Designing a Single Protein-Chain Reporter for Opioid Detection at Cellular Resolution
Designing a Single Protein-Chain Reporter for Opioid Detection at Cellular Resolution
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DOI：
10.1002/anie.202101262
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发表时间：
2021-04-07
影响因子：
16.6
通讯作者：
Wang, Wenjing
中科院分区：
文献类型：
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作者：
Kroning, Kayla E.;Wang, Wenjing
μ-阿片受体（莫尔）信号传导调节多种神经元通路，包括参与疼痛、奖赏和呼吸的通路。为了促进对莫尔在疼痛调节中的作用的理解，需要体外阿片样物质的高通量筛选方法和脑中阿片样物质的高分辨率映射。为了满足这一需求，我们设计并表征了一种基因编码的荧光报告基因，称为用于莫尔的基于单链蛋白质的阿片类药物传输指示工具（M-SPOTIT）。M-SPOTIT代表了一种新的独特的荧光报告分子设计机制，可以检测莫尔激活，留下持久的绿色荧光标记用于图像分析。M-SPOTIT显示出高达12.5的阿片类药物依赖性信噪比（S/N），并且能够在HEK 293 T细胞培养物中检测到与30秒阿片类药物暴露一样快的速度。此外，它在神经元培养物中显示出高达4.6的阿片样物质依赖性S/N，并检测到芬太尼的EC 50为15 nM。M-SPOTIT将有可能用于细胞培养物中阿片类药物的高通量检测和体内阿片类药物的细胞分辨率检测。M-SPOTIT的新机制可以用作设计其他基于G蛋白偶联受体的传感器的平台。
Mu-opioid receptor (MOR) signaling regulates multiple neuronal pathways, including those involved in pain, reward, and respiration. To advance the understanding of MOR's roles in pain modulation, there is a need for high-throughput screening methods of opioids in vitro and high-resolution mapping of opioids in the brain. To fill this need, we designed and characterized a genetically encoded fluorescent reporter, called Single-chain Protein-based Opioid Transmission Indicator Tool for MOR (M-SPOTIT). M-SPOTIT represents a new and unique mechanism for fluorescent reporter design and can detect MOR activation, leaving a persistent green fluorescence mark for image analysis. M-SPOTIT showed an opioid-dependent signal to noise ratio (S/N) up to 12.5 and was able to detect as fast as a 30-second opioid exposure in HEK293T cell culture. Additionally, it showed an opioid-dependent S/N up to 4.6 in neuronal culture and detected fentanyl with an EC50 of 15 nM. M-SPOTIT will potentially be useful for high-throughput detection of opioids in cell cultures and cellular-resolution detection of opioids in vivo. M-SPOTIT's novel mechanism can be used as a platform to design other G-protein-coupled receptor-based sensors.