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

Area of Science:

• Molecular Biology
• RNA Biology
• Biochemistry

Background:

• Riboswitches are natural RNA molecules that regulate gene expression in response to cellular metabolites.
• Diverse classes of riboswitches have been identified, controlling responses to various small molecules.
• The structural switching mechanism of natural riboswitches is key to their function.

Purpose of the Study:

• To develop a new class of genetically encoded metabolite sensors called Spinach riboswitches.
• To enable direct fluorescence readouts upon metabolite binding.
• To facilitate the discovery of riboswitch agonists and antagonists and visualize intracellular metabolite dynamics.

Main Methods:

• Engineered Spinach riboswitches by swapping Spinach into the expression platforms of natural riboswitches.
• Utilized the thiamine 5'-pyrophosphate (TPP) riboswitch from Escherichia coli thiM as a model system.
• Tested the TPP Spinach riboswitch for TPP binding affinity and selectivity.
• Expressed the TPP Spinach riboswitch in Escherichia coli for live-cell imaging.
• Converted other natural riboswitches (guanine, adenine, S-adenosyl-methionine-I) into Spinach riboswitches.

Main Results:

• Demonstrated that Spinach can be directly induced to fluoresce upon metabolite binding when integrated into riboswitch scaffolds.
• The TPP Spinach riboswitch exhibited TPP binding affinity and selectivity comparable to the endogenous riboswitch.
• Successfully used the TPP Spinach riboswitch for live imaging of intracellular TPP concentrations in E. coli.
• Showed that other riboswitches can be converted into functional Spinach riboswitches.

Conclusions:

• Spinach riboswitches represent a novel class of RNA-based fluorescent metabolite sensors.
• This technology leverages the natural diversity of ligand-binding riboswitches for sensing.
• Spinach riboswitches offer a powerful tool for metabolite discovery and real-time cellular imaging.