Novel genetically encoded biosensors using firefly luciferase.
Genetically encoded biosensors have proven valuable for real-time monitoring of intracellular phenomena, particularly FRET-based sensors incorporating variants of green fluorescent protein. To increase detection sensitivity and response dynamics, we genetically engineered firefly luciferase to detect specific intermolecular interactions through modulation of its luminescence activity. This concept has been applied in covalent, noncovalent, and allosteric design configurations. The covalent design gives sensitive detection of protease activity through a cleavage-dependent increase in luminescence. The noncovalent and allosteric designs allow reversible detection of the small molecules rapamycin and cAMP, respectively. These sensors allow detection of molecular processes within living cells following addition of the luciferin substrate to the growth medium. For example, the cAMP sensor allows monitoring of intracellular signal transduction associated with G-protein coupled receptor function. These and other luminescent biosensors will be useful for the sensitive detection of cellular physiology in research and drug discovery.
Fan
Frank
F
Binkowski
Brock F
BF
Butler
Braeden L
BL
Stecha
Peter F
PF
Lewis
Martin K
MK
Wood
Keith V
KV
D000494:Allosteric Regulation; D015374:Biosensing Techniques / Q000379:methods*; D002477:Cells / Q000166:cytology* / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000032:analysis / Q000378:metabolism; D010450:Endopeptidases / Q000032:analysis / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D005815:Genetic Code; D006801:Humans; D049409:Luciferases, Firefly / Q000737:chemistry / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods*; D043562:Receptors, G-Protein-Coupled / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D020123:Sirolimus / Q000032:analysis / Q000378:metabolism
D005456:Fluorescent Dyes; D043562:Receptors, G-Protein-Coupled; D000242:Cyclic AMP; D049409:Luciferases, Firefly; D010450:Endopeptidases; D020123:Sirolimus