Red-Shifted Aequorin Variants Incorporating Non-Canonical Amino Acids: Applications in In Vivo Imaging

• Published in: PloS one Vol. 11; no. 7; p. e0158579
• Main Authors: Grinstead, Kristen M, Rowe, Laura, Ensor, Charles M, Joel, Smita, Daftarian, Pirouz, Dikici, Emre, Zingg, Jean-Marc, Daunert, Sylvia
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 01.07.2016; Public Library of Science (PLoS)
• Subjects: Aequorin - chemistry; Aequorin - genetics; Aequorin - metabolism; Amino Acid Substitution; Amino acids; Animal models; Animals; Biochemistry; Biocompatibility; Biology and Life Sciences; Bioluminescence; Calcium; Chemistry; Color; Emission; Emissions; Eye; Female; Gene expression; Imaging; Imaging systems; In vivo methods and tests; Laboratories; Light; Luciferin; Luminescence; Medical imaging; Medicine; Medicine and Health Sciences; Mice; Models, Molecular; Molecular biology; Molecular Imaging; Mutagenesis; Mutants; Mutation; NMR; Nuclear magnetic resonance; Photoproteins; Physical Sciences; Physiological aspects; Properties; Protein binding; Protein Conformation; Proteins; Renilla-luciferin 2-monooxygenase; Research and Analysis Methods; Technology application; Tomography; United States; United States > US
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0158579

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.