DNA四面体纳米探针介导的胞外囊泡内microRNA原位多重检测技术研究

Extracellular vesicles (EVs) can mediate intercellular communication and regulate the expressions of downstream molecules. Therefore, the specific microRNA (miRNA) expression profiling is considered as a new biomarker that can reflect various physiological and pathological processes. However, most existing detection methods need the process of extraction of the internal components of EVs, which often leads to lower analytical result than the real expression levels of miRNAs within the EVs. Supported by grant from the national science foundation committee, we found that carbon quantum dots (CQD)-labeled fluorescent probes can be used for low concentration miRNA detection and the fluorescence on-off strategy of molecular beacons is particularly suitable for in-situ molecular detection in cells. However, the above-mentioned probe is difficult to penetrate into the nanoscale EVs membrane and is prone to be degraded by intracellular enzyme. This project aims to detect four pancreatic cancer-related miRNAs using multicolor CQD-modified molecular beacons. Then the well penetrated DNA pyramid will be assembled by DNA origami technology as a carrier to deliver the fluorescent molecular probe into the EVs. And these intracellular miRNAs could be accurately analyzed. The fulfillment of the project will realize extracellular vesicle microRNA direct and multiplexed visualization in situ, which would provide technical support for the early diagnosis, treatment and monitoring, and prognosis evaluation of various diseases.

胞外囊泡（EVs）内miRNA的时空表达谱可反映多种生理病理过程，明确其丰度变化对于提升临床疾病诊治水平具有重要价值。但目前尚缺乏EVs内miRNA原位表征手段，现有技术中RNA提取这一共性环节常导致结果均一性差且低于真实水平。申请人前续项目发现上转换碳量子点可提高miRNA检测灵敏度，且分子信标（MB）的荧光“开/关”策略尤其适合细胞内分子示踪。然而，MB探针难以穿入EVs胞膜且易被酶降解，故仍无法实现EVs内小分子原位检测。本项目拟以胰腺癌EVs内四种特异性miRNA为靶标，设计装配四条末端分别修饰不同颜色碳量子点的MB探针；采用DNA折纸技术自组装DNA四面体骨架，利用其良好穿膜特性递送荧光分子探针进入EVs胞内，记录杂交后荧光光谱改变以实现EVs胞内多个miRNA靶标的同步检测，最终建立EVs内miRNA表达谱丰度的原位多重检测技术，为疾病早期诊断、治疗监控和预后评估提供技术支撑。

胞外囊泡（EVs）内miRNA的时空表达谱可反映多种生理病理过程，明确其丰度变化对于提升临床疾病诊治水平具有重要价值。但目前尚缺乏EVs内miRNA原位表征手段，现有技术中RNA提取这一共性环节常导致结果均一性差且低于真实水平。项目组前期研究发现量子点自组装技术可对DNA进行标记性信号放大直接检测。基于此，本项目开展基于DNA自组装及时和碳量子点标记的荧光信号放大技术进行EVs内多联microRNA直接检测技术研究。本研究在研究过程中，严格按照预定计划开展为期4年的实验研究，以完成了所有预定研究目标：构建碳量子点纳米颗粒、表征并搭建成熟的表面功能化技术平台，优化了分子新表探针好量子点的偶联，构建了量子点标记的荧光信号放大系统。目研究发现DNA自组装技术结合荧光信号标记放大策略可有效实现靶标分子的直接检测。通过合理建模和 DNA 折纸技术设计并构建 DNA 四面体 3D 微结构，实现多重分子信标探针在 EVs 胞膜内的可控导入，并阐明其穿膜效应机制；建立一种能够实时动态检测 EVs 内多种 miRNA 时空表达丰度的分析技术，实现对胰腺癌等癌症相关EVs内miRNA组合的准确检测，以期用于肿瘤早期诊断和预后分析.本课题历时四年，共发表标注有基金号的文章16篇，其中SCI论文15篇，IF＞10分7篇，共申请发明专利4件。本项目的顺利实施为后续的基于DNA纳米探针的生物应用及EVs作为液体活检标志物的临床诊断应用研究奠定了坚实的理论和实验基础，对于研发相应的检验试剂和设备具有重要意义，为拓展纳米生物医学在临床诊断医学中的临床应用奠定了基础。