基于DNA纳米结构的活细胞内lncRNA的可视化分析及其在肿瘤诊疗中的应用研究

Recent studies in functional genomics manifest that long noncoding RNAs (lncRNA) play important roles in the regulation of multiple cellular developmental processes, including (post-)transcription, translation, and epigenesis. More than that, lncRNA have been validated to be a new family of cancer biomarkers. However, conventional in vitro RNA detection techniques could hardly reveal the spatiotemporal specificity and the subcellular localization diversity of the intracellular lncRNA expression, the functional information in which correlate with the occurrence and the development of tumors intimately. This project intends to make the best of DNA nanostructures in terms of their spontaneous assembly, easy transfection and convertible conformation, by fusing binding sites and reporter moieties, to develop novel strategy for visualizing and analyzing lncRNA molecules in the single living cell. The in situ real-time monitoring would be realized on the activity, population and distribution of lncRNA under conditions of drug stimulation; so it would be for the adsorption of multiplexed miRNA biomarkers onto lncRNA as “molecular sponge” and their mutual regulations. Both would elucidate the characteristics and the capability of lncRNA in cellular signal transduction as well as tumorigenesis and development. The implementation of this project will provide practical paradigm for the application of DNA nanostructures in highly efficient lncRNA determination, input statistical data of theoretical value for the illumination of lncRNA-mediated signaling pathways, and lastly pave a series of brand-new technological approaches for lncRNA-targeted tumor theranostics.

功能基因组学的最新研究表明，长非编码RNA(简称lncRNA)在转录(后)、翻译、表观遗传等多个调控过程中发挥重要作用，是一类新的癌症诊断标志物。传统胞外RNA检测技术难以揭示lncRNA胞内表达的时空特异性和亚细胞位置多样性，这些与肿瘤发生发展密切相关的功能信息。本项目拟利用DNA纳米结构自组装、易转染、可变构等优点，融合靶点和信标，发展单细胞内多元lncRNA分子的可视化分析新策略，实现对“分子海绵”lncRNA与多组分miRNA的吸附与相互调节研究，以及抗癌药物刺激下肿瘤细胞内lncRNA丰度分布、运动状态等关键参数的实时监测，从而揭示lncRNA在细胞信号转导和肿瘤发生发展中的特性功用。该项目的实施将为复合DNA探针面向lncRNA的高效检测应用提供实践范例，为阐明lncRNA介导的细胞信号通路输入有理论价值的一般规律，并为发展以lncRNA为靶标的肿瘤诊疗开辟一系列新的技术途径。

本研究以框架核酸（Framework nucleic acids, FNA）为核心部件，构建了可变构的DNA纳米复合探针，利用其易组装、构架稳定且变构可控的优点，发展细胞内、外RNA 的识别、定量及可视化分析新方法。项目按照项目任务书计划执行。首先，合理设计、制备并筛选出结构新颖、功能丰富的FNA组装体：高对称柏拉图多面体DNA四面体兼具几何刚性和形状持久性，通过编码棱边或修饰其顶点的改造，产生多样化的变构模式；生物可切割的DNA八面体，其中含有特异性响应肿瘤细胞中GSH而断裂的S-S 键；基于非模板依赖的等温扩增调制的枝状DNA构建的三维DNA水凝胶。随后，以FNA为关键组件，设计和构造多种纳米结构探针，通过开关变构、串联复用和级联置换等方式实现信号的通断或增减，并探索了该结构在生物标记和信号传导方面的潜力。最后以刚性的FNA作为支脚架，将DNA纳米探针递送至细胞内，实现活细胞中ncRNA成像分析，并可区分肿瘤细胞分化程度，为其在肿瘤诊断中的应用奠定基础。