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Optical Imaging Probes
Published in Martin G. Pomper, Juri G. Gelovani, Benjamin Tsui, Kathleen Gabrielson, Richard Wahl, S. Sam Gambhir, Jeff Bulte, Raymond Gibson, William C. Eckelman, Molecular Imaging in Oncology, 2008
Measuring emitted bioluminescence photons using photon counting devices or CCDs provides one of the most sensitive means of detecting the exogenous marker protein expression enabling the detection of single transfected cells in vitro (34). Likewise, because of the extremely low levels of background bioluminescence in mammals one can detect a very low number of luciferase-positive cells in vivo. This is very useful if the evidence of tumor proliferation or firefly (Photinus pyralis beetle) luciferase marker protein expression in HeLa cells enabled the detection of only about 1000 cells injected in peritoneal cavity of rats (35). Firefly luciferase (61 kDa) and Renilla (sea pansy) luciferase (36 kDa) are both monomeric proteins that do not require posttranslational processing for enzymatic activity. Unlike fluorescent proteins, luciferases do not require a maturation period and newly translated products are already catalytically active. Firefly luciferase chemiluminescence has a range of approximately 550 to 570 nm, whereas click beetle (Pyrophorus sp.) red-shifted luciferase emits photons with a spectral maximum of 635 nm. The latter enables spectral separation of luminescence peaks and dual reporter detection, which is usually performed by using click beetle luciferase mutants. However, dual detection of Renilla and firefly luciferases more convenient since the enzymes utilize chemically distinct substrate systems. Beetle luciferase photon emission results from the oxidation of beetle luciferin to oxyluciferin in the presence of ATP, Mg2+, and oxygen, which occurs via luciferyl-AMP intermediate and results in a very rapidly decaying intense release of light. Firefly luminescence in vivo can have more favorable kinetics, which depend on coenzyme A concentration. Wild-type Renilla luciferase (RLuc) as well as its improved mutant forms catalyze oxidation of coelenterazine into coelenteramide, proceeds in the absence of ATP, and results in a release of blue light (480-nm maximum). Because of the high level of light output, mutant RLuc is indispensable as a source of excitation of EGFP in emerging in vivo BRET assays (36). The mainstay of luciferase use as tumor marker is the noninvasive imaging of tumor cell numbers (37), which shows excellent correlation with tumor volumes determined by using high-resolution magnetic resonance imaging (38,39). Recently, luciferase imaging found applications for determining kinetics of cell proliferation using transgenic models (40) as well as for performing promoter analysis in vivo (41). These applications are feasible due to the fact that luciferases mature rapidly and have a relatively short intracellular life (about 3 hours in mammals, spanning from translation to degradation) (42).
Advances in luminescence-based technologies for drug discovery
Published in Expert Opinion on Drug Discovery, 2023
Bolormaa Baljinnyam, Michael Ronzetti, Anton Simeonov
Coelenterazine reporters Renilla luciferase (RLuc) is another commonly deployed luciferase reporter derived from the octocoral, Renilla reniformis (Table 1). Interestingly, RLuc shares only minimal primary sequence homology with FLuc, and light production is independent of ATP cofactor [15,16]. RLuc produces luminescence through the oxidative decarboxylation of coelenterazine to coelenteramide, resulting in the concomitant emission of blue light [17].Gaussia luciferase (GLuc) is a smaller (20 kDa) secreted luminescent protein found in small crustaceans called copepods (Gaussia princeps). Unlike other coelenterazine-based reporters, GLuc requires the formation of disulfide bonds integral to the activity of the enzyme, precluding its use in systems that require reducing conditions [18,19]. Interestingly, GLuc, once secreted, has a considerably longer half-life (6 days) than either FLuc or RLuc [19].NanoLuc luciferase (NLuc) is an engineered version of shrimp Oplophorus gracilirostris luciferase. It reacts with furimazine, a coelenterazine derivative, with a specific activity over 150 times that of RLuc or FLuc [20]. In addition to NLuc’s enhanced brightness, it also has improved thermal stability, pH stability, and an unbiased distribution in cells as compared to other luciferases, leading to its rapid adoption into many HTS assays.