基于小环DNA的核酸自组装技术

发布时间：2018-09-18 08:29

【摘要】：DNA分子是自然界普遍存在的一种分子,是储存遗传信息的重要载体,此外基于各种配对方式形成双链结构,DNA已经被证明可以作为一种强大的基础材料构建各种纳米结构。1982年,美国科学家Seeman教授受生物重组过程中霍利迪结(Holiday junction)的启发,第一个仅仅利用可编码的碱基互补配对来构建二维和三维的DNA纳米结构,标志DNA纳米技术的开始。此后,由于DNA有合适的大小、明确的配对结构加之合成方便,无数的几何图案的DNA纳米结构和功能迥异的各种DNA纳米器件被设计和开发利用,目前该领域的研究人员已经构建了静态结构如二维和三维的纳米晶格、纳米管、多面体和任意形状的功能器件如分子机器和DNA计算机,并正在纳米医学、分子电子学等领域得到应用。在自然界中,环状DNA的扭曲、成环、弯曲、扭转等现象具有复杂的生物作用,如调控复合物、影响基因表达等。在DNA纳米技术研究中,刚性的DNA模块是构建纳米结构的基础,最近我们课题组使用酶法合成的小环DNA来代替直链,构建线性排列的DNA刚性模块自组装得到DNA纳米结构。本论文以小环DNA为基元构建不同方式和连接的DNA tile,并自组装得到了多种形态的DNA纳米结构,具体工作如下:1.以小环DNA为中心,辅以其他直链DNA构建DNAtile,然后使用两种常用的设计,分别是 SAE(semicrossover,antiparallel,and even half-turns;半交叉,反向平行,偶数倍半周期)和 DAE(doublecrossover,antiparallel,and even half-turns;双交叉,反向平行,偶数被半周期),以很短的距离(11/16 bp)来连接DNA tile后完成模块化自组装。此外,借助物理上的同相和非同相(In-phase/out of-phase)的概念,我们发现了两种同相生长的分子模块SAE-E(SAE型的DNA tile,tile间的连接距离为偶数个半周期)和DAE-O(DAE型的DNA tile,tile间的连接距离为奇数个半周期)。实验结果,我们用SAE-E的设计结构可以自组装产生均一大小的DNA纳米管,宽度约16-20nm,长度逾14μμm;DAE-E的设计结构可以产生较细长的多层结构,观察得到类似毛线团的图案(25-30nm宽)、类似围巾的纳米片(100-300 nm宽)以及纳米带(约100 nm宽)。此外我们还观察到了令人振奋的组装过程,一种从凌乱的毛线团逐渐形成纳米片的现象。最后,我们根据小环DNA的旋转方向推测纳米结构的弯曲现象,并解释了 DNA纳米管和波浪形纳米线的形成机制。2.利用DAE-O的设计、以小环DNA为核心构建二维纳米结构,其中DAE-O即Doublecrossover,Antiparallel,and Even half-turns tiles with Odd half-turns connection,双交叉、反向平行、DNA tile内距离为偶数个半周期、DNA tile间为奇数个半周期,我们设计了三种尺寸(42 nt,64 nt,84 nt)小环DNA分别构建DAE。实验结果表明,以42 nt或64 nt为中心的DNA链(直链DNA或小环DNA)构建DAE可以自组装得到规则的纳米结构;对于含有84 nt小环DNA的DAE也会自组装得到二维单晶结构,而含有84 nt直链DNA的DAE则只能自组装得到非典型态的网状结构即二维多晶结构。在DNA自组装过程中,柔韧性和刚性之间的平衡至关重要,以64 nt DNA相对来讲有最好的柔性和刚性,所以带有64 nt DNA的tile能够得到最好的结果;84 nt小环DNA有最低的刚性,至于84 nt或更长的线性DNA在构建分子瓦时则减少了刚性增加了柔性,不利于形成多晶纳米结构。不同DAE tile的在侧面的刚性差别是导致出现不同形态结构的主要原因。3.利用32 nt的小环DNA为中心设计了一个三臂模块、利用42 nt的小环DNA为中心设计了一个四臂模块,顶点处采用半交叉结构、连接处采用一个双螺旋和26 bp的连接距离,自组装后观察到了六边形和四边形状小孔的格子结构,但是没有得到大片规则结构。三臂结构自组装形成六边形的小孔结构,并有延展得到蜂窝状规则图案的趋势,目前我们获得的结构中,横向上有连续的3-5个规则小孔,而纵向上的连续排列可以达10个左右,经过部分结构的剖面图分析可得每个小孔内径为23.4 nm,接近六边形的理论估算;四臂结构自组装多为线条,这些结构大多为生长方向基本一致的线条,少部分会靠在一起形成双条或三条结构,线条长100-200 nm,线条宽20 nm,同时在局部可发现小的2×2或2×3的网格结构,平均内径13.9 nm,接近四边形的理论估计。电泳分析结果表明我们可以成功构建三臂和四臂结构,但是其他的杂交、错配的副产物可能会影响整体大结构的形成,且单个双螺旋的连接和小尺寸小环DNA会使整体结构缺乏刚性束缚,难以形成大片规律结构。
[Abstract]:DNA molecule is a ubiquitous molecule in nature and an important carrier for storing genetic information. In addition, DNA has been proved to be a powerful basic material for constructing nanostructures based on the formation of double-stranded structures by various mating methods. In 1982, American scientist Professor Seeman was subjected to biological recombination of Holiday Ju knot. Inspired by the idea that the first two-dimensional and three-dimensional DNA nanostructures were constructed using only coded base complementary pairings, marking the beginning of DNA nanotechnology. Researchers in this field have constructed static structures such as two-dimensional and three-dimensional nanolattices, nanotubes, polyhedrons and functional devices with arbitrary shapes, such as molecular machines and DNA computers, and are being used in nanomedicine, molecular electronics and other fields. In the research of DNA nanotechnology, rigid DNA modules are the basis of constructing nanostructures. Recently, our research group used enzymatic synthesis of small ring DNA instead of straight chain to construct linear array of DNA rigid modules to self-assemble DNA nanostructures. In this paper, we construct DNA tiles with different modes and connections based on small-loop DNA, and self-assemble DNA nanostructures with various morphologies. The main work is as follows: 1. We construct DNA tiles with small-loop DNA as the center and other linear DNA as the supplement. Then we use two commonly used designs, namely SAE (semi-crossover, anti-parallel, and even half-turns). In addition, with the help of physical in-phase and out-of-phase concepts, we have developed a modular self-assembly method for connecting DNA tiles at very short distances (11/16 bp). Two kinds of molecular modules, SAE-E (SAE-type DNA tiles, with even half-cycle connection distance between tiles) and DAE-O (DAE-type DNA tiles, with odd half-cycle connection distance between tiles), were synthesized. The experimental results show that we can self-assemble DNA nanotubes of uniform size with SAE-E design structure. The width is about 16-20 nm and the length is over 14 nm. The design structure of DAE-E can produce slim and long layers of structure. The patterns of wool-like clusters (25-30 nm wide), scarf-like nanosheets (100-300 nm wide) and nanoribbons (100-300 nm wide) are observed. In addition, the exciting assembly process, a gradual formation of nanosheets from disordered wool clusters, is also observed. Then, we speculate the bending of nanostructures according to the rotation direction of the small-ring DNA, and explain the formation mechanism of DNA nanotubes and wavy nanowires. 2. Using DAE-O design, we construct two-dimensional nanostructures with small-ring DNA as the core, in which DAE-O is Doublecrossover, Antiparallel, and Even half-turns tiles with Odd half-turns conne. We designed three kinds of small ring DNA (42 nt, 64 nt, 84 nt) to construct DAE. The experimental results showed that the DAE with 42 nt or 64 NT centered DNA strands (linear DNA or small ring DNA) could self-assemble into regular nanostructures. In the process of DNA self-assembly, the balance between flexibility and rigidity is very important, and 64 NT DNA has the best flexibility and rigidity. So tiles with 64 NT DNA have the best results; 84 NT DNA has the lowest stiffness, while 84 nt or longer linear DNA reduces stiffness and increases flexibility in building molecular tiles, which is not conducive to the formation of polycrystalline nanostructures. 3. A three-arm module is designed with 32 NT ring DNA as the center. A four-arm module is designed with 42 NT ring DNA as the center. A semi-crossed structure is used at the apex and a double helix and 26 BP connection distance are used at the junction. The lattice structure of hexagonal and quadrilateral small holes is observed after self-assembly, but not large. Three-arm structure self-assembles to form hexagonal pore structure, and has the tendency to extend to form honeycomb-like regular pattern. At present, we have obtained the structure, there are 3-5 regular pores in the horizontal direction, and the vertical continuous arrangement can be up to 10 or so, after section analysis of some structures, we can get the inner diameter of each pore as follows 23.4 nm, close to the theoretical estimate of the hexagon; most of the four-arm structure self-assembly lines, most of which are basically the same direction of growth lines, a few of which will form two or three structures together, lines 100-200 nm long, line width 20 nm, at the same time in the local can be found small 2 x 2 or 2 x 3 grid structure, the average diameter of 13.9 nm, close to The results of electrophoresis analysis show that we can successfully construct three-arm and four-arm structures, but other hybridization, mismatch by-products may affect the formation of the overall large structure, and a single double helix connection and small size ring DNA will make the overall structure lack of rigid binding, it is difficult to form large regular structure.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q52

【相似文献】