基于超电荷荧光蛋白的核酸传感新方法

Homogeneous nucleic acid assay is crucial for high-throughput screening and clinic point-of-care testing. Nowadays, although nanomaterials are widely used in such assays, the development of nanomaterial-independent strategies is still highly desirable because of the potential nanotoxicity. This project intends to develop a novel strategy of homogeneous nucleic acid assay based on the supercharged fluorescent protein (ScFP). Several new ScFPs will be constructed, and the interaction between ScFP and DNA will be thoroughly studied. Based on such interaction, a novel ScFP-based sensing principle of nucleic acid assays will be established and several new singly-labeled or label-free homogeneous methods for the detection of nucleic acid-recognized targets will be developed. Moreover, the methods will be extended to multi-targets measurements with different kinds of ScFPs, and ultrasensitive detections will be achieved by coupling with nucleic acid amplification techniques. Integrating the fluorescent characteristics and multiple fluorescences and charges of ScFP, the selective interaction between ScFP and DNA, with the highly designable sequence and recognition capability of DNA, and the nucleic acid amplification techniques, this project is expected to build a novel platform of homogeneous nucleic acid assays with multiple targets, high sensitivity and quick detection. Furthermore, to address the requirement of epigenetic modification study and early diagnosis of cancer, novel ScFPs-based assays will be created for specific and sensitive measurements of DNA methylation and other new nucleic acid epigenetic modifications, such as DNA hydroxymethylation, hydroformylation, and carboxylation.

均相核酸传感对于高通量筛查与临床即时诊断具有重要意义。目前纳米材料被广泛用于该领域，然而纳米生物毒性越来越引起关注，亟需发展非纳米材料依赖的均相核酸传感新原理、新技术。本项目拟发展基于新型超电荷荧光蛋白（supercharged fluorescence protein, ScFP）的均相核酸传感新策略：构建系列ScFP及系统研究ScFP/核酸相互作用，提出基于该相互作用的核酸传感新原理，开发多种单标记或免标记均相检测新方法，进一步发展多目标检测体系及超敏传感新技术。本课题将ScFP的荧光性质、荧光/电荷多样性及对DNA的选择性结合作用，与DNA的高度序列可设计性、分子识别能力以及核酸信号放大技术相结合，以期建立多对象、高灵敏的均相核酸传感新平台。根据表观遗传学研究和肿瘤早期诊断的需求，针对DNA甲基化及羟甲基化、醛基化、羧基化等新兴表观遗传修饰类型，发展基于ScFP的特异性灵敏检测方法。

均相核酸传感对于高通量筛查与临床即时诊断具有重要意义。目前纳米材料被广泛用于该领域，然而纳米生物毒性越来越引起关注，亟需发展非纳米材料依赖的均相核酸传感新原理、新技术。本项目提出了基于新型超电荷荧光蛋白（supercharged fluorescence protein, ScFP）的均相核酸传感新策略。设计构建了多种ScFP 蛋白， 为新型传感和成像研究提供新材料、新思路。在此基础上，研究了ScFP的荧光、电荷性质、细胞穿透性，及与核酸的相互作用。利用ScFP与核酸之间的相互作用，与DNA的高度序列可设计性、分子识别能力以及核酸信号放大技术相结合，发展了一系列高灵敏、高特异性的均相检测新方法，可分别用于复杂样品中多种关键酶如乙酰胆碱酯酶(AChE)、碱基切除修复酶(UDG)、蛋白激酶(PKA)、转肽酶(SrtA)、PARP-1等的活性检测及其抑制剂筛选，以及食物样品中农残和革兰氏阳性细菌的检测。进一步开发了基于富含组氨酸的HisnFP的运载系统，为功能核酸及相关蛋白的可控细胞转运提供了新方法。最后，通过将ScFP与多肽逻辑门、纳米材料以及DNA纳米结构等相关研究进行结合，为荧光蛋白的应用领域开辟出新的道路。本项目在相关领域发表 SCI 论文 22篇（其中影响因子大于 10 的 1 篇，影响因子大于 5 的 11篇），申请相关专利 1 项（201610272231.7），培养博士研究生 6 名、硕士研究生 9 名，完成课题预期目标。