Red-Shifted Aequorin Variants Incorporating Non-Canonical Amino Acids: Applications in In Vivo Imaging.
Red-Shifted Aequorin Variants Incorporating Non-Canonical Amino Acids: Applications in In Vivo Imaging.
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DOI：
10.1371/journal.pone.0158579
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Daunert S
中科院分区：
文献类型：
--
作者：
Grinstead KM;Rowe L;Ensor CM;Joel S;Daftarian P;Dikici E;Zingg JM;Daunert S
体内诊断重要性的增加对成像技术提出了新的要求。在这方面，需要能够扩展当前最先进的体内诊断成像的应用的成像分子。为此，需要能够提供强信号、无毒并且可以定制以诊断或监测许多疾病的进展的新报告分子。水母发光蛋白是一种无毒的发光蛋白，可用作生物发光体内成像的敏感标记物。水母发光蛋白的敏感性是由于生物发光在自然界中是罕见的现象，因此，它不受自发荧光的影响，自发荧光有助于背景发射。只有当钙和腔肠素中的钙离子与蛋白质结合并引发导致光产生的生化反应时，水母发光蛋白才会在光谱的蓝色区域发射生物发光。正是这种反应赋予水母发光蛋白独特的特性，使其非常适合生物分析和成像领域的许多应用。在这里，我们报告了非典型或非天然氨基酸和几种腔肠素类似物的位点特异性掺入，从而产生了72种无半胱氨酸的水母发光蛋白变体的目录，这些变体通过提供几种更适合于体内使用的红移突变体来扩展这些发光蛋白的潜在应用。使用眼睛的透明组织在小鼠模型中进行的体内研究证实了在注射到眼睛中并局部添加腔肠素后掺入L-4-碘苯丙氨酸和L-4-甲氧基苯丙氨酸的水母发光蛋白变体的活性。该信号也保持在眼睛内。这是第一次，水母发光蛋白的变体纳入非典型的氨基酸已被证明是活跃的，在体内和有用的报告生物发光成像。
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.