DNA纳米结构的功能化及其生物学应用

发布时间：2018-01-08 06:31

本文关键词：DNA纳米结构的功能化及其生物学应用　出处：《中国科学院研究生院(上海应用物理研究所)》2017年博士论文　论文类型：学位论文

【摘要】：DNA纳米结构具有精确的碱基配对能力,良好的生物相容性和稳定性,较高的膜渗透性和可控靶向释放能力,在生物传感、生物检测、药物转运载体以及诱导高阶纳米材料自组装等方面得到广泛应用。本论文构建了两种DNA纳米结构,并研究了其生物学应用和多功能化。工作主要分为两部分:第一部分:DNA纳米结构在智能载药和药物转运载体中的应用,包括第二至四章。第二章中,我们构建了以6-helix DNA纳米结构为基础的智能药物转运系统,该结构具有两个重要元素:1.智能型“开关”,可被特定分子识别并打开;2.共价连接多个药物分子。研究发现6-helix结构可被癌细胞有效摄取,在细胞内被特殊的钥匙链打开,并暴露内部药物位点;药物分子美登素干扰微管形成,最终导致细胞凋亡。智能药物转运系统实现了药物分子在靶向位置的可控释放,并且显著提升药效、降低副作用,这对于疾病治疗具有重要意义。6-helix因具有中空管状结构且机械稳定性高,常被用作小分子跨膜通道、药物转运载体和分子胶囊等,其内径和结构疏松程度决定了待转运分子的尺寸。在第三章中我们采用理论模拟和X射线小角散射(SAXS)技术联合表征不同离子强度以及小分子荷载对6-helix结构的影响。结果表明随着离子强度提高,6-helix结构变得紧密,内径减小;对DNA骨架进行乙基化修饰,以屏蔽磷酸基团负电荷后,修饰区域的DNA之间碱基排斥力减小,从而使得内径减小;同时利用SAXS技术能够较真实地反映6-helix在生理状态下的尺寸信息。该研究将有助于实现对DNA纳米结构的形貌和机械性能等的调控,为DNA纳米结构的生物学应用提供了理论基础。转运基因进入细胞核的效率是决定基因治疗的重要因素,高效且高安全性的转运载体能显著提高基因治疗的效果。第四章中,我们通过“点击化学”构建了DNA四面体与核定位序列(NLS)的复合结构,得到了稳定的DNA-NLS复合物,解决了简单混合带来的体内容易解离的问题;提高了细胞核摄取DNA四面体的效率。为获得高纯度、高产率的DNA-NLS复合物,我们将经典的碱性NLS_(12)延伸为酸性NLS29,显著降低了NLS与DNA四面体的非特异性结合,提高了连接反应效率至72.3%,同时也保留了NLS靶向细胞核的能力。该研究为临床基因治疗奠定了基础。第二部分:DNA四面体介导的纳米颗粒自组装,为第五章。第五章中,我们用DNA四面体装载了纳米金和量子点纳米颗粒。随后,采用“砖块-水泥”的思想,我们以构建DNA四面体超结构的方式,组装制备了同质和异质纳米颗粒团簇。这种方法简便地实现了纳米颗粒可控价态的组装,且具有高普适性。而且,DNA四面体笼形结构对维持纳米颗粒的稳定性具有重要作用;更重要的是,超结构的构建影响了纳米颗粒的性质。这一研究为发展具有新性质的纳米结构提供了新途径。综上所述,我们以DNA纳米结构为基础,构建了智能载药和药物转运载体新平台,并以简便、通用的方式组装了纳米颗粒超结构,这对DNA纳米结构的生物学应用和功能化具有重要意义。
[Abstract]:DNA nanostructures with precise base pairing ability, good biocompatibility and stability, higher membrane permeability and controllable target release ability in biological sensing, biological detection, widely used by drug transporters and higher order induced self-assembly of nanometer materials and so on. This paper constructed two kinds of DNA nanostructures, and study its biological applications and multifunctional. The work is mainly divided into two parts: the first part: the application of DNA nanostructures in intelligent drug loading and drug delivery carrier, including the second chapter to the four chapter. The second chapter, we construct the intelligent drug delivery system based on 6-helix DNA nano structure as the foundation, the the structure has two important elements: 1. intelligent switch, which can be a specific molecular recognition and open; 2. covalently connecting multiple drug molecules. The results indicated that the 6-helix structure can be effective uptake of cancer cells is in the cell The special key chain opened and exposed internal drug sites; drug molecules maytansine interfere with microtubule formation, eventually lead to apoptosis. Intelligent drug delivery system can release drug molecules to the position of the target and a significant increase in control, efficacy, reduce side effects, which is important because.6-helix has a hollow tubular structure and mechanical stability for the treatment of the disease, is often used as a small transmembrane channel, drug delivery carrier and molecular capsule, the diameter and the loose structure determines the stay of the transporter molecule size. In the third chapter we use the theory and Simulation of small angle X ray scattering (SAXS) technology combined with characterization of different ionic strength and small molecule load the structure of 6-helix. The results showed that with the increase of ionic strength, 6-helix structure becomes close, diameter decreases; ethyl modification of DNA skeleton, to shield the negative phosphate groups After the charge, between the modified region DNA base repulsion is reduced, so that the inner diameter decreases; while using SAXS technology can reflect real 6-helix in the physiological state of the size information. This study will be helpful to realize the control of DNA nano structure morphology and mechanical properties, and provides the theoretical basis for the biological applications of DNA nano structure. The efficiency of transporter gene into the nucleus of the cell is an important determinant of gene therapy, high efficiency and high safety transporters can significantly improve the effect of the gene therapy. In the fourth chapter, we construct a DNA tetrahedron and a nuclear localization sequence by "click chemistry" (NLS) of the composite structure, the DNA-NLS complex stable, simple solution to the mixed problem brought in easy dissociation; improve the efficiency of nuclear uptake of DNA tetrahedron. To achieve high purity, high yield of DNA-NLS complex, I Who will be the classic alkaline NLS_ (12) extended to acidic NLS29, significantly reduced the nonspecific NLS and DNA tetrahedron combination, improve the connection reaction efficiency to 72.3%, while retaining the NLS nuclear targeting ability. This study lays a foundation for clinical gene therapy. The second part: the nanoparticle mediated DNA tetrahedron the self assemble into fifth chapters. The fifth chapter, we loaded the nano gold nanoparticles and quantum dots with DNA tetrahedra. Then, using the "bricks and cement", we set DNA tetrahedral super structure, homogeneous and heterogeneous nanoparticle clusters by the assembly system. This method easily realized the assembly of nanoparticles controllable valence, and has high universality. Moreover, plays an important role in the stability of DNA tetrahedral cage structure to maintain the nano particle; more importantly, the construction of super structure properties of nanometer particle effects . provides a new way for the study of nano structure with new properties for development. In summary, based on our DNA nano structure, constructed the intelligent drug loading and drug delivery carrier of the new platform, with simple, generic way of assembled nanoparticles super structure, which is of great significance to the application of biology and function of DNA nano structure.

【学位授予单位】：中国科学院研究生院(上海应用物理研究所)
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TB383.1;Q523

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