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Imaging metabolite dynamics in living cells using a Spinach-based riboswitch

Significance Developing sensors to image cellular metabolites and signaling molecules in living cells is challenging. Here we describe Spinach riboswitches, a novel class of genetically encoded metabolite sensor based on riboswitches, a group of naturally occurring ligand-binding RNAs. Spinach riboswitches use Spinach, an RNA aptamer that binds and activates the fluorescence of an otherwise nonfluorescent small-molecule fluorophore. Drawing upon structural insights into the mechanism of structural switching in riboswitches, we show that Spinach can be swapped into various riboswitches, allowing metabolite binding to induce Spinach fluorescence directly. Expression of Spinach riboswitches in cells allows metabolite levels to be imaged in real time in live bacterial cells. Spinach riboswitches thus provide a novel approach to image cellular metabolites in living cells. Riboswitches are natural ligand-sensing RNAs typically that are found in the 5′ UTRs of mRNA. Numerous classes of riboswitches have been discovered, enabling mRNA to be regulated by diverse and physiologically important cellular metabolites and small molecules. Here we describe Spinach riboswitches, a new class of genetically encoded metabolite sensor derived from naturally occurring riboswitches. Drawing upon the structural switching mechanism of natural riboswitches, we show that Spinach can be swapped for the expression platform of various riboswitches, allowing metabolite binding to induce Spinach fluorescence directly. In the case of the thiamine 5′-pyrophosphate (TPP) riboswitch from the Escherichia coli thiM gene encoding hydroxyethylthiazole kinase, we show that insertion of Spinach results in an RNA sensor that exhibits fluorescence upon binding TPP. This TPP Spinach riboswitch binds TPP with affinity and selectivity similar to that of the endogenous riboswitch and enables the discovery of agonists and antagonists of the TPP riboswitch using simple fluorescence readouts. Furthermore, expression of the TPP Spinach riboswitch in Escherichia coli enables live imaging of dynamic changes in intracellular TPP concentrations in individual cells. Additionally, we show that other riboswitches that use a structural mechanism similar to that of the TPP riboswitch, including the guanine and adenine riboswitches from the Bacillus subtilis xpt gene encoding xanthine phosphoribosyltransferase, and the S-adenosyl-methionine-I riboswitch from the B. subtilis yitJ gene encoding methionine synthase, can be converted into Spinach riboswitches. Thus, Spinach riboswitches constitute a novel class of RNA-based fluorescent metabolite sensors that exploit the diversity of naturally occurring ligand-binding riboswitches.