超猝灭多功能分子信标的构建及应用研究

Molecular beacon (MB) has wide application prospect in molecular biology and molecular medicine for its outstanding advantages, including easy synthesis, high selectivity, and real-time detection. While MB has been applied successfully in many fields, its utility for quantification in the practical application has some limitations. In existing MBs, some have high background emission, some have bad repeatability because of their complex preparation process and poor stability, and some other have slow reaction speed because of steric hindrance caused by large nanoparticles. In addition, the detection sensitivity of MB needs to be further improved. To address these problems of MB for quantitative detection, this project plans to carry out research in the following three aspects. Multifunctional super-quencher molecular beacon will be designed using simultaneous quenching effect of organic quencher and G bases to fluorophore, and achieve quantification detection of HBV. Different super-quencher MBs will be designed, the fluorophores of these MBs have same or similar Stokes shift, and achieve simultaneous quantification detection of two nucleic acids using synchronous scanning fluorescence spectrometry. Ultra high sensitive detection methods of HBV and thrombin will be developed based on high selective super-quencher molecular beacon and high sensitive nucleic acid dye. It not only can enhance significantly the detection sensitivity, but also can guarantee the selectivity of detection method. If the sequence of the loop of the super-quencher MB is changed, it can also be applied to detect other nucleic acid and protein.

分子信标（MB）合成容易、选择性好且能实时检测，在分子生物学及分子医学等方面具有广泛应用前景。MB在很多领域都获得了成功应用，但现有的MB在定量分析中各自存在一些不足：有的荧光背景高，有的制备过程复杂且稳定性差，检测重复性不好，有的存在较大粒径的纳米粒子，空间位阻大，与目标物反应速度慢。另外，现有MB的灵敏度都相对较低。这些缺陷使MB很难应用于实际样品的定量分析中。针对这些问题，本项目拟从以下几个方面开展研究：利用有机猝灭基团和鸟嘌呤对荧光基团的双重猝灭作用，构建结构简单的超猝灭MB，实现乙肝病毒的定量检测；构建两种具有相近Stock's位移荧光基团的超猝灭MB，采用同步荧光分析法，实现两种核酸的同步定量检测；利用超猝灭MB和高灵敏核酸染料，建立超高灵敏乙肝病毒及凝血酶检测新方法，在保证方法选择性的同时，显著提高检测灵敏度。改变MB中环的序列，还可用于其它核酸和蛋白的检测。

基于有机猝灭基团及鸟嘌呤（G碱基）对有机荧光基团的双猝灭作用，构建了一系列结构简单的超猝灭分子信标。通过筛选，得到了一种荧光背景低、信背比很高的分子信标。该分子信标以TAMRA为荧光基团，BHQ-2为有机猝灭基团，三个具有G碱基的核苷酸与BHQ-2相连。将该分子信标的环设计成与乙肝病毒（HBV）寡核苷酸片段互补的DNA片段，实现了对野生HBV寡核苷酸片段（目标DNA）的定量检测。 .以野生HBV寡核苷酸片段和艾滋病毒RNA片段的反转录序列为目标DNA设计了两种结构简单的双重猝灭分子信标，FAM和TAMRA分别作为两种分子信标的荧光基团，BHQ-1和BHQ-2分别作为两种分子信标的猝灭基团，三个具有G碱基的核苷酸分别与BHQ-1和BHQ-2相连。利用这两种分子信标，结合同步分析法建立了一种高灵敏的两种DNA同时检测方法。.以艾滋病毒RNA片段的反转录序列为目标DNA，设计了一种双重猝灭分子信标。该分子信标中，荧光基团设计为FAM，有机猝灭基团设计为BHQ-1，分子信标的茎完全设计为C-G碱基对，与BHQ-1相连接的为3个带有G碱基的核苷酸，分子信标的环设计为目标DNA的互补序列。利用该分子信标，结合核酸染料Hoechst 33258，建立了一种高灵敏单链核酸（ssDNA）的双色荧光定量检测方法。.以凝血酶为检测对象，设计了一种双重猝灭分子信标。该分子信标中，荧光基团设计为FAM，有机猝灭基团设计为BHQ-1，BHQ-1连接3个带有G碱基的核苷酸，分子信标的环设计为凝血酶的核酸适配体。利用该分子信标，建立了一种凝血酶的简单检测方法。.Hg2+及Pb2+为检测对象，设计了两种结构简单的双重猝灭分子信标，FAM和TAMRA分别作为两种分子信标的荧光基团，BHQ-1和BHQ-2分别作为两种分子信标的猝灭基团，三个具有G碱基的核苷酸分别与BHQ-1和BHQ-2相连， 两种分子信标的环和茎的一部分设计为Hg2+及Pb2+的适配体。利用这两种分子信标，用同步分析法建立了一种高灵敏的Hg2+及Pb2+同时检测方法。