基于SELEX技术的特异荧光DNA酶筛选和机制研究

发布时间：2019-03-07 11:31

【摘要】：目前癌症是世界上最致命的人类疾病之一。当前,在世界范围内的所有癌症当中,乳腺癌是妇女当中被诊断最多的,大约占所有癌症的四分之一。而且随着全球经济的持续发展以及随着而来的西方生活方式的流行,发展中国家的乳腺癌发病率在持续上升。在医学临床研究中,不同阶段的乳腺癌有相应不同的临床表现和治疗方案。尽管目前已经有一些早期诊断方法,诸如乳房X射线放射造影、核磁共振成像等,然而在发展中国家,大部分的乳腺癌在发现时已经处于乳腺癌发展后期。这是因为这些传统的方法具有价格昂贵、检测设备依赖度较高、以及检查过程繁琐等缺点。因此开发一种简易并且快捷的乳腺癌检测手段对于提高乳腺癌患者的生存率是非常有意义的。所以利用乳腺癌细胞外分泌混合物建立的体外血清检测无疑是最好的方法。基于这种需要,我们尝试开发一种新型的乳腺癌检测方法,其主要依赖具有催化活性的单链脱氧核糖核苷酸分子(DNAzyme)或者称之为适配子。利用指数富集的配基系统进化技术(SELEX),这种催化DNA分子能够从一个DNA随机序列库中筛选得到,目前已经在很多领域有所应用。所以,在实验室,我们假定具有荧光催化能力的DNA分子能够检测由特殊癌症细胞所产生的胞外分泌混合物,进而其能够被开发为癌症早期检测的探针。我们决定选用的具有荧光催化能力的探针是基于能对RNA切割并产生荧光信号的催化DNA。该种DNA分子在其嵌入了一个单独的RNA残基,并且在该残基的两端分别修饰了一个荧光分子集团和一个淬灭分子集团。这些巧妙的结构特征的设计目的是:在同一条链上的DNAzyme催化切割断自身的RNA残基后,能够释放出强烈的荧光信号。最终,我们开发出了具有RNA剪切功能的催化DNA分子——LYF5,该催化DNA分子能够特异地识别人类乳腺癌细胞T47D的胞外培养液,在体外,其荧光检测信号最低能检测500个细胞每毫升T47D培养浓度。经过DNA一级序列和二级结构优化后,第二代的荧光探针分子(2G-LYF5)是第一代探针LYF5的催化活性的约两倍。此外,我们还发现第二代荧光分子的二级发卡结构在其催化切割功能中发挥了决定性的作用。因此,该分子将有希望开发为临床上作为乳腺癌早期诊断的探针。
[Abstract]:Cancer is one of the world's most deadly human diseases. Currently, among all the cancers worldwide, breast cancer is the most diagnosed among women, accounting for about a quarter of all cancers. The incidence of breast cancer in developing countries has continued to rise with the continued development of the global economy and the prevalence of the way of life in the West. In the medical clinical study, different stages of breast cancer have different clinical manifestations and treatment options. Although there have been some early diagnostic methods, such as mammography, nuclear magnetic resonance imaging, and the like, most of the breast cancer in developing countries has been in the late stage of breast cancer development at the time of discovery. This is because these traditional methods have the disadvantages of high price, high dependence of the detection equipment, and the complicated checking process. Therefore, it is of great significance to develop a simple and rapid method of breast cancer detection to improve the survival rate of breast cancer patients. The in vitro serum detection established using the extracellular secretion mixture of breast cancer is no doubt the best method. Based on this need, we try to develop a new method of detecting breast cancer, which relies primarily on a single-stranded deoxyribose nucleic acid molecule (DNAzyme) with a catalytic activity or called an aptamer. This catalytic DNA molecule can be screened from a DNA random sequence library using an exponential-enriched ligand system evolution technique (SELEX), which has been applied in many fields. Therefore, in the laboratory, we assume that a DNA molecule with a fluorescence catalytic capability can detect an extracellular secretion mixture produced by a particular cancer cell, and further it can be developed as a probe for early detection of cancer. Our decision to select a probe with a fluorescence catalytic capability is based on the catalytic DNA that can cut the RNA and produce a fluorescence signal. The DNA molecule has a single RNA residue embedded therein, and a fluorescent molecule group and a quencher molecule group are modified at both ends of the residue, respectively. These ingenious structural features are designed to release a strong fluorescence signal after cleavage of its own RNA residue with the DNAzyme on the same strand. Finally, we developed a catalytic DNA molecule _ LYF5 with an RNA shearing function, which can specifically identify the extracellular medium of human breast cancer cell T47D. In vitro, its fluorescence detection signal can detect the minimum of 500 cells per milliliter of T47D culture concentration. The second generation of the fluorescence probe molecules (2G-LYF5) is about twice the catalytic activity of the first-generation probe LYF5 after the DNA-level sequence and the secondary-structure optimization. In addition, we have also found that the secondary hairpin structure of the second generation of fluorescent molecules plays a decisive role in its catalytic cutting function. Thus, the molecule will have the desire to develop a probe that is clinically useful as an early diagnosis of breast cancer.
【学位授予单位】：清华大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.9

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