長鏈端粒DNA的結(jié)構(gòu)研究
發(fā)布時(shí)間：2019-06-17 20:34
【摘要】：端粒是真核生命線性染色體保護(hù)性末端,它的長度決定著細(xì)胞的壽限。隨著有絲分裂端粒長度不斷縮短,短至Hayflick極限后細(xì)胞周期停止,并開始邁向衰老。若能激活端粒酶維持端粒的長度,細(xì)胞則可無限擴(kuò)增不老也不死,而這也是絕大部分惡性腫瘤的擴(kuò)增機(jī)制。端粒末端有一段單鏈DNA突出部分可以折疊成G4聯(lián)體結(jié)構(gòu),這一結(jié)構(gòu)能夠抑制端粒酶的活性,因而成為了潛在的抗癌靶點(diǎn)。許多抗癌藥物的設(shè)計(jì)思路就是出于利用小分子配體穩(wěn)定G4聯(lián)體構(gòu)象從而達(dá)到限制腫瘤細(xì)胞擴(kuò)增的目的。人們對(duì)端粒DNA高級(jí)結(jié)構(gòu)的認(rèn)識(shí)從單個(gè)G4聯(lián)體模型開始,隨著研究的深入,重心逐漸向長鏈端粒DNA序列轉(zhuǎn)移,而這也更接近生物體內(nèi)的真實(shí)情況。長鏈端粒序列中能夠形成多種復(fù)雜結(jié)構(gòu),并且不同構(gòu)象的G4聯(lián)體之間會(huì)有不同的相互作用。由于長鏈端粒DNA序列難以合成并且構(gòu)象復(fù)雜,一度是困擾這里領(lǐng)域研究的最大難題。本文將采用滾環(huán)復(fù)制(RCA,Rolling Circle Replication)方法合成長鏈端粒DNA序列并對(duì)其結(jié)構(gòu)與性質(zhì)展開研究:在第一章中,簡要介紹了端粒與端粒酶的起源與發(fā)現(xiàn)和生物進(jìn)化上的意義與生物學(xué)功能,并從微觀到宏觀上對(duì)癌癥的產(chǎn)生、生物體的衰老機(jī)制進(jìn)行了闡述。另外系統(tǒng)論述了G4聯(lián)體結(jié)構(gòu)的分子生物學(xué)認(rèn)識(shí)和發(fā)展現(xiàn)狀。最后,在本章末尾介紹了基于原子力顯微鏡(AFM)的掃描成像與單分子力譜技術(shù)的原理、工作模式、儀器構(gòu)造與適用范圍及其在現(xiàn)代生物學(xué)領(lǐng)域的應(yīng)用。第二章中主要介紹了長鏈端粒DNA的合成方案,并通過AFM掃描成像、紫外熔融、圓二色譜(CD)等多種手法對(duì)產(chǎn)物進(jìn)行表征。結(jié)果表明我們合成的長鏈DNA序列在一定條件下能夠形成高級(jí)結(jié)構(gòu),序列折疊成G4聯(lián)體并呈串珠狀,然而構(gòu)象與短鏈G4模型有些不同,我們推測(cè)可能是由于長鏈中G4聯(lián)體結(jié)構(gòu)單元末端不再自由,導(dǎo)致優(yōu)勢(shì)構(gòu)象改變。通過引物修飾的方法,我們成功地使RCA反應(yīng)在基片上進(jìn)行,得到共價(jià)鏈接在基底的長鏈端粒DNA產(chǎn)物方便后續(xù)研究。另外我們還建立了一套合成雙鏈DNA重復(fù)序列的方法,突破了以往文獻(xiàn)中條帶彌散、產(chǎn)率低、退火后互補(bǔ)混亂的局限,為這一領(lǐng)域更深入的研究提供了參考和選擇。第三章中我們基于已有實(shí)驗(yàn)事實(shí)提出了G4聯(lián)體不完整折疊的模型,這一模型解釋了隨DNA鏈段增長Tm值降低的熔融行為,并解決了長鏈序列變溫紫外實(shí)驗(yàn)中熱穩(wěn)定性減弱與G4聯(lián)體相互作用(Quadruplex-Quadruplex Interaction,QQI)增強(qiáng)端粒DNA結(jié)構(gòu)穩(wěn)定性之間的矛盾�，F(xiàn)有的紫外熔融實(shí)驗(yàn)與CD測(cè)試結(jié)果都支持這一猜想。另外,根據(jù)不同突變序列的長鏈端粒DNA的CD表征結(jié)果,我們推測(cè)不完整折疊結(jié)構(gòu)中最外側(cè)4個(gè)鳥嘌呤在長鏈G4聯(lián)體結(jié)構(gòu)中折疊相對(duì)困難。這可能是由于長鏈端粒DNA重復(fù)序列中臨近鏈段的束縛與分子熱運(yùn)動(dòng)對(duì)G4聯(lián)體結(jié)構(gòu)產(chǎn)生了破壞。與之前發(fā)現(xiàn)的G3聯(lián)體不同的是我們的模型中G4聯(lián)體兩端的破壞是對(duì)稱的。這一模型的提出深化了對(duì)于長鏈端粒DNA序列結(jié)構(gòu)的認(rèn)識(shí),并為一些基于G4聯(lián)體結(jié)構(gòu)來設(shè)計(jì)小分子抗癌藥物的研究提供了新的思路。第四章中我們通過基于原子力顯微鏡的單分子力譜技術(shù)考察了長鏈端粒DNA的力學(xué)穩(wěn)定性,結(jié)果表明基于QQI形成的更高級(jí)結(jié)構(gòu)在拉伸曲線中表現(xiàn)為55p N的平臺(tái)。往復(fù)拉伸曲線之間有明顯的滯后,而滯后區(qū)域所包含的面積△E與拉伸過程中長度變化△L呈線性相關(guān),這說明不同拉伸程度下破壞的是相同的結(jié)構(gòu)。從該線性關(guān)系的斜率可以衡量端粒DNA高級(jí)結(jié)構(gòu)的穩(wěn)定性,我們發(fā)現(xiàn)在40%PEG中擬合線的斜率比水中大,表明40%PEG中G4聯(lián)體結(jié)構(gòu)更加穩(wěn)定,這與之前文獻(xiàn)中的觀點(diǎn)一致。該單分子方法還可以用于非常直觀地考察各種其它因素,比如p H、離子環(huán)境、配體等對(duì)G4聯(lián)體穩(wěn)定性的影響規(guī)律。系統(tǒng)的對(duì)比實(shí)驗(yàn)結(jié)果還顯示只有端粒DNA的拉伸與松弛曲線之間能夠產(chǎn)生滯后,若當(dāng)序列中插入無規(guī)部分,將端粒DNA序列中相鄰G4聯(lián)體結(jié)構(gòu)單元彼此隔開(間隔基序列),則不出現(xiàn)明顯的滯后。這說明單個(gè)G4聯(lián)體結(jié)構(gòu)的形成對(duì)其鄰近片段的折疊有促進(jìn)作用,加速其余部位G4聯(lián)體結(jié)構(gòu)的形成;間隔基的引入將大大削弱該協(xié)同效應(yīng)。該研究加深了我們對(duì)端粒DNA高級(jí)結(jié)構(gòu)形成機(jī)制的認(rèn)識(shí)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q523
本文編號(hào)：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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q523
【引證文獻(xiàn)】
相關(guān)博士學(xué)位論文 前1條
1 呂秀娟;鋸齒型構(gòu)象高分子單晶納米力學(xué)性質(zhì)單分子力譜研究[D];吉林大學(xué);2018年
，本文編號(hào)：2501248
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