuator domain relies on kingdom-specific gene expression control mechanisms. For instance, the mechanisms that regulate the initiation of protein translation are different in bacteria and eukaryotes, while splicing or miRNA-based regulation occurs only in eukaryotes. Ideally, programmable RNA-based gene-regulatory devices should rely on a _portable_ mechanism of actuation, that is, a transduction mechanism that is independent of cell-specific machinery [54]. Fast-cleaving variants of the hammerhead ribozyme (HHRz) have been widely utilized as actuator domains in RNA devices. These HHRz-based RNA devices are functional across different organisms, where they showed to respond to diverse ligands [58; 59; 60; 50], generating varied responses, including the regulation of complex cell phenotypes [53; 61]. The architecture of such devices consists of a small molecule-binding RNA aptamer grafted into the HHRz in place of one of its stems II or III (Figure 1B). To implement the allosteric ribozyme (or _aptazyme_), linker sequences are rationally designed to relay ligand-binding into a secondary structure change [54] or randomized and screened for ligand-dependent impact on self-cleaving activity [58]. An alternative approach aims to select synthetic allosteric ribozymes where ligand binding interferes with or contributes to stabilizing the tertiary interactions between the two loops of the HHRz (Figure 1B). In this case, aptamer-coupled ribozyme libraries are constructed by varying the sequence of the loop opposite to the stem-loop where the sensing domain is grafted [59; 60; 62]. Figure 1: RNA-based sensor frameworks implemented to detect intracellular metabolites. (**A**) Basic design of a sensor. (**B**) Attazyme-based sensor. Hammerhead (HH) endonucleolytic activity depends  
on the binding of the metabolite (Orange; FBP, fructose-1,6-bisphosphate). Active ribozyme destabilizes the mRNA [62]. (**C-F**) Light-up sensors. Folding of a fluorogenic aptamer (Grey; Spinach, Broccoli) depends on the binding of the metabolite to the sensing aptamer (blue; ADP, TPP, SAM aptamers). When folded, the fluorogenic aptamer can bind a fluorophore (DFHBI, DFHBI-1T), generating fluorescence. Three-way junction motif: 3 WJ. DFHBI-1T: 1T.