【作者】 张玲；

【导师】 肖守军；

【作者基本信息】 南京大学 ， 化学工程（专业学位）， 2020， 硕士

【摘要】 众所周知,DNA是生物系统中储存和传递遗传信息的重要分子,因其具有双螺旋结构且始终遵循碱基互补配对原则,且具备生物相容性、可编程性和可寻址性等特性,DNA被认为是纳米技术领域理想且功能丰富的纳米材料。自美国纽约大学Seeman教授于1982年第一次提出利用DNA模块构建有序的结构开始,DNA纳米技术的时代正式来临。随着DNA纳米技术的进一步发展,DNA折纸技术（DNA Origami）、DNA单链分子瓦（Single-Stranded Tiles,SSTs）等DNA纳米自组装技术愈发成熟,自组装得到的纳米结构的尺寸、功能以及基元结构的种类都在不断扩大和丰富。从纳米量级到微米量级、甚至到三维晶体的毫米量级,从一维纳米线、二维纳米平面到三维纳米结构,复杂的DNA纳米结构设计更加容易,应用范围亦更加广泛,药物运载、生物传感、生物计算机、逻辑计算等领域均为其用武之地。在DNA纳米自组装技术中,使用的DNA链多为线性DNA,而首尾相连的小环DNA因其具有特殊的生物学和拓扑学性质而备受关注。若利用小环DNA分子构建DNA纳米结构,这将进一步提升DNA纳米技术对自组装结构形状的控制能力并扩展DNA纳米结构的应用范围。本论文中,我们以84nt和64nt的小环DNA（circular DNA of 84 nucleotides,简称c84nt DNA;circular DNA of 64 nucleotides,简称c64nt DNA）为基元构建不同的分子瓦自组装成二维和三维纳米结构,并对其左右螺旋构象转变的影响因素进行探索。在线性DNA纳米结构中,霍利迪结（Holliday Junction,HJ）对增强纳米结构的刚性起到了至关重要的作用,于是我们将HJ应用到c84nt和c64nt小环DNA中,增强其结构的刚性,分别得到84HJ（84:c84nt,HJ:Holliday Junction）和64HJ分子瓦。在c84nt DNA中,我们将84HJ分子瓦的小环两侧分别留出10nt与10nt的辅助短链互补配对,得到84t HJ（t:triangle）分子瓦,该分子瓦通过构建三臂结（Three-way junction,简称TX）和双螺旋臂与其它相邻分子瓦连接。在c64nt DNA中,我们在64HJ分子瓦的两侧构建HJ和双螺旋臂与其它相邻分子瓦连接。首先,我们开展了小环DNA分子瓦构建纳米管的研究。基于84t HJ和84t HJ-3T分子瓦构建层交叉分子瓦（Layered-crossover Tiles,简称LX Tiles）,将其命名为84LX_n（84:c84nt,LX:Layered-crossover,n:LX所在的碱基位置）分子瓦,探索位点及分子瓦内在性质的调整对组装纳米管的影响。这里,84t HJ-3T分子瓦是指84t HJ分子瓦中小环DNA的四个角分别插入三个胸腺嘧啶（TTT）;本论文中用于构建纳米管的LX,主要分为四大类别,分别被称为84LX_n-S（84:c84nt,LX:Layered-crossover,n:位点,S:Sticky ends）,84LX_n-3T-S（84:c84nt,LX:Layered-crossover,n:位点,3T:插入的TTT碱基,S:Sticky ends）,84LXD_n-A（84:c84nt,LX:Layered-crossover,D:Diagonal,n:位点,A:Adjacent）和84LXD_n-I（84:c84nt,LX:Layered-crossover,D:Diagonal,n:位点,I:Interval）分子瓦。接着,我们探索利用c84nt和c64nt DNA组装的分子瓦构建二维和三维纳米结构,本文中涉及单层、双层以及三层纳米结构的自组装。在单层纳米结构自组装的研究中,我们设计将三个84t HJ分子瓦通过21nt序列组装成三角星分子瓦,称为three 84t HJ分子瓦,探索利用平末端堆积和粘性末端的互补配对设计不同的连接方式使其组装成以六边形为单元格的平面结构;在双层纳米结构自组装的研究中,我们以84LX_n分子瓦为基元探索3至16位点双层分子瓦的稳定性及其平末端堆积作用,通过原子力显微镜表征组装成的纳米结构;在三层纳米结构自组装的研究中,我们使用两个64LX₈分子瓦通过21nt序列连接在一起构建two 64LX₈分子瓦,该分子瓦用于组装有限宽度的纳米结构,通过构建有限宽度的纳米结构我们对64LX₈分子瓦的认识加深。最后,基于逻辑推理,c64nt DNA构建的三维纳米结构中存在右手螺旋DNA转变为左手螺旋DNA的可能性,因此我们以64LX₈分子瓦为基元设计了一系列I型分子瓦,探索结构中插入特殊序列、增加阳离子浓度、以及改变样品溶液所处环境温度等因素对右手螺旋DNA转变为左手螺旋DNA的影响。总之,这些实验结果帮助我们对小环DNA构建的分子瓦有了进一步了解,为构建更大更复杂的纳米结构提供有力的支撑,为加快小环DNA纳米结构投入到实际应用中奠定基础。更多还原

【Abstract】 DNA is well known as the important molecule for heredity information storage and transmission in biological systems.Because of its double helix geometry and the principle of complementary base pairing,DNA molecule is equipped with the features of biological compatibility,programmability and addressability.Hence DNA is regarded as an ideal and functional nanomaterial in the field of DNA nanotechnology.In 1982,Professor Nadrian C.Seeman,at New York university,first proposed using DNA modules to build ordered nanostructures,which means the start of DNA nanotechnology.Since then,DNA Origami,single-stranded tiles（SSTs）and other nanotechnologies have been developed.The size of nanostructures built from many kinds of tiles is continuously expanding,from nanometer scale to microns,and even to millimeter scale.DNA nanostructures include 1D nanowires and nanotubes,2D nanoribbons,and 3D nanoarrays and single crystals.The design and self-assembly of complex DNA nanostructures become easier.Nowadays DNA nanostructures are widely applied in drug delivery,biosensing,biological imaging,logic computing,and so on.In DNA nanotechnology,the linear DNAs are mostly used;however,the small circular DNAs,which have special biological and topological properties,have yet been investigated until we started using them.We assume that the application of small circular DNAs in DNA nanotechnology will obviously improve the rigidity of DNA tiles,enhance the control-ability of the geometric complexity of nanostructures,and further expand their applications.Herein,we explored the construction of 2D and 3D nanostructures based on 84 nucleotides and 64nucleotides circular DNA（c84nt DNA,c64nt DNA,c:circular）and the influencing factors for transforming the double-helix from right-handed to left-handed.Traditionally,Holliday junctions（HJs）play an important role in enhancing the rigidity of nanostructures,so we constructed HJs in c84nt and c64nt DNAs to form tiles,called 84HJ（84:c84nt,HJ:Holliday Junction）and 64HJ,respectively.In 84HJ tiles,we set aside 2×10nt from c84nt at both end sides,which will pair with helper strand,and form 84t HJ（t:triangle）tiles.84t HJ tiles can be connected together to form 2D arrays via constructing three-way junctions（TX）and double helix arms.In 64t HJ,polar HJs and extruding double helix arms can be formed at both sides for joining together to nanostructures.First,we constructed nanotubes via small circular DNA tiles.Based on 84t HJ and 84t HJ-3T tiles,we constructed Layered-crossover（LX）tiles,named 84LX_n（84:c84nt,LX:Layered-crossover,n:site number）,which were used to explore the influence of the different sites and tiles’intrinsic properties in the assembly of nanotubes.Herein,the 84t HJ-3T tile means that each of a 84t HJ tile’s four corners is inserted with three bases of thymine（TTT）.In this work,we constructed four categories of layered-crossover tiles for forming nanotubes:84LX_n-S（84:c84nt DNA,LX:Layered-crossover,n:site number,S:Sticky ends）,84LX_n-3T-S（84:c84nt DNA,LX:Layered-crossover,n:site number,3T:three T bases,S:Sticky ends）,84LXD_n-A（84:c84nt DNA,LX:Layered-crossover,D:Diagonal,n:site number,A:Adjacent）and84LXD_n-I（84:c84nt DNA,LX:Layered-crossover,D:Diagonal,n:site number,I:Interval）.Next,we explored the construction of 2D and 3D nanostructures of one-layer,two-layers and three-layers based on c84nt DNA and c64nt DNA.For single-layer structures,we designed and assembled three kinds of nanostructures from 84t HJ tiles through 21bp connections,in order to form planar structures with hexagon lattices.For two-layer nanostructures,we explored the stability of bilayer tiles from 3 to 16 sites and the assembly size by the base stacking force based on 84LX_ntiles.For three-layer nanostructures,we connected two 64LX₈ tiles through 21nt intertile arms.We also tried the assembly of finite nanostructures of 2-tiles wide using two64LX₈ tiles.Finally,we proposed that the right-handed helix in 3D DNA nanostructures could transfer into the left-handed helix conformation.Therefore,we designed a series of tiles based on 64LX₈ to explore the influencing factors that make double helix conformation conversion from right-handed to left-handed by CD spectrometer.We explored the following parameters,inserting special sequences into different locations,increasing the cationic concentrations,and changing the environment temperatures.Above all,these experimental results help us learn more about the physical-chemical properties of tiles constructed by small circular DNAs,provide another strategy for construction of larger and more complex nanostructures,and lay a foundation for applying DNA nanostructures into reality.更多还原