Directed evolution of a magnetic resonance imaging contrast agent for noninvasive imaging of dopamine.
Directed evolution of a magnetic resonance imaging contrast agent for noninvasive imaging of dopamine.
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DOI：
10.1038/nbt.1609
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发表时间：
2010-03
影响因子：
46.9
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中科院分区：
文献类型：
--
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开发分子探针，使在体内成像的神经信号传导过程具有高的时间和空间分辨率仍然具有挑战性。在这里，我们应用定向进化技术来创建对神经递质多巴胺敏感的磁共振成像（MRI）造影剂。传感器来自细菌细胞色素P450-BM 3（BM 3 h）的血红素结构域。配体结合到BM 3 h的顺磁性血红素铁附近的位点导致MRI信号增强的下降和光吸收的偏移。使用基于吸光度的筛选，我们将BM 3 h的特异性从其天然配体进化到多巴胺，产生多巴胺解离常数为3.3-8.9 μM的传感器。这些分子被用于成像去极化触发的神经递质从PC 12细胞和活体动物的大脑中释放。我们的研究结果表明，分子水平的功能性磁共振成像使用神经活动依赖的传感器的可行性，我们的蛋白质工程方法可以推广到创建其他目标的探针。
The development of molecular probes that allow in vivo imaging of neural signaling processes with high temporal and spatial resolution remains challenging. Here we applied directed evolution techniques to create magnetic resonance imaging (MRI) contrast agents sensitive to the neurotransmitter dopamine. The sensors were derived from the heme domain of the bacterial cytochrome P450-BM3 (BM3h). Ligand binding to a site near BM3h’s paramagnetic heme iron led to a drop in MRI signal enhancement and a shift in optical absorbance. Using an absorbance-based screen, we evolved the specificity of BM3h away from its natural ligand and toward dopamine, producing sensors with dissociation constants for dopamine of 3.3–8.9 μM. These molecules were used to image depolarization-triggered neurotransmitter release from PC12 cells and in the brains of live animals. Our results demonstrate the feasibility of molecular-level functional MRI using neural activity–dependent sensors, and our protein engineering approach can be generalized to create probes for other targets.
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通讯作者：
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