基于功能核酸串联的诊疗一体化探针的构建与应用研究

Simultaneous detection and regulation disease-related mRNA level is a key research task in the field of theranostics. In this project, we propose a novel tandem functional nucleic acids-based theranostic platform for mRNA detection and regulation in tumor cells by taking advantage of the unique properties of each type of functional nucleic acids. To overcome the limitations of conventional molecular beacon (MB) during in vivo applications (e.g. poor cellular uptake and susceptible to exonucleases digestion), a tumor targeting aptamer (AP) is coupled to MB sequence to form AP-MB tandem structure, which could actively internalize into cancer cells with the aid of AP. In addition, the circular AP-MB sensor is designed to improve thermal stability and nuclease resistance. Finally, a RNA-cleaving DNAzyme (DZ) was inserted into AP-MB structure to construct circular AP-MB-DZ multi-functional theranostic sensor. Each sensor will be systematically characterized for their theranostic performances as well as biocompatibility in vitro and in vivo. This study will provide a new approach to design of convenient and efficient probes for cancer theranostics.

检测与调控胞内疾病相关mRNA水平是医学诊疗研究的重要内容。本项目以肿瘤相关mRNA为靶点，结合多种功能核酸的特性，构建基于功能核酸串联的新型肿瘤诊疗探针，应用于胞内mRNA的实时监测与水平调控。项目旨在解决传统分子信标（molecular beacon, MB）无法自由进入细胞、易被核酶降解及无法实时调控mRNA水平等缺陷，1）将特异性识别肿瘤细胞的核酸适配体（aptamer, AP）与MB串联，通过AP介导入胞，实现胞内mRNA检测；2）构建环状串联信标，以期增加信标在生理环境下的稳定性；3）在MB的结构中引入脱氧核酶（DNAzyme, DZ）序列，构建环状三串联核酸信标，分别利用MB及DZ对mRNA的识别及切割能力，同步监测与调控mRNA水平，实现肿瘤的诊疗一体化。本项目拟从体外及细胞水平全面评价信标的诊疗性能及生物安全性，研究结果将为新型肿瘤的诊疗一体化探针的开发提供基础。

本课题构建了一系列基于功能核酸（如核酸适配体、DNA酶）的肿瘤诊疗探针，此类探针能在功能核酸的介导下特异性进入肿瘤细胞，对肿瘤特异性的核酸分子标志物进行识别，产生荧光信号，用于肿瘤早期诊断。与此同时，信标结构内部的DNA酶可对肿瘤相关mRNA与microRNA进行切割，实现肿瘤基因治疗。在此基础上，扩展功能核酸的应用领域，并结合纳米载体在药物递送、信号转导、自身生物活性等方面的特性，构建基于功能核酸的多模块纳米诊疗体系，用于肿瘤的治疗。通过上述工作的开展，申请人以通讯作者的身份，在Biomaterials, Materials Horizons, Nano Letters, Theranostics, Journal of Controlled Release等杂志发表高水平SCI论文19篇（均标注基金号），其中IF大于10分论文5篇，授权国家发明专利。