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.
In recent years, luminescent proteins have been studied for their potential application in a variety of detection systems. Bioluminescent proteins, which do not require an external excitation source, are especially well-suited as reporters in analytical detection. The photoprotein aequorin is a bioluminescent protein that can be engineered for use as a molecular reporter under a wide range of conditions while maintaining its sensitivity. Herein, the characteristics of aequorin as well as the engineering and production of aequorin variants and their impact on signal detection in biological systems are presented. The structural features and activity of aequorin, its benefits as a label for sensing and applications in highly sensitive detection, as well as in gaining insight into biological processes are discussed. Among those, focus has been placed on the highly sensitive calcium detection in vivo, in vitro DNA and small molecule sensing, and development of in vivo imaging technologies. Graphical Abstract.
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