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Methods Enzymol. 2016;572:315-33. doi: 10.1016/bs.mie.2016.03.021. Epub 2016 Apr 19.

Developing Fluorogenic Riboswitches for Imaging Metabolite Concentration Dynamics in Bacterial Cells.

Author information

1
Tri-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.
2
Weill Medical College, Cornell University, New York, NY, United States.
3
Tri-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. Electronic address: srj2003@med.cornell.edu.

Abstract

Genetically encoded small-molecule sensors are important tools for revealing the dynamics of metabolites and other small molecules in live cells over time. We recently developed RNA-based sensors that exhibit fluorescence in proportion to a small-molecule ligand. One class of these RNA-based sensors are termed Spinach riboswitches. These are RNAs that are based on naturally occurring riboswitches, but have been fused to the Spinach aptamer. The resulting RNA is a fluorogenic riboswitch, producing fluorescence upon binding the cognate small-molecule analyte. Here, we describe how to design and optimize these sensors by adjusting critical sequence elements, guided by structural insights from the Spinach aptamer. We provide a stepwise procedure to characterize sensors in vitro and to express sensors in bacteria for live-cell imaging of metabolites. Spinach riboswitch sensors offer a simple method for fluorescence measurement of a wide range of metabolites for which riboswitches exist, including nucleotides and their derivatives, amino acids, cofactors, cations, and anions.

KEYWORDS:

Cellular imaging; Dynamics; Fluorescence; Metabolite; RNA probes; Riboswitch; Sensor

PMID:
27241761
PMCID:
PMC5540731
DOI:
10.1016/bs.mie.2016.03.021
[Indexed for MEDLINE]
Free PMC Article

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