长链端粒DNA的结构研究
发布时间：2019-06-17 20:34
【摘要】：端粒是真核生命线性染色体保护性末端,它的长度决定着细胞的寿限。随着有丝分裂端粒长度不断缩短,短至Hayflick极限后细胞周期停止,并开始迈向衰老。若能激活端粒酶维持端粒的长度,细胞则可无限扩增不老也不死,而这也是绝大部分恶性肿瘤的扩增机制。端粒末端有一段单链DNA突出部分可以折叠成G4联体结构,这一结构能够抑制端粒酶的活性,因而成为了潜在的抗癌靶点。许多抗癌药物的设计思路就是出于利用小分子配体稳定G4联体构象从而达到限制肿瘤细胞扩增的目的。人们对端粒DNA高级结构的认识从单个G4联体模型开始,随着研究的深入,重心逐渐向长链端粒DNA序列转移,而这也更接近生物体内的真实情况。长链端粒序列中能够形成多种复杂结构,并且不同构象的G4联体之间会有不同的相互作用。由于长链端粒DNA序列难以合成并且构象复杂,一度是困扰这里领域研究的最大难题。本文将采用滚环复制(RCA,Rolling Circle Replication)方法合成长链端粒DNA序列并对其结构与性质展开研究:在第一章中,简要介绍了端粒与端粒酶的起源与发现和生物进化上的意义与生物学功能,并从微观到宏观上对癌症的产生、生物体的衰老机制进行了阐述。另外系统论述了G4联体结构的分子生物学认识和发展现状。最后,在本章末尾介绍了基于原子力显微镜(AFM)的扫描成像与单分子力谱技术的原理、工作模式、仪器构造与适用范围及其在现代生物学领域的应用。第二章中主要介绍了长链端粒DNA的合成方案,并通过AFM扫描成像、紫外熔融、圆二色谱(CD)等多种手法对产物进行表征。结果表明我们合成的长链DNA序列在一定条件下能够形成高级结构,序列折叠成G4联体并呈串珠状,然而构象与短链G4模型有些不同,我们推测可能是由于长链中G4联体结构单元末端不再自由,导致优势构象改变。通过引物修饰的方法,我们成功地使RCA反应在基片上进行,得到共价链接在基底的长链端粒DNA产物方便后续研究。另外我们还建立了一套合成双链DNA重复序列的方法,突破了以往文献中条带弥散、产率低、退火后互补混乱的局限,为这一领域更深入的研究提供了参考和选择。第三章中我们基于已有实验事实提出了G4联体不完整折叠的模型,这一模型解释了随DNA链段增长Tm值降低的熔融行为,并解决了长链序列变温紫外实验中热稳定性减弱与G4联体相互作用(Quadruplex-Quadruplex Interaction,QQI)增强端粒DNA结构稳定性之间的矛盾。现有的紫外熔融实验与CD测试结果都支持这一猜想。另外,根据不同突变序列的长链端粒DNA的CD表征结果,我们推测不完整折叠结构中最外侧4个鸟嘌呤在长链G4联体结构中折叠相对困难。这可能是由于长链端粒DNA重复序列中临近链段的束缚与分子热运动对G4联体结构产生了破坏。与之前发现的G3联体不同的是我们的模型中G4联体两端的破坏是对称的。这一模型的提出深化了对于长链端粒DNA序列结构的认识,并为一些基于G4联体结构来设计小分子抗癌药物的研究提供了新的思路。第四章中我们通过基于原子力显微镜的单分子力谱技术考察了长链端粒DNA的力学稳定性,结果表明基于QQI形成的更高级结构在拉伸曲线中表现为55p N的平台。往复拉伸曲线之间有明显的滞后,而滞后区域所包含的面积△E与拉伸过程中长度变化△L呈线性相关,这说明不同拉伸程度下破坏的是相同的结构。从该线性关系的斜率可以衡量端粒DNA高级结构的稳定性,我们发现在40%PEG中拟合线的斜率比水中大,表明40%PEG中G4联体结构更加稳定,这与之前文献中的观点一致。该单分子方法还可以用于非常直观地考察各种其它因素,比如p H、离子环境、配体等对G4联体稳定性的影响规律。系统的对比实验结果还显示只有端粒DNA的拉伸与松弛曲线之间能够产生滞后,若当序列中插入无规部分,将端粒DNA序列中相邻G4联体结构单元彼此隔开(间隔基序列),则不出现明显的滞后。这说明单个G4联体结构的形成对其邻近片段的折叠有促进作用,加速其余部位G4联体结构的形成;间隔基的引入将大大削弱该协同效应。该研究加深了我们对端粒DNA高级结构形成机制的认识。
[Abstract]:The telomere is the protective end of the linear chromosome of the eukaryote, and its length determines the cell's life limit. With the continuous shortening of the mitotic telomere length, the cell cycle stops after the short to Hayflick limit and begins to move towards aging. If the telomerase is activated to maintain the length of the telomere, the cells can be amplified indefinitely or not, and this is the amplification mechanism of the vast majority of the malignant tumors. The end of the telomere has a single-stranded DNA protruding part which can be folded into a G4 concatemer structure, and the structure can inhibit the activity of the telomerase, thereby being a potential anti-cancer target. Many anti-cancer drugs are designed for the purpose of limiting the amplification of tumor cells by using small molecular ligands to stabilize the G4 concatemer conformation. The recognition of the high-level structure of the telomere DNA begins with a single G4-linked model, and with the depth of the study, the center of gravity is gradually transferred to the long-chain telomere DNA sequence, which is closer to the real situation in the organism. A variety of complex structures can be formed in the long-chain telomere sequences, and there will be different interactions between the G4 concatemers of different conformations. As long-chain telomere DNA sequences are difficult to synthesize and are complex in conformation, one time is the biggest challenge to the research in the field. In this paper, the long-chain telomere DNA sequence is synthesized by rolling-ring replication (RCA) and its structure and properties are studied. In the first chapter, the origin and discovery of the telomere and the telomerase and the significance and biological function of the biological evolution are briefly introduced. The mechanism of the generation of cancer and the aging mechanism of the organism are discussed from the microscopic to the macroscopic. In addition, the molecular biology and development of the structure of the G4 conjuncted structure are discussed in this paper. At the end of this chapter, the principle, working mode, instrument structure and application range of atomic force microscope (AFM) and its application in the field of modern biology are introduced at the end of this chapter. In the second chapter, the synthesis of long-chain telomere DNA was introduced, and the products were characterized by AFM, UV-melting and CD. The results show that the synthesized long-chain DNA sequence can form a high-grade structure under certain conditions, and the sequence is folded into the G4 concatemer and is in the form of a bead. However, the conformation is somewhat different from that of the short-chain G4 model. Resulting in an advantageous conformational change. With the method of primer modification, the RCA reaction was successfully carried out on the substrate to obtain a long-chain telomere DNA product covalently linked to the substrate for subsequent study. In addition, we have set up a set of synthetic double-stranded DNA repeated sequence method, which breaks through the limitation of strip dispersion, low yield and complementary disorder after annealing in the past, and provides the reference and choice for the more in-depth research in this field. In the third chapter, we put forward the model of incomplete folding of the G4 conjunct based on the existing experimental facts, which explains the melting behavior with the decrease of the Tm value with the growth of the DNA segment, and solves the problem that the thermal stability of the long-chain-sequence variable-temperature ultraviolet experiment is weakened and the G4-linked interaction (Quadruplex-Quadruplex Direction) is reduced. QQI) enhances the contradiction between the stability of telomere DNA structures. The results of the existing UV-melting and CD-test support this conjecture. In addition, according to the CD characterization of the long-chain telomere DNA of different mutation sequences, we are speculating that the most out of four of the most out of the incomplete folded structure is relatively difficult to fold in the long-chain G4 conjoined structure. This may be due to the disruption of the binding and molecular thermal movement of the adjacent segment in the long-chain telomere DNA repeat sequence to the G4 conjoined structure. Unlike the previously discovered G3 conjuncts, the destruction of both ends of the G4 conjunct in our model is symmetrical. The development of this model deepens the understanding of the structure of long-chain telomere DNA, and provides a new way for the design of small-molecule anti-cancer drugs based on the G4-linked structure. In the fourth chapter, we examined the mechanical stability of long-chain telomere DNA by a single-molecular force spectrum technique based on the atomic force microscope. The results show that the higher-level structure formed on the basis of the QQI is the platform of 55p N in the tensile curve. There is a significant lag between the reciprocating stretching curves, while the area of the hysteresis included in the hysteresis region is linearly related to the length change length L during the stretching process, which indicates that the same structure is destroyed under different stretching levels. The slope of the linear relationship can be used to measure the stability of the high-level structure of the telomere DNA, and we find that the slope of the fitting line in 40% PEG is larger than that in water, indicating that the structure of the G4 concatemer in 40% PEG is more stable, which is consistent with the point of view in the previous literature. The single-molecule method can also be used to examine the influence of various other factors, such as p H, ionic environment, ligand, and the like on the stability of the G4 conjunct. The results of the comparison of the system also show that there is a lag between the stretching of the telomere DNA and the relaxation curve, and if the random part is inserted in the sequence, no significant hysteresis occurs when the adjacent G4-linked structural units in the telomere DNA sequence are separated from each other (spacer-based sequence). This indicates that the formation of a single G4 concatemer structure can promote the folding of its adjacent segment, and accelerate the formation of the combined structure of the remaining part G4; the introduction of the spacer will greatly weaken the synergistic effect. The study deepens our understanding of the formation mechanism of the high-level structure of the telomere DNA.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q523
本文编号：2501248
[Abstract]:The telomere is the protective end of the linear chromosome of the eukaryote, and its length determines the cell's life limit. With the continuous shortening of the mitotic telomere length, the cell cycle stops after the short to Hayflick limit and begins to move towards aging. If the telomerase is activated to maintain the length of the telomere, the cells can be amplified indefinitely or not, and this is the amplification mechanism of the vast majority of the malignant tumors. The end of the telomere has a single-stranded DNA protruding part which can be folded into a G4 concatemer structure, and the structure can inhibit the activity of the telomerase, thereby being a potential anti-cancer target. Many anti-cancer drugs are designed for the purpose of limiting the amplification of tumor cells by using small molecular ligands to stabilize the G4 concatemer conformation. The recognition of the high-level structure of the telomere DNA begins with a single G4-linked model, and with the depth of the study, the center of gravity is gradually transferred to the long-chain telomere DNA sequence, which is closer to the real situation in the organism. A variety of complex structures can be formed in the long-chain telomere sequences, and there will be different interactions between the G4 concatemers of different conformations. As long-chain telomere DNA sequences are difficult to synthesize and are complex in conformation, one time is the biggest challenge to the research in the field. In this paper, the long-chain telomere DNA sequence is synthesized by rolling-ring replication (RCA) and its structure and properties are studied. In the first chapter, the origin and discovery of the telomere and the telomerase and the significance and biological function of the biological evolution are briefly introduced. The mechanism of the generation of cancer and the aging mechanism of the organism are discussed from the microscopic to the macroscopic. In addition, the molecular biology and development of the structure of the G4 conjuncted structure are discussed in this paper. At the end of this chapter, the principle, working mode, instrument structure and application range of atomic force microscope (AFM) and its application in the field of modern biology are introduced at the end of this chapter. In the second chapter, the synthesis of long-chain telomere DNA was introduced, and the products were characterized by AFM, UV-melting and CD. The results show that the synthesized long-chain DNA sequence can form a high-grade structure under certain conditions, and the sequence is folded into the G4 concatemer and is in the form of a bead. However, the conformation is somewhat different from that of the short-chain G4 model. Resulting in an advantageous conformational change. With the method of primer modification, the RCA reaction was successfully carried out on the substrate to obtain a long-chain telomere DNA product covalently linked to the substrate for subsequent study. In addition, we have set up a set of synthetic double-stranded DNA repeated sequence method, which breaks through the limitation of strip dispersion, low yield and complementary disorder after annealing in the past, and provides the reference and choice for the more in-depth research in this field. In the third chapter, we put forward the model of incomplete folding of the G4 conjunct based on the existing experimental facts, which explains the melting behavior with the decrease of the Tm value with the growth of the DNA segment, and solves the problem that the thermal stability of the long-chain-sequence variable-temperature ultraviolet experiment is weakened and the G4-linked interaction (Quadruplex-Quadruplex Direction) is reduced. QQI) enhances the contradiction between the stability of telomere DNA structures. The results of the existing UV-melting and CD-test support this conjecture. In addition, according to the CD characterization of the long-chain telomere DNA of different mutation sequences, we are speculating that the most out of four of the most out of the incomplete folded structure is relatively difficult to fold in the long-chain G4 conjoined structure. This may be due to the disruption of the binding and molecular thermal movement of the adjacent segment in the long-chain telomere DNA repeat sequence to the G4 conjoined structure. Unlike the previously discovered G3 conjuncts, the destruction of both ends of the G4 conjunct in our model is symmetrical. The development of this model deepens the understanding of the structure of long-chain telomere DNA, and provides a new way for the design of small-molecule anti-cancer drugs based on the G4-linked structure. In the fourth chapter, we examined the mechanical stability of long-chain telomere DNA by a single-molecular force spectrum technique based on the atomic force microscope. The results show that the higher-level structure formed on the basis of the QQI is the platform of 55p N in the tensile curve. There is a significant lag between the reciprocating stretching curves, while the area of the hysteresis included in the hysteresis region is linearly related to the length change length L during the stretching process, which indicates that the same structure is destroyed under different stretching levels. The slope of the linear relationship can be used to measure the stability of the high-level structure of the telomere DNA, and we find that the slope of the fitting line in 40% PEG is larger than that in water, indicating that the structure of the G4 concatemer in 40% PEG is more stable, which is consistent with the point of view in the previous literature. The single-molecule method can also be used to examine the influence of various other factors, such as p H, ionic environment, ligand, and the like on the stability of the G4 conjunct. The results of the comparison of the system also show that there is a lag between the stretching of the telomere DNA and the relaxation curve, and if the random part is inserted in the sequence, no significant hysteresis occurs when the adjacent G4-linked structural units in the telomere DNA sequence are separated from each other (spacer-based sequence). This indicates that the formation of a single G4 concatemer structure can promote the folding of its adjacent segment, and accelerate the formation of the combined structure of the remaining part G4; the introduction of the spacer will greatly weaken the synergistic effect. The study deepens our understanding of the formation mechanism of the high-level structure of the telomere DNA.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q523
【引证文献】
相关博士学位论文 前1条
1 吕秀娟;锯齿型构象高分子单晶纳米力学性质单分子力谱研究[D];吉林大学;2018年
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