title: Red-Shifted Aequorin Variants Incorporating Non-Canonical Amino Acids: Applications in In Vivo Imaging.
abstract: The increased importance of in vivo diagnostics has posed new demands for imaging technologies. In that regard, there is a need for imaging molecules capable of expanding the applications of current state-of-the-art imaging in vivo diagnostics. To that end, there is a desire for new reporter molecules capable of providing strong signals, are non-toxic, and can be tailored to diagnose or monitor the progression of a number of diseases. Aequorin is a non-toxic photoprotein that can be used as a sensitive marker for bioluminescence in vivo imaging. The sensitivity of aequorin is due to the fact that bioluminescence is a rare phenomenon in nature and, therefore, it does not suffer from autofluorescence, which contributes to background emission. Emission of bioluminescence in the blue-region of the spectrum by aequorin only occurs when calcium, and its luciferin coelenterazine, are bound to the protein and trigger a biochemical reaction that results in light generation. It is this reaction that endows aequorin with unique characteristics, making it ideally suited for a number of applications in bioanalysis and imaging. Herein we report the site-specific incorporation of non-canonical or non-natural amino acids and several coelenterazine analogues, resulting in a catalog of 72 cysteine-free, aequorin variants which expand the potential applications of these photoproteins by providing several red-shifted mutants better suited to use in vivo. In vivo studies in mouse models using the transparent tissue of the eye confirmed the activity of the aequorin variants incorporating L-4-iodophehylalanine and L-4-methoxyphenylalanine after injection into the eye and topical addition of coelenterazine. The signal also remained localized within the eye. This is the first time that aequorin variants incorporating non-canonical amino acids have shown to be active in vivo and useful as reporters in bioluminescence imaging.
authors.affiliation: Department of Biochemistry and Molecular Biology, R. Bunn Gautier Bldg., 1011 NW 15th Street, Miller School of Medicine, University of Miami, Miami, FL, 33136, United States of America.
authors.forename: Kristen M
authors.initials: KM
authors.lastname: Grinstead
authors.affiliation: Department of Chemistry, 1610 Campus Drive East, Valparaiso University, Valparaiso, IN, 46385, United States of America.
authors.forename: Laura
authors.initials: L
authors.lastname: Rowe
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY, 40536, United States of America.
authors.forename: Charles M
authors.initials: CM
authors.lastname: Ensor
authors.affiliation: Department of Biochemistry and Molecular Biology, R. Bunn Gautier Bldg., 1011 NW 15th Street, Miller School of Medicine, University of Miami, Miami, FL, 33136, United States of America.
authors.forename: Smita
authors.initials: S
authors.lastname: Joel
authors.affiliation: Department of Biochemistry and Molecular Biology, R. Bunn Gautier Bldg., 1011 NW 15th Street, Miller School of Medicine, University of Miami, Miami, FL, 33136, United States of America.
authors.forename: Pirouz
authors.initials: P
authors.lastname: Daftarian
authors.affiliation: Department of Biochemistry and Molecular Biology, R. Bunn Gautier Bldg., 1011 NW 15th Street, Miller School of Medicine, University of Miami, Miami, FL, 33136, United States of America.
authors.forename: Emre
authors.initials: E
authors.lastname: Dikici
authors.affiliation: Department of Biochemistry and Molecular Biology, R. Bunn Gautier Bldg., 1011 NW 15th Street, Miller School of Medicine, University of Miami, Miami, FL, 33136, United States of America.
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Zingg
authors.affiliation: Department of Biochemistry and Molecular Biology, R. Bunn Gautier Bldg., 1011 NW 15th Street, Miller School of Medicine, University of Miami, Miami, FL, 33136, United States of America.
authors.forename: Sylvia
authors.initials: S
authors.lastname: Daunert
mesh_terms: D000331:Aequorin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D003116:Color; D005260:Female; D049449:Luminescence; D051379:Mice; D008958:Models, Molecular; D057054:Molecular Imaging; D011487:Protein Conformation
chemical_list: D000331:Aequorin