基于酶扩增的新型DNA水凝胶的构建及其应用

发布时间：2018-05-29 16:54

  本文选题：DNA水凝胶 + 末端脱氧核苷酸转移酶　； 参考：《湖南大学》2016年硕士论文

【摘要】：功能水凝胶作为一种新型生物材料,由于其物理机械性能与生物组织的类似性,已经成为生物医学领域一种重要的生物材料。近几年来,DNA水凝胶得到了快速发展,由于其优异的生物相容性、可降解性以及易于功能化等优点,DNA水凝胶在生物传感、药物的运载与释放、蛋白的体外表达以及单细胞操控等方面都有着广泛的应用。但是以往DNA水凝胶的合成方法中,往往需要大量的DNA作为原料,极大增大了合成成本,限制了DNA凝胶的发展。因此,本文中,我们基于末端脱氧核苷酸转移酶(TdT)的催化聚合性能,以二维或者三维DNA纳米材料作为基本结构单元,开发了两种简便、快捷且成本低的方法构建DNA水凝胶,并且将其应用于物质的包裹及可控释放,蛋白的富集,和多酶反应体系的构建,具体如下：1.基于TdT无模板扩增DNA的性能,以十字交叉型DNA结构(X-DNA)作为基本结构单元,构建了一种合成DNA水凝胶的新方法。该方法中,以X-DNA为引物,通过TdT扩增将三磷酸脱氧胸苷(dTTP)和三磷酸脱氧腺苷(dATP)添加至X-DNA的3’-羟基末端,合成四端带有富含胸腺嘧啶(T)的X-DNA-Tn和富含腺嘌呤(A)的X-DNA-An,然后利用A-T碱基互补配对,自组装形成DNA水凝胶。本方法中,TdT的引入极大的减少了水凝胶合成中初始DNA的用量,由原来的数十微摩级别降低到几个微摩,从而降低了成胶成本。另外,我们考察了这种凝胶形成的机理,发现带有富A序列或者带有富T序列X-DNA无法单独形成凝胶,且以线性DNA为引物合成的富A序列与富T序列的杂交以及错配碱基(如A/C)的扩增产物都无法合成水凝胶。这些结果表明,该凝胶的合成中以十字交叉型DNA作为中心点,而延伸臂作为交联剂,二者缺一不可。接着,利用流变仪、扫描电子显微镜、原子力显微镜分别表征其力学性能、表面形貌以及内部结构。最后,我们探究了该DNA水凝胶在大分子物质的封装以及可控释放方面的应用。2.利用上述通用型的合成方法构建了一种功能DNA水凝胶——DNA酶水凝胶(DNAzyme hydrogel).将一段DNAzyme(Dz)序列连接于上述X-DNA结构上,形成Dz-X.DNA。该结构在一些阳离子(K+)存在的情况下,能够形成鸟嘌呤-四链体,与氯化血红素(hemin)结合后具有模拟过氧化物酶活性,能够催化H2O2氧化ABTS2产生绿色产物ABTS-。首先,我们以Dz-X-DNA作为结构单元合成DNAzyme水凝胶,探索了其过氧化物酶活性,并且将其用于H2O2的可视化检测。基于这一现象,我们发展了两种生物酶串联体系：结合葡萄糖氧化酶(GOx)构建了双酶串联体系；将GOx以及半乳糖苷酶(β-Gal)固定于凝胶中即可构成三酶串联反应体系。在这些酶联体系中,我们合成的凝胶不仅充当生物酶的支架,更为重要的是,它还是串联反应中的一个不可或缺的催化反应单元,参与整个酶联反应的过程。这些有效的酶联反应为葡萄糖、乳糖提供了一种潜在的检测方法。3.以三维DNA纳米材料——DNA纳米管(DNA-nanotube)为基本结构单元,构建了一种结构更为稳定,机械性能更强的DNA水凝胶。该方法中,DNA-nanotube在TdT的催化下延四周扩增产生多条富T或者富A侧链,这些侧链的杂交即可形成DNA水凝胶。流变性能测试证实了其凝胶的形成,且发现以DNA-nanotube为单体合成的凝胶,其机械强度更大,需要的初始DNA量更少。此外,我们初步探索了该凝胶的应用范围,实验发现其可用于蛋白以及多种生物酶的富集,并且能够保持其生物活性,实现多酶串联反应。
[Abstract]:As a new biological material, functional hydrogels have become an important biological material in the field of biomedicine because of their physical and mechanical properties similar to biological tissues. In recent years, DNA hydrogels have developed rapidly. Due to their excellent biocompatibility, biodegradability and ease of functionalization, DNA hydrogels are in the field. Biosensors, drug delivery and release, protein expression in vitro and single cell manipulation are widely used. However, in the previous synthesis methods of DNA hydrogels, a large number of DNA were often needed as raw materials, which greatly increased the cost of synthesis and limited the development of DNA gel. Therefore, in this paper, we based on the terminal deoxy nucleoside. The catalytic polymerization of acid transferase (TdT), using two or three dimensional DNA nanomaterials as the basic structural units, developed two simple, fast and low cost methods to construct DNA hydrogels, and applied it to the encapsulation and controlled release of material, the accumulation of protein and the construction of the multi enzyme reaction system, as follows: 1. based on the TdT model The performance of DNA, using cross type DNA structure (X-DNA) as the basic structural unit, is a new method to synthesize DNA hydrogels. In this method, X-DNA is used as primers to add TdT three deoxylated thymidine (dTTP) and three phosphoric acid deoxy adenosine (dATP) to the 3 '- hydroxyl terminal of X-DNA, and the four ends are rich in thymus rich gland. The X-DNA-Tn of pyrimidine (T) and X-DNA-An rich in adenine (A) and then the complementary pairing of A-T bases and self-assembly to form DNA hydrogels. In this method, the introduction of TdT greatly reduced the amount of initial DNA in the hydrogel synthesis, reduced to a few micro frictional levels from the original tens of micro frictional levels, thus reducing the cost of the gelation. In addition, we examined it. The formation of this gel shows that a rich A sequence or a rich T sequence X-DNA can not form a gel alone, and the rich A sequence synthesized by the linear DNA primers and the rich T sequence and the mismatched base (such as A/C) can not synthesize the hydrogel. These results show that the synthesis of the gel is made of cross type DNA in the synthesis of the gel. As the center point and the extension of the arm as a crosslinker, the two are indispensable. Then, using rheometer, scanning electron microscope and atomic force microscope to characterize their mechanical properties, surface morphology and internal structure. Finally, we explored the application of the DNA hydrogel in the encapsulation and controlled release of macromolecules by.2.. A functional DNA hydrogel, DNA hydrogel (DNAzyme hydrogel), is constructed with a functional synthesis method. A DNAzyme (Dz) sequence is connected to the above X-DNA structure to form a Dz-X.DNA. that can form guanine four chain in the presence of some cations (K+), which is combined with hemin (hemin) to simulate peroxises. Enzyme activity of chemical compounds can catalyze H2O2 oxidation of ABTS2 to produce green product ABTS-. first. We synthesized DNAzyme hydrogel with Dz-X-DNA as a structural unit, and explored its peroxidase activity and used it for the visual detection of H2O2. Based on this phenomenon, we developed two kinds of biological enzyme series system: combined with glucose oxidase (GOx). A double enzyme series system was constructed, and GOx and galactosidase (beta -Gal) were immobilized in the gel to form a three enzyme series reaction system. In these enzymes, our synthesized gel not only acts as a scaffold for biological enzymes, but also, more importantly, is an indispensable catalytic unit in the series reaction and participates in the whole enzyme. These effective enzyme reactions are glucose, and lactose provides a potential detection method,.3., with a three-dimensional DNA nanomaterial, DNA nanotube (DNA-nanotube) as the basic structural unit. A more stable and more mechanical DNA hydrogel has been constructed. In this method, DNA-nanotube extends four under the catalysis of TdT. A number of rich T or A rich side chains were produced by week amplification. The hybridization of these side chains could form DNA hydrogels. The rheological properties test confirmed the formation of its gel and found that the gel with DNA-nanotube as a monomer was more mechanical and needed less initial DNA. In addition, we initially explored the application range of the gel. Experimental discovery It can be used for protein and enzyme enrichment, and can maintain its biological activity and achieve multiple enzyme tandem reactions.
【学位授予单位】：湖南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q523;O648.17
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本文编号：1951675