基于DNA自组装的microRNA及ATP的放大检测

发布时间：2018-04-13 11:03

本文选题：DNA自组装 + 电化学生物传感　；参考：《山东师范大学》2017年硕士论文

【摘要】：MicroRNA(miRNA)和ATP等重要生物分子是生物体和生命现象的结构基础和功能基础,它们的含量和活性直接关系到生物体的健康。其中,miRNA在调控基因表达中发挥着重要的生物学作用。建立这些生物分子快速、简便、准确、灵敏的分析方法,对攻克许多疑难病症以及促进信息科学的发展都有重要的意义,是当前生物分析化学研究的前沿和热点。在本论文中,利用DNA自组装及核酸外切酶的循环放大技术发展了miRNA及ATP的检测方法,对肿瘤的早期诊断具有重大意义,主要内容如下:1.运用DNA自组装原理,发展了一种用于let-7d RNA超敏检测的无标记电化学生物传感器,该传感器是基于切口核酸内切酶(NEase)辅助级联模板增强杂交过程(TEHP)和滚环扩增(RCA),诱导形成G-四链体-血红素(hemin)复合物。发夹探针(H1)通过Au-S键固定在金电极的表面,在辅助DNA的帮助下,引入目标RNA let-7d,形成了切口核酸内切酶(NEase)的剪切位点,剪开发夹探针并释放目标RNA。发夹探针(H_2)的引入引发了另外两个级联循环过程。被剪开的片段作为滚环扩增(RCA)反应的引物,产生了大量hemin的适体,适体在钾离子的帮助下,可以形成G-四链体-血红素(hemin)复合物,而hemin则是电化学检测的响应物。最新设计的超灵敏电化学检测方法使得let-7d RNA的检测限达0.42 fM,并能从let-7RNA家族中特异性的识别目标RNA。这种检测手段在相关基因疾病的早期诊断应用中有着巨大的潜力。2.设计了ATP适体-羧基荧光素(FAM)/氧化石墨烯纳米片(GO-nanosheets)纳米复合物,结合核酸外切酶的剪切循环放大作用,以调查其在活细胞中的分子探测的能力。结果证明细胞对适体-FAM/GO-nanosheets纳米复合物的摄取以及细胞内靶标ATP循环放大监测成功实现。GO-nanosheets在活细胞中的递送,保护和感测能力表明氧化石墨烯可以是许多生物学领域的有力候选物,例如DNA和蛋白质分析,基因和药物递送以及细胞内跟踪等。3.展示了一种DNA纳米自组装结构用以携带治疗型mi RNA进入并杀死肿瘤细胞,而且在DNA自组装的初期结构可以用于检测mi RNA。该体系主要由FAM修饰的三个茎环结构和一条直链DNA组成。当遇到靶标miRNA时,发生杂交链式反应,三个茎环结构交替打开形成星状DNA自组装结构,未反应的茎环结构被MnO_2荧光猝灭,通过检测星状DNA自组装结构的荧光信号可以用以检测miRNA。以星状DNA自组装结构为前体,加入直链DNA,在T4连接酶的作用下,直链DNA在星状DNA自组装结构上连接成环,随后在T7 RNA聚合酶的作用下,以直链DNA为模板发生滚环转录反应,最终形成基于星状DNA纳米结构的CXCR4茎环和三螺旋串联的DNA自组装纳米结构,经离心后,形成DNA纳米水凝胶,其携带CXCR4和三螺旋RNA作为治疗型miRNA进入并杀死三阴性乳腺癌细胞。
[Abstract]:MicroRNAs miRNAs, ATP and other important biological molecules are the structural and functional basis of biological and life phenomena. Their contents and activities are directly related to the health of organisms.Among them, miRNA plays an important biological role in regulating gene expression.The establishment of rapid, simple, accurate and sensitive analytical methods for these biomolecules is of great significance for solving many difficult problems and promoting the development of information science, and is the frontier and hot spot of bioanalytical chemistry research at present.In this paper, the detection methods of miRNA and ATP were developed by using DNA self-assembly and cyclic amplification of nucleic acid exonuclease, which is of great significance for the early diagnosis of tumor. The main contents are as follows: 1.The biosensor is based on the cleavage endonuclease (NEase) -assisted cascade template enhancement hybridization (TEHP) and ring-amplification (RCAA) to induce the formation of a G-quadruplex hemin complex.The hairpin probe H1 was immobilized on the surface of the gold electrode by Au-S bond. With the help of DNA, the target RNA let-7d was introduced to form the cleavage site of the incision endonuclease. The hairpin probe was cut open and the target DNA was released.The introduction of hairpin probe H _ 2) triggered two other cascading cycles.The clipped fragments were used as primers for the rca reaction, resulting in a large number of aptamers of hemin. With the help of potassium ions, the aptamer could form the G-quadruplex heme hemin complex, while hemin was the responder of electrochemical detection.The newly designed hypersensitive electrochemical detection method enables the detection limit of let-7d RNA to reach 0.42 fM, and can specifically identify the target from the let-7RNA family.This method has great potential in the early diagnosis of related genetic diseases.The ATP aptamer, carboxyl fluorescein (Fam) / graphene oxide nanocrystalline (GO-nanosheets) nanocomposites were designed and amplified by the shear cycle of nucleic acid exonuclease in order to investigate their ability to detect molecules in living cells.The results showed that cell uptake of aptamer-FAM / GO-nanosheets nanosheets and intracellular target ATP cycle amplification were successful in delivering, protecting and sensing graphene oxide in living cells, suggesting that graphene oxide could be a potent candidate in many biological fields.Examples include DNA and protein analysis, gene and drug delivery, and intracellular tracking.A DNA nano-self-assembly structure was demonstrated to carry therapeutic mi RNA into and kill tumor cells, and it could be used to detect mi rna in the early stages of DNA self-assembly.The system consists of three stem rings modified by FAM and a straight chain DNA.When the target miRNA is encountered, the hybrid chain reaction occurs, and the three stem ring structures open alternately to form the stellate DNA self-assembly structure. The unreacted stem ring structure is quenched by MnO_2 fluorescence. The fluorescence signal of the stellate DNA self-assembly structure can be used to detect the miRNAA by detecting the fluorescence signal of the stellate DNA self-assembly structure.The stellate DNA self-assembly structure was used as the precursor, and the linear DNA was added into the straight strand DNA. Under the action of T4 ligase, the linear DNA was connected to the stellate DNA self-assembly structure to form a ring. Then, under the action of the T7 RNA polymerase, the ring-ring transcription reaction occurred using the straight-stranded DNA as the template.Finally, CXCR4 stem rings based on stellate DNA nanostructures and DNA self-assembled nanostructures in tandem with trihelix were formed. After centrifugation, DNA nanogels were formed, which carried CXCR4 and trihelix RNA as therapeutic miRNA to enter and kill triple-negative breast cancer cells.
【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TP212.3;R730.4

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