本文關(guān)鍵詞：DNA納米結(jié)構(gòu)的功能化及其生物學(xué)應(yīng)用　出處：《中國科學(xué)院研究生院(上海應(yīng)用物理研究所)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： DNA納米結(jié)構(gòu) 六螺旋 DNA四面體 藥物轉(zhuǎn)運載體 超結(jié)構(gòu)

【摘要】：DNA納米結(jié)構(gòu)具有精確的堿基配對能力,良好的生物相容性和穩(wěn)定性,較高的膜滲透性和可控靶向釋放能力,在生物傳感、生物檢測、藥物轉(zhuǎn)運載體以及誘導(dǎo)高階納米材料自組裝等方面得到廣泛應(yīng)用。本論文構(gòu)建了兩種DNA納米結(jié)構(gòu),并研究了其生物學(xué)應(yīng)用和多功能化。工作主要分為兩部分:第一部分:DNA納米結(jié)構(gòu)在智能載藥和藥物轉(zhuǎn)運載體中的應(yīng)用,包括第二至四章。第二章中,我們構(gòu)建了以6-helix DNA納米結(jié)構(gòu)為基礎(chǔ)的智能藥物轉(zhuǎn)運系統(tǒng),該結(jié)構(gòu)具有兩個重要元素:1.智能型“開關(guān)”,可被特定分子識別并打開;2.共價連接多個藥物分子。研究發(fā)現(xiàn)6-helix結(jié)構(gòu)可被癌細(xì)胞有效攝取,在細(xì)胞內(nèi)被特殊的鑰匙鏈打開,并暴露內(nèi)部藥物位點;藥物分子美登素干擾微管形成,最終導(dǎo)致細(xì)胞凋亡。智能藥物轉(zhuǎn)運系統(tǒng)實現(xiàn)了藥物分子在靶向位置的可控釋放,并且顯著提升藥效、降低副作用,這對于疾病治療具有重要意義。6-helix因具有中空管狀結(jié)構(gòu)且機(jī)械穩(wěn)定性高,常被用作小分子跨膜通道、藥物轉(zhuǎn)運載體和分子膠囊等,其內(nèi)徑和結(jié)構(gòu)疏松程度決定了待轉(zhuǎn)運分子的尺寸。在第三章中我們采用理論模擬和X射線小角散射(SAXS)技術(shù)聯(lián)合表征不同離子強(qiáng)度以及小分子荷載對6-helix結(jié)構(gòu)的影響。結(jié)果表明隨著離子強(qiáng)度提高,6-helix結(jié)構(gòu)變得緊密,內(nèi)徑減小;對DNA骨架進(jìn)行乙基化修飾,以屏蔽磷酸基團(tuán)負(fù)電荷后,修飾區(qū)域的DNA之間堿基排斥力減小,從而使得內(nèi)徑減小;同時利用SAXS技術(shù)能夠較真實地反映6-helix在生理狀態(tài)下的尺寸信息。該研究將有助于實現(xiàn)對DNA納米結(jié)構(gòu)的形貌和機(jī)械性能等的調(diào)控,為DNA納米結(jié)構(gòu)的生物學(xué)應(yīng)用提供了理論基礎(chǔ)。轉(zhuǎn)運基因進(jìn)入細(xì)胞核的效率是決定基因治療的重要因素,高效且高安全性的轉(zhuǎn)運載體能顯著提高基因治療的效果。第四章中,我們通過“點擊化學(xué)”構(gòu)建了DNA四面體與核定位序列(NLS)的復(fù)合結(jié)構(gòu),得到了穩(wěn)定的DNA-NLS復(fù)合物,解決了簡單混合帶來的體內(nèi)容易解離的問題;提高了細(xì)胞核攝取DNA四面體的效率。為獲得高純度、高產(chǎn)率的DNA-NLS復(fù)合物,我們將經(jīng)典的堿性NLS_(12)延伸為酸性NLS29,顯著降低了NLS與DNA四面體的非特異性結(jié)合,提高了連接反應(yīng)效率至72.3%,同時也保留了NLS靶向細(xì)胞核的能力。該研究為臨床基因治療奠定了基礎(chǔ)。第二部分:DNA四面體介導(dǎo)的納米顆粒自組裝,為第五章。第五章中,我們用DNA四面體裝載了納米金和量子點納米顆粒。隨后,采用“磚塊-水泥”的思想,我們以構(gòu)建DNA四面體超結(jié)構(gòu)的方式,組裝制備了同質(zhì)和異質(zhì)納米顆粒團(tuán)簇。這種方法簡便地實現(xiàn)了納米顆�？煽貎r態(tài)的組裝,且具有高普適性。而且,DNA四面體籠形結(jié)構(gòu)對維持納米顆粒的穩(wěn)定性具有重要作用;更重要的是,超結(jié)構(gòu)的構(gòu)建影響了納米顆粒的性質(zhì)。這一研究為發(fā)展具有新性質(zhì)的納米結(jié)構(gòu)提供了新途徑。綜上所述,我們以DNA納米結(jié)構(gòu)為基礎(chǔ),構(gòu)建了智能載藥和藥物轉(zhuǎn)運載體新平臺,并以簡便、通用的方式組裝了納米顆粒超結(jié)構(gòu),這對DNA納米結(jié)構(gòu)的生物學(xué)應(yīng)用和功能化具有重要意義。
[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.

【學(xué)位授予單位】：中國科學(xué)院研究生院(上海應(yīng)用物理研究所)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：TB383.1;Q523

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