核酸适配体功能化上转换纳米探针用于神经元内蛋白激酶的成像和光控活性调控

The mitogen-activated protein kinases (MAPKs) play an important role in the pathological mechanism of neurodegenerative diseases. However, due to inability to image MAPKs spatiotemporally and regulate its activity efficiently, it remains a challenge to understand the specific mechanism of MAPKs in neurons. Herein, we propose to use aptamer-functionalized upconversion nanoprobes to realize real-time in situ imaging and high-efficiency activity regulation of MAPKs in neurons. Upconversion nanoparticles can harvest the biocompatible NIR light and convert it into UV and visible light, which can be used in MAPKs imaging with excellent advantages, such as enhanced tissue penetration, an essentially elimination of background fluorescence and low damage to neurons. In addition, the light conversion can also be used to design valves to control the interactions between MAPKs and regulators, further regulating the MAPKs activity. Aptamer can simultaneously bind and inhibit MAPKs with high specificity and high efficiency, and thus is an ideal candidate for imaging and activity regulation of MAPKs. We seek support of this project for real-time in situ imaging and activity regulation of MAPKs in neurons by using aptamer-functionalized upconversion nanoprobes. This research proposal puts forward a strategy to investigate the specific mechanism of MAPKs in neurons and provide keen insights into the pathology of neurodegenerative diseases.

神经元内丝裂原活化蛋白激酶（MAPKs）信号通路与神经退行性疾病的发病机制有密切联系。目前神经元内MAPKs的具体作用机制尚不明确，其深入研究受到缺乏实时原位成像方法和激酶活性调控效率低等因素的限制。上转换纳米材料吸收近红外光并发射紫外光和可见光，这一性质使其具有组织穿透深、可抑制背景荧光干扰等显著特点，在生物成像方面具有极大优势。此外，利用上转换材料的光信号转变，还可以设计光控分子开关实现蛋白激酶与调控因子的相互作用调节进而调控蛋白激酶的活性。核酸适配体同时具有酶调控因子和分子识别的双重功能，能够实现MAPKs靶向识别和活性调控的目的。本申请项目提出将核酸适配体功能化的上转换纳米探针用于神经元内MAPK信号通路蛋白激酶的靶向、高效的实时原位成像和光控活性调控，研究MAPK蛋白激酶的具体作用机制，更加深入地了解其与神经退行性疾病发病机制的联系，有望为神经退行性疾病的治疗提供帮助。

申请人按照拟定计划合成了CaF2:Yb,Er，NaYF4:Yb,Tm，NaYF4:Yb,Er/Mn和NaYF4:Yb/Er/Nd@NaYF4:Nd等上转换纳米颗粒。进一步，申请人对上转换纳米颗粒进行功能组装，修饰核酸适配体实现靶向成像和标志物分子检测等应用。相关研究成果已被撰写成论文并发表（Quan Yuan* et al. Chinese Science Bulletin. 2019, 64, 1067-1075；Quan Yuan* et al. Chem. Eur. J. 2017, 23, 10683 – 10689；Quan Yuan* et al. CCS Chemistry, 2019, 1, 490-501）。另外，申请人对核酸适配体探针进行设计和构建，实现了功能蛋白的活性调控。神经相关的Aβ蛋白的折叠与阿尔兹海默症、帕金森症等神经退行性疾病密切相关，抑制Aβ蛋白的折叠对于相关疾病的预防与治疗有重要的意义。申请人设计了一种双核酸适配体探针，能够有效抑制Aβ蛋白的折叠。同时，该探针还能实现Zn2+浓度的检测，探究Aβ蛋白的折叠与该区域Zn2+浓度的关系。这类集检测与调节双功能的探针对神经退行性疾病的诊断与病理研究具有重要的意义。（Quan Yuan* et al. Analytical chemistry, 2019, 91, 1, 823-829）。另外，申请人还设计了核酸适配体复合纳米探针，结合核酸适配体的靶向作用与纳米材料的调控功能来实现蛋白活性的特异性调控，促进了碱性磷酸酶、骨钙蛋白和Ⅰ型胶原等重要蛋白的表达。（Quan Yuan* et al. ACS Nano. 2020, 14, 16085–16095；Quan Yuan* et al. Nature Communications, 2019, 10, 2829）。在此基础上，针对现有核酸适体库种类有限、应用受限的问题，申请人在核酸适配体中引入了人工合成碱基，扩大了核酸适配体的容量，为筛选高特异性和亲和力的核酸适配体提供了可能，筛选得到了蛋白活性调控性能更优的核酸适配体序列，有望进一步拓宽核酸适配体纳米探针的应用领域（Quan Yuan* et al. Angew. Chem. Int. Ed. 2019, 58, 1621 –1625.）。