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研究生: 沈洋逸
Shen, Yang-I
論文名稱: 利用單分子技術研究與染色體易碎症相關的d(CGG)重複序列及其抑制疾病的變異序列之構型動態學
Single-Molecule Study on the Conformational Dynamics of d(CGG) Tandem Repeats Associated with Fragile X Syndrome and Their Disease Inhibition Mutations
指導教授: 李以仁
Lee, I-Ren
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2018
畢業學年度: 106
語文別: 中文
論文頁數: 68
中文關鍵詞: 單分子螢光共振能量轉移DNA擴張CGG三核苷酸重複序列
英文關鍵詞: smFRET, DNA expansion, CGG trinucleotides
DOI URL: http://doi.org/10.6345/THE.NTNU.DC.048.2018.B05
論文種類: 學術論文
相關次數: 點閱:104下載:1
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  • 染色體易碎症為一種常見的遺傳性疾病,其發病原因可以歸咎於人體中FMR1中CGG/CCG三核苷酸重複序列的擴張。在正常人體中,CGG/CCG的重複次數落在5至44之間;然而,在病人中,重複次數則會擴大至200以上。若CGG三核苷酸重複序列在FMR1基因中過度的擴張,將會觸發一種不正常的甲基化,稱為過甲基化;當基因過甲基化後,會造成組蛋白去乙醯化。經由這一系列不正常的生物修飾後,就會使FMR1基因失效。相較之下,在正常人體中的CGG/CCG序列之間會差入AGG/CCT序列。插入的頻率大約為9至10個CGG會出現一個AGG序列。
    然而CGG三核苷酸重複序列的構型以及AGG序列會對一般的CGG序列所照成影響仍是未知的。因此我們利用單分子螢光共振能量轉移光譜研究插入AGG以及未插入AGG之CGG序列的構型。我們的研究結果顯示,CGG序列不能在生理條件下折疊成鳥嘌呤四具體聚體。僅有在重複次數少於12的CGG序列能夠在有鳥嘌呤四具體聚體誘導劑的環境下,才能夠形成鳥嘌呤四具體聚體結構。在一般情況下,CGG序列則是會穩定地摺疊成對齊型的髮夾型構型。插入AGG的CGG的序列則會使這種使這種穩定的髮夾型結構改變,變化成另一種髮夾型結構。以先前研究所提出的重複序列的擴張理論為基礎,我們提出一個AGG三重複序列可能在DNA擴張中所扮演的角色。在具有AGG插入之序列含有傾向於形成帶有露出核苷酸之髮夾型結構,此一結構較不利於序列擴張,且在插入AGG後並未觀察到此結構活動至較利於被擴張的對齊型髮夾型結構。我們認為此一結構與結構動態學上的指標性與AGG插入抑制DNA擴張的原因。

    The expansion of CGG/CCG trinucleotides in the fragile X mental retardation (FMR1) gene leads to Fragile X syndrome (FXS), one of the most common genetic disorders. The number of CGG tract is in between 5 and 44 tandem repeat units in the healthy humans, while in the pathological samples, more than 200 repeat units were found in FMR1 gene. The overexpansion of CGG repeat would trigger hypermethylation, an abnormal DNA methylation and lead to inhibition of histone modification and epigenetic gene of FMR1 silence. In contrast, healthy individuals show that AGG/CCT interruptions exist in every 9-10 CGG/CCG trinucleotides. CGG repeat can fold into hairpin-like structures or G-quadruplexes, which are still under debating.
    Moreover, how AGG interruption affect to d(CGG)n structure is remained elusive. We use single-molecule fluorescence resonance energy transfer (smFRET) spectroscopy to study the conformation of CGG repeat with and without the interruption by AGG insertions. Our results show that d(CGG) repeats do not form a G-quadruplex under the physiological condition until the G-quadruplex inducer (pyridostatin) was added to the short (<16 repeat units) CGG sequences. The stable d(CGG) hairpin structure is interfered by the AGG insertion and lead to become another hairpin structure. Based on the DNA expansion model from our previous study, we propose a structure and structural dynamics role of AGG insertion in DNA preventing expansion. The insertion of AGG makes the majority of the sequencing folded into a hairpin with overhang configuration, which has less tendency to be expanded. When the CGG triplet fold into the hairpin structure, the new generated CGG repeat would enter the hairpin region and the DNA polymerase won’t work. Once the DNA replication restart, it’ll cause the DNA expansion. According to our result, CGG repeat interrupted by AGG triplet tend to fold into the hairpin structure with overhang. Furthermore, this configuration failed to slip to a expansion-favored blunt-end hairpin configuration. The unique structure and structural dynamics caused by the AGG insertion to tandem (CGG) repeat prevents the disease caused by error-prone expansion.

    致謝 i 摘要 ii Abstract iii 圖目錄 vii 表目錄 xi 第一章、緒論 1 1-1前言 1 1-2染色體易碎症 5 1-3致病原因 7 1-4已解出的CGG/CCG之二級結構 9 1-4.1髮夾型結構 9 1-4.2鳥嘌呤四聚體 11 1-5研究動機 14 第二章、實驗方法與儀器 15 2-1 單分子實驗技術 15 2-1.1螢光共振能量轉移 17 2-1.2全內反射螢光顯微鏡 19 2-2實驗樣品的製備 21 2-2.1實驗樣品槽製作 21 2-2.2樣品槽的組裝 24 2-2.3實驗與DNA序列的設計 25 2-2.4螢光分子的標記 27 2-2.5DNA黏合反應 28 2-2.6分子內二聚體去除反應 29 2-2.7將分子固定於玻片表面 30 2-2.8光漂白與閃爍現象 31 2-2.9影像緩衝溶液 33 2-3數據處理與分析 35 2-3.1數據處理 35 2-3.2擬合分析 38 第三章、實驗結果 40 3-1實驗設計 40 3-2結構鑒定 41 3-3髮夾型結構鑑定 42 3-4長時間軌跡圖 44 3-5鳥嘌呤四聚體結構鑑定 45 3-6插入AGG之CGG序列 50 3-6.1插入單組AGG之CGG序列 50 3-6.2插入雙組AGG之CGG序列 55 第四章、結論 58 4-1d(CGG)n之結構 58 4-2AGG對CGG序列之影響 59 4-3DNA在人體中的擴張模型 61 參考文獻 63

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