核酸特殊二级结构在DNA甲基转移酶和乳腺癌检测方面的应用

发布时间：2018-05-09 15:21

本文选题：甲基化转移酶 + DNAzyme　；参考：《武汉大学》2016年博士论文

【摘要】：在本论文中,我们对核酸的一些特殊二级结构(脱氧核酶和适配子)及其特有的功能进行了探讨,并通过利用这些特殊二级结构及其特有的功能,建立了两种与癌症疾病诊断相关的新方法,并对这些检测方法进行了深入的研究。本论文的研究内容主要分为两个大的部分,第一部分是利用DNAzyme能够选择性切割底物序列的特点,设计了一种基于DNAzyme切割信号放大来对DamDNA甲基转移酶活性进行检测的新方法。在第二部分工作中,我们利用核酸适配子对靶标的选择性,建立了一种结合适配子靶向和光激活荧光信号输出的新方法,并成功运用于乳腺癌的相关检测中。第一部分的内容包括：DNA甲基化是通过甲基转移酶的作用,将供体SAM上的甲基转移到受体胞嘧啶或腺嘌呤上的,它是一种非常重要表观遗传学修饰。由于DNA甲基化与基因激活、基因印记、染色质稳定性、女性中X-染色体失活和众多疾病的发生密切相关,DNA甲基化的研究在近些年中备受关注。有报道表明DNA甲基化的程度在不同的物种和不同的器官各不相同,且均有自身准确的调控。由于甲基化的失调会造成各种疾病发生,DNA甲基化被视为是临床诊断中一种潜在的重要标志物。因为甲基转移酶的活性直接影响着DNA甲基化程度,所以甲基转移酶活性的检测对基础研究和临床诊断都至关重要。我们利用DNAzyme这种特殊的核酸二级结构,并设计了一种新的发卡结构的探针,用于Dam甲基转移酶的检测。一旦探针中的特定序列被Dam甲基转移酶所识别,序列中的腺嘌呤会被甲基化,而甲基化后的序列可以被DpnI限制性内切酶识别并切割,从而释放出含有8-17 DNAzyme序列的单链,使DNAzyme的切割活性恢复,对分子信标结构底物进行切割,使荧光强度提高。同时,切割底物后的DNAzyme序列可以再与新的底物结合并将其切割,产生循环信号放大。最终,输出的荧光信号即为Dam甲基转移酶的活性。我们通过荧光动力学的方法验证了该策略的可行性,并且通过凝胶电泳实验证明了甲基化后的探针被成功切割,最后通过甲基转移酶的抑制剂实验证实了该策略得到的信号即为甲基转移酶的活性。因此,我们这种基于8-17 DNAzyme来检测Dam甲基转移酶的方法具有简单、灵敏、高效的特点。第二部分的主要内容包括：癌症被认为世界范围内发病和死亡的主要原因之一。恶性肿瘤能够无限制地增长,进而干预到消化系统、神经系统和循环系统。更糟糕的是,它们能通过释放激素来改变身体的机能。癌症难以检测,因为它生长于器官本身,人体的免疫系统不能将其识别和清除。大部分癌症患者在病发前,并没有明显的临床症状。然而一旦恶性肿瘤形成,病人的条件会急剧恶化。因此,早期癌症诊断对于癌症的治疗异常重要。传统的癌症检测方法需要从肿瘤形态学变化上来进行诊断,不能满足癌症早期检测的要求。发展从分子水平上区分癌症部位与正常部位的方法,对于癌症的早期诊断和及时治疗都有着非常重要的意义。我们选取了AS1411这种具有特殊二级结构的核酸适配子序列,结合光激活荧光成像技术,建立起了一种对该适配子的靶标——核仁素高特异性和高灵敏度的检测方法,并将其运用到乳腺癌的检测中。在这项工作中,我们首先合成了具有光激活响应的荧光小分子,并将其成功标记到AS1411核酸适配子上,制备出具有光激活响应的适配子荧光探针。然后,我们通过荧光检测、凝胶电泳和DNA质谱证明了该探针具有光激活荧光响应的性质。最后,我们通过细胞实验和组织切片实验证明了该策略对乳腺癌细胞具有高灵敏度和高选择性,能够达到和临床上一些金标准诊断方法相同的准确程度,通过对比光激活前后的荧光图像,可以得到更加准确和持续性的信号。
[Abstract]:In this paper, we explored some special two structures of nucleic acid (deoxy ribozyme and aptamer) and their specific functions. Through the use of these special two grade structures and their specific functions, we have established two new methods related to the diagnosis of cancer diseases, and studied these methods in depth. The main part of the study is divided into two major parts. The first part is the design of a new method for detecting the activity of DamDNA methyltransferase based on DNAzyme cutting signal amplification by using DNAzyme to selectively cut the substrate sequence. In the second part, we use the selectivity of the nucleic acid aptamer to target the target. A new method combining aptamer targeting and light activated fluorescence signal output is successfully applied to the detection of breast cancer. The first part includes: DNA methylation is a very important epigenetic inheritance of methyl transferase on the receptor cytosine or adenine through the role of methyl transferase on the donor SAM. The study of DNA methylation, gene activation, gene imprinting, chromatin stability, X- chromosome inactivation in women is closely related to the occurrence of many diseases. The study of DNA methylation has attracted much attention in recent years. It has been reported that the degree of DNA methylation is different in different species and different organs, and has its own accuracy. Regulation. Due to maladjustment of methylation can cause a variety of diseases, DNA methylation is regarded as a potential important marker in clinical diagnosis. As the activity of methyltransferase directly affects the degree of DNA methylation, the detection of methyltransferase activity is essential for both basic and clinical diagnosis. We use DNAzyme Special nucleic acid two structure and a new hairpin structure probe is designed for the detection of Dam methyltransferase. Once the specific sequence in the probe is identified by Dam methyltransferase, the adenine in the sequence can be methylated, and the sequence after methylation can be identified and cut by the DpnI restrictive endonuclease. The single strand of 8-17 DNAzyme sequences restores the cleavage activity of DNAzyme, cutting the molecular beacon structure substrate and improving the fluorescence intensity. At the same time, the DNAzyme sequence after the cutting substrate can be combined with the new substrate and cut it to produce the cyclic signal amplification. Finally, the output fluorescence signal is the activity of the Dam methyltransferase. We verified the feasibility of the strategy by fluorodynamic method and proved that the methylation probe was successfully cut through the gel electrophoresis experiment. Finally, the signal obtained by the methyltransferase inhibitor was proved to be the activity of the methyltransferase. Therefore, we detect Dam based on 8-17 DNAzyme. The methylation enzyme method has a simple, sensitive, and efficient feature. The main contents of the second part include: cancer is considered to be one of the major causes of disease and death worldwide. Malignant tumors can grow unrestricted, and then interfere with the digestive system, the nervous system and the loop system. And, worse, they can release hormones. It is difficult to detect the function of the body. Cancer is difficult to detect because it grows in the organ itself, the immune system of the human body can not identify and remove it. Most cancer patients have no obvious clinical symptoms before the onset of the disease. However, once the malignant tumor is formed, the condition of the patient will be drastically evil. Therefore, early cancer diagnosis of cancer treatment The traditional cancer detection methods need to be diagnosed from the morphological changes of cancer and can not meet the requirements of the early detection of cancer. It is very important for the early diagnosis and timely treatment of cancer to develop the methods to distinguish the cancer sites from the normal parts at the molecular level. We have selected the AS1411. With a special two stage structure of nucleic acid aptamer sequence, combined with light activated fluorescence imaging, a detection method for the high specificity and sensitivity of the target, the target of the aptamer, is established and applied to the detection of breast cancer. In this work, we first synthesized a small fluorescent molecule with light activation response. By labeling its success to the AS1411 nucleic acid aptamer, the aptamer fluorescent probe with light activation response was prepared. Then, we demonstrated the properties of the photoactivated fluorescence response by fluorescence detection, gel electrophoresis and DNA mass spectrometry. Finally, we proved the strategy to breast cancer by cell test and tissue section experiment. The cells have high sensitivity and selectivity, and can achieve the same accuracy as some of the clinical gold standard diagnostic methods. By comparing the fluorescence images before and after light activation, more accurate and persistent signals can be obtained.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：O657.3;R737.9

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