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.

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Notes/Citation Information

Published in PLOS ONE, v. 11, no. 7, e0158579, p. 1-17.

© 2016 Grinstead et al.

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Funding Information

This study was funded by the National Institutes of Health, grant number 7R01GM047915, and by the Lucille P. Markey Chair.

journal.pone.0158579.s001.DOC (77 kB)
S1 Fig. Plasmids for expression of aequorin variant proteins.

journal.pone.0158579.s002.DOC (154 kB)
S2 Fig. SDS Gel Showing Purified Aequorin and Aequorin with L-4-iodophenylalanine at position 82 and 86.

journal.pone.0158579.s003.DOC (42 kB)
S1 Table. Mass spectrometry data for the aequorin mutants.

journal.pone.0158579.s004.DOC (45 kB)
S2 Table. Specific activity of aequorin variants: (A) Single substitution at position 86 and (B) Double substitution aequorin variants at positions 82 and 86.

journal.pone.0158579.s005.DOC (34 kB)
S3 Table. Emission wavelengths of aequorin variants.

journal.pone.0158579.s006.DOC (43 kB)
S4 Table. Emission half-lives of aequorin variants.

journal.pone.0158579.s007.DOC (24 kB)
S5 Table. Mass Spectrometry Data for AminoPhe86AEQ.

journal.pone.0158579.s008.DOC (24 kB)
S6 Table. Mass Spectrometry Data for BromoPhe86AEQ.

journal.pone.0158579.s009.DOC (24 kB)
S7 Table. Mass Spectrometry Data for IodoPhe86AEQ.

journal.pone.0158579.s010.DOC (24 kB)
S8 Table. Mass Spectrometry Data for MethoxyPhe86AEQ.

journal.pone.0158579.s011.DOC (24 kB)
S9 Table. Mass Spectrometry Data for BromoPhe8286AEQ.

journal.pone.0158579.s012.DOC (24 kB)
S10 Table. Mass Spectrometry Data for IodoPhe8286AEQ.

journal.pone.0158579.s013.DOC (24 kB)
S11 Table. Mass Spectrometry Data for MethoxyPhe8286AEQ.