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.
Highlights
Imaging is a critical component of medical diagnostics and biomedical research
We propose the direct administration of engineered red-shifted emission variants of the photoprotein aequorin, a protein employed previously in deep tissue calcium measurements [24]
Our data demonstrate that aequorin variants containing non-natural amino acids in place of the tryptophan residue at position 86 and the tyrosine at position 82 showed red-shifted bioluminescence activity when complexed with native coelenterazine
Summary
X-Ray computed tomography, ultrasonography, magnetic resonance imaging (MRI), and positron emission tomography imaging (PET), and optical methods based on fluorescence or bioluminescence are used as stand-alone or in-tandem in diagnostic imaging to provide information on morphological, anatomical, or organ function. Despite their wide use, the majority of these methods do not have the ability to monitor specific molecular events linked to disease states and provide a complete picture of a biochemical process. Bioluminescence does not require external illumination, making it suitable for light sensitive organs and tissues such as the retina
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