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研究生: 王思蓓
Wang, Si-Bei
論文名稱: 精胺對 GGCCTG 重複序列之髮夾型結構的滑動重組動態學的影響
Influence of Spermine on the Slippage Hairpin Reconfiguration Dynamics of Tandem GGCCTG Repeat
指導教授: 李以仁
Lee, I-Ren
口試委員: 侯明宏
Hou, Ming-Hon
詹智強
Chan, Chih-Chiang
余建泓
Yu, Chien-hung
李以仁
Lee, I-Ren
口試日期: 2021/07/20
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2022
畢業學年度: 110
語文別: 中文
論文頁數: 56
中文關鍵詞: 單分子螢光共振能量轉移脊髓小腦萎縮症 36 型六核苷酸重複序列擴張GGCCTG 重複序列精胺
英文關鍵詞: single-molecule fluorescence resonance energy transfer (smFRET), spinocerebellar ataxia 36 (SCA36), hexanucleotide repeat expansion, GGCCTG repeat sequence, spermine
研究方法: 實驗設計法觀察研究
DOI URL: http://doi.org/10.6345/NTNU202201157
論文種類: 學術論文
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  • 很多神經退化性疾病都是由重複序列異常擴張所引起,包含脊髓小腦萎縮症 36 型 (Spinocerebellar ataxia 36, SCA36)。他是由位於 Nop56 基因中的 GGCCTG 六核苷酸重複序列擴張所造成的,會造成一些神經功能障礙,像是肢體不協調及小腦萎縮等症狀。此類重複序列通常會折疊成二級結構,而這些二級結構被認為是在複製、修復或重組過程中造成不正常序列擴張的原因。
    我們使用單分子螢光共振能量轉移 (single-molecule fluorescence resonance energy transfer, smFRET) 研究重複次數為 3 ~ 8、11 及位於預突變 (premutation) 範圍的 17 的 (GGCCTG)n 重複序列的結構動態學,以及在精胺 (為與 GGCCTG 具有特異性結合的多電荷胺類) 影響下的動力學。我們發現偶數重複的 (GGCCTG)n 會穩定形成 2 個核酸 (2-nt) 突出髮夾結構 (2-nt overhang hairpin);而奇數重複的 (GGCCTG)n 會在 2-nt 突出髮夾結構與 8-nt 突出髮夾結構 (8-nt overhang hairpin) 之間做轉換,且平衡利於 2-nt 突出髮夾結構。精胺的添加使平衡傾向於 2-nt 突出髮夾結構,使其可能具有更高的重複序列擴張的趨勢。

    Abnormal repeat expansion is a major cause for many neurodegenerative diseases, including Spinocerebellar Ataxia 36 (SCA36). GGCCTG hexanucleotide repeat expansion in the Nop56 gene is responsible for nervous system dysfunctions, including incoordination and cerebellar atrophy. These tandem repeats DNA usually fold into a secondary structure, which is believed to be crucial in abnormal expansion during replication, repair or recombination process.
    We used single-molecule fluorescence resonance energy transfer (smFRET) microscopy to study conformational dynamics of (GGCCTG)n sequences with n = 3 ~ 8, 11 and 17, as well as the dynamics under the influence of spermine, a polycharged amine that binds to GGCCTG tandem repeat specifically. We found that even-numbered repeats of (GGCCTG)n form stable 2-nt overhang hairpin structure while odd-numbered repeats form hairpin interconverting between a 2-nt and 8-nt overhang structures, with the equilibrium favors a 2-nt overhang hairpin structure. The addition of spermine makes the equilibrium futher lean toward the 2-nt overhang hairpin structure, which may have higher tendency to undergo repeat expansions.

    摘要 i Abstract ii 目錄 iii 表目錄 v 圖目錄 vi 第一章、 緒論 1 1.1 脊髓小腦萎縮症 36 型之簡介 1 1.2 RNA 相關研究 3 1.3 非典型二級結構 5 1.4 重複序列的擴張機制 6 1.5 多胺的功能 10 1.6 研究動機 11 第二章、 實驗儀器與方法 12 2.1 實驗儀器與原理 12 2.1.1 單分子實驗技術 12 2.1.2 螢光共振能量轉移 13 2.1.3 全內反射螢光顯微鏡 16 2.2 實驗方法與樣品製備 18 2.2.1 實驗樣品槽的製備 18 2.2.2 實驗樣品槽的組裝 22 2.2.3 DNA序列之設計 23 2.2.4 螢光分子標記 25 2.2.5 DNA黏合反應 26 2.2.6 實驗樣品固定於玻片表面上 27 2.2.7 成像緩衝溶液 28 2.3 數據處理與分析 30 2.3.1 數據處理 30 2.3.2 擬合分析 32 第三章、 實驗結果與討論 34 3.1 單分子實驗設計 34 3.2 (GGCCTG)n 結構鑑定 35 3.2.1 G-四聯體結構鑑定 35 3.2.2 髮夾結構鑑定 36 3.2.3 長時間軌跡圖 39 3.2.4 轉換密度分析 41 3.3 精胺對 (GGCCTG)n 之影響 42 3.4 (GGCCTG)n 構型轉換之動力學分析 45 3.5 精胺對 (GGCCTG)n 構型結構穩定性 50 第四章、 結論與未來展望 52 參考文獻 53

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