# 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  
_Scalability_ is a paramount design principle of RNA-based sensors because of their modular architecture. It refers to the possibility of generating new sensors by implementing different small molecule-binding aptamers into standardized sensor frameworks, such as those relying on the aptazyme or light-up actuator domains [54]. However, the diversity of available RNA-based sensors for intracellular metabolites available today is strikingly scant. To my knowledge, there are less than 15 RNA-based sensors with different specificities (Table 1). This indicates that although RNA-based sensors are, in principle, scalable, there are still important challenges that halt the implementation of sensors with novel specificities. Most light-up RNA sensors have been constructed utilizing metabolite-binding aptamers from naturally-occurring riboswitches (Table 1). This has three major advantages. First, the affinity and selectivity of the aptamer have naturally evolved to interact with the target metabolite under physiological conditions. Second, the switching activity of in vivo \begin{table}
\begin{tabular}{c c c c c c} \hline
**Target** & **Metabolite 1** & **Aptamer 2** & **Type of Sensor 3** & **Output 4** & **Cell Type** & **Reference** \\ \hline
5-HT & SELEX & Light-up (Broccoli) & GF & Bacteria & [PERSON] et al., 2017 [75] \\ \hline ADP & SELEX & Light-up (Spinach) & GF & Bacteria & [PERSON] et al., 2012 [63] \\ \hline c-AMP-GMP & Natural & Light-up (Spinach) & GF & Bacteria & [PERSON] et al., 2013 [76] \\ \hline c-di-AMP & Natural & Light-up (Spinach2) & GF & Bacteria & [PERSON] et al., 2015 [77] \\ \hline c-di-GMP & Natural & Aptazyme & RF/BF & Mammalian & [PERSON] et al., 2019 [60] \\ \hline c-di-GMP & Natural & Light-up (Spinach) & GF & Bacteria & [PERSON] et al., 2013 [76] \\ \hline c-di-GMP & Natural & Light-up (Spinach2) & GF & Bacteria & [PERSON] et al., 2016 [78] \\ \hline FBP & SELEX & Aptazyme (HHRz) & GF/RF & Yeast & [PERSON] et al., 2021 [62] \\ \hline FBP & SELEX & Light-up (Baby Spinach) & GF & Mammalian & [PERSON] et al., 2022 [79] \\ \hline L-DOPA & SELEX & Light-up (Broccoli) & GF & Bacteria & [PERSON] et al., 2017 [75] \\ \hline (p)ppGpp & Natural & Light-up (Broccoli) & GF & Bacteria & [PERSON] et al., 2021 [80] \\ \hline (p)ppGpp & Natural & BRET (nlLuc donor and Pepper acceptor) & GF & Bacteria & [PERSON] et al., 2023 [81] \\ \hline SAH & Natural & Light-up (cpSPinach2) & GF & Bacteria & [PERSON] et al., 2016 [82] \\ \hline SAM & Natural & BRET (nlLuc donor and Pepper acceptor) & GF & Bacteria & [PERSON] et al., 2023 [81] \\ \hline SAM & Natural & Light-up (Broccoli) & GF & Mammalian & [PERSON] et al., 2019 [66] \\ \hline SAM & Natural & Light-up (Broccoli) & GF & Mammalian & [PERSON] et al., 2021 [65] \\ \hline SAM & Natural & Light-up (Corn) & YF & Mammalian & [PERSON] et al., 2019 [71] \\ \hline SAM & Natural & Light-up (Red Broccoli) & RF & Mammalian & [PERSON] et al., 2020 [83] \\ \hline SAM & Natural & Light-up (Spinach) & GF & Bacteria & [PERSON] et al., 2012 [63] \\ \hline SAM & Natural & Light-up (Squash and Broccoli) & YF/GF & Mammalian & [PERSON] et al., 2022 [72] \\ \hline TPP & Natural & Aptazyme & GF & Bacteria & [PERSON] et al., 2009 [67] \\ \hline TPP & Natural & Aptazyme + Light-up (Broccoli) & GF & Bacteria & [PERSON] et al. 2019 [74] \\ \hline TPP & Natural & Light-up (Spinach) & GF & Bacteria & [PERSON] et al., 2015 [64] \\ \hline Xanthine & SELEX & Aptazyme & RF/BF & Mammalian & [PERSON] et al., 2019 [60] \\ \hline \end{tabular}  
* 1 Intracellular metabolite (no exogenous compounds). 2 Natural: metabolite-sensing domain of a riboswitch; SELEX: in vitro-selected aptamer. 3 Only portable actuators (trans-kingdom actuation mechanisms). 4 GF: green fluorescence; YF: yellow fluorescence; RF: red fluorescence; YF, RF/BF, GF/RF: ratio of fluorescence intensity.  
\end{table} 
Table 1: RNA-based sensors for intracellular metabolites validated in cells.