FRET-based optical assay for monitoring riboswitch activation.
Riboswitches are regulatory RNAs located in the 5'-untranslated region of mRNA sequences that recognize and bind to small molecules and regulate the expression of downstream genes. Creation of synthetic riboswitches to novel ligands depends on the ability to monitor riboswitch activation in the presence of analyte. In our work, we have coupled a synthetic riboswitch to an optical reporter assay based on fluorescence resonance energy transfer (FRET) between two genetically encoded fluorescent proteins. The theophylline-sensitive riboswitch was placed upstream of the Tobacco Etch Virus (TEV) protease coding sequence. Our FRET construct was composed of eGFP and a nonfluorescent yellow fluorescent protein mutant called REACh (for resonance energy-accepting chromoprotein) connected with a peptide linker containing a TEV protease cleavage site. Addition of theophylline to the E. coli cells activates the riboswitch and initiates the translation of mRNA. Synthesized protease cleaves the linker in the FRET-based fusion protein causing a change in the fluorescence signal. By this method, we observed an 11-fold increase in cellular extract fluorescence in the presence of theophylline. The advantage of using an eGFP-REACh pair is the elimination of acceptor fluorescence. This leads to an improved detection of FRET via better signal-to-noise ratio, allowing us to monitor riboswitch activation in a wide range of analyte concentrations from 0.01 to 2.5 mM.
Harbaugh
Svetlana
S
Applied Biotechnology Branch, Human Effectiveness Directorate, Air Force Research Laboratory, Wright-Patterson Air Force Base, Dayton, Ohio 45433, USA.
Kelley-Loughnane
Nancy
N
Davidson
Molly
M
Narayanan
Latha
L
Trott
Sandra
S
Chushak
Yaroslav G
YG
Stone
Morley O
MO
D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013806:Theophylline / Q000737:chemistry / Q000378:metabolism
D008024:Ligands; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012313:RNA; D013806:Theophylline; D010450:Endopeptidases; C099488:TEV protease