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Luciferin analogs for improved genetic reporter assays
Author(s) -
Kovic Yumi,
Rosenberg Justin C.,
Behney Curran E.,
Southworth Tara L.,
Branchini Bruce R.,
Woodroofe Carolyn C.,
Meisenheimer Poncho L.,
Klaubert Dieter H.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.574.3
Subject(s) - bioluminescence , benzothiophene , luciferase , luciferin , substrate (aquarium) , chemistry , benzothiazole , benzoxazole , indole test , benzofuran , light emission , fluorescence , stereochemistry , biophysics , biochemistry , biology , materials science , organic chemistry , ecology , transfection , optoelectronics , gene , thiophene , physics , quantum mechanics
In an effort to produce new firefly luciferin analogs for application to dual reporter assays, we prepared and evaluated novel substrate analogs. The benzothiazole ring structure of the natural substrate that produces light with firefly luciferase and ATP was replaced with the benzimidazole, benzofuran, benzothiophene, benzoxazole and indole rings. In vitro testing with P. pyralis luciferase revealed that the benzothiophene‐containing analog BtLH 2 had particularly interesting kinetic, bioluminescence and fluorescence properties. Particularly, bioluminescence reactions initiated with BtLH 2 displayed slow decay glow kinetics that resulted in the substrate emitting more than four times the light compared to the natural substrate. In addition, the substrate sustains a blue‐shifted emission maximum at 523 nm over a pH range of 6.2 to 9.4 compared to its natural substrate counterpart, which shifts from green to red with increasing pH. The stable light emission signal and absence of color shift observed with BtLH 2 demonstrates a potential for improved dual luciferase‐based genetic reporter applications using available red emitting luciferins.