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

Authors

Kristen M. Grinstead, University of Miami
Laura Rowe, Valparaiso University
Mark Ensor, University of KentuckyFollow
Smita Joel, University of Miami
Pirouz Daftarian, University of Miami
Emre Dikici, University of Miami
Jean-Marc Zingg, University of Miami
Sylvia Daunert, University of Miami

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.

Document Type

Article

Publication Date

7-1-2016

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.

Digital Object Identifier (DOI)

https://doi.org/10.1371/journal.pone.0158579

Funding Information

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

Repository Citation

Grinstead, Kristen M.; Rowe, Laura; Ensor, Mark; Joel, Smita; Daftarian, Pirouz; Dikici, Emre; Zingg, Jean-Marc; and Daunert, Sylvia, "Red-Shifted Aequorin Variants Incorporating Non-Canonical Amino Acids: Applications in In Vivo Imaging" (2016). Pharmaceutical Sciences Faculty Publications. 79.
https://uknowledge.uky.edu/ps_facpub/79