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Developing Fluorogenic Riboswitches for Imaging Metabolite Concentration Dynamics in Bacterial Cells
J L Litke1, M You2, S R Jaffrey1
1Tri-Institutional Chemical Biology Program at Weill-Cornell Medical College, Rockefeller University, Memorial Sloan-Kettering Cancer Center, New York, NY, United States; Weill Medical College, Cornell University, New York, NY, United States.
Methods in Enzymology
|June 1, 2016
Summary
We developed novel RNA-based sensors, Spinach riboswitches, that fluoresce when binding small molecules. This allows for live-cell imaging and measurement of various metabolites.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Genetically encoded small-molecule sensors are crucial for understanding cellular dynamics.
- RNA-based sensors offer a promising avenue for real-time metabolite monitoring.
- Naturally occurring riboswitches can be engineered into functional biosensors.
Purpose of the Study:
- To describe the design and optimization of RNA-based sensors, specifically Spinach riboswitches.
- To provide a method for characterizing these sensors in vitro and in vivo.
- To enable live-cell imaging of metabolites using these novel sensors.
Main Methods:
- Engineering RNA-based sensors by fusing natural riboswitches with the Spinach aptamer.
- Optimizing sensor performance by adjusting critical RNA sequence elements.
- In vitro characterization and bacterial expression for live-cell imaging applications.
Main Results:
- Development of fluorogenic Spinach riboswitch sensors that exhibit fluorescence proportional to small-molecule binding.
- Demonstration of a stepwise procedure for sensor design, optimization, and characterization.
- Successful application of these sensors for live-cell imaging of metabolites in bacteria.
Conclusions:
- Spinach riboswitch sensors provide a simple and versatile platform for fluorescence-based metabolite measurement.
- These sensors are applicable to a wide range of analytes, including nucleotides, amino acids, and ions.
- The described methods facilitate the development and application of RNA-based biosensors for cellular research.
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