基于SELEX技術(shù)的特異熒光DNA酶篩選和機(jī)制研究
[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.
【學(xué)位授予單位】:清華大學(xué)
【學(xué)位級別】:碩士
【學(xué)位授予年份】:2015
【分類號】:R737.9
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