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研究生: 陳巧穎
Chen, Chiao-Ying
論文名稱: B 細胞淋巴瘤基因啟動子區域中 DNA 四股結構之構型間轉換的單分子研究
Interconversion between G-quadruplex Conformations in B-cell Lymphoma 2 Promoter Region
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: 78
中文關鍵詞: Bcl-2前致癌基因Pu39DNA 二級結構G4 結構單分子螢光共振能量轉移
英文關鍵詞: Pu39, proto-oncogene, interconversion, single-molecule
DOI URL: http://doi.org/10.6345/THE.NTNU.DC.055.2018.B05
論文種類: 學術論文
相關次數: 點閱:165下載:2
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  • B 細胞淋巴瘤基因 2 (簡稱 Bcl-2) 在人體內是一負責控制細胞凋亡的前致癌基因,其過表達現象和人體多種癌症皆有高度的關聯性。在 Bcl-2 的啟動子 P1 區域上有一富含 G、C 的 39 個鹼基對的序列被指出會參與此基因的基因表達,名為 Pu39,且其在特定的鹽離子濃度與 pH 值下會形成特殊的 DNA 二級結構 G-quadruplex (G4結構),此結構可以抑制基因轉錄進而阻止癌細胞增生。

    先前 Yang 團隊使用 Pu39 的縮短版序列 midG4 和 Pu30 鑑定出兩種不同的 G4 結構,且認為這兩種結構會互相轉換,但並沒有找到相關的直接證據。在本篇研究中,我們的目標物為完整版的 Pu39 序列,並且使用單分子螢光共振能量轉移光譜技術觀察即時的 G4 結構互相轉換的動態學與動力學。

    我們發現 midG4 和 Pu30 兩種結構會經由一未知的中間狀態進行互相轉換,有趣的是互相轉換的現象來自於 Pu39 上的第六組連續 G 片段,表示第六組連續 G 片段對於基因的調節與表達而言相當重要,然而這組片段在先前 Yang 團隊的實驗中皆是被去除的。此外我們也在 Pu39 序列上發現了先前的研究團隊沒有鑑定出來的G4 結構 (3456G4)。

    B-cell Lymphoma 2 (Bcl-2) is a proto-oncogene that is responsible for cell apoptosis. The overexpression of Bcl-2 gene is highly correlated to human cancer of many kinds. A 39-nucleotide (nt) GC-rich region (Pu39) in promoter P1 of Bcl-2 gene is believed to be involved in the gene modulation. This 39nt GC-rich region may form multiple G-quadruplex (G4) structures at specific cation concentration and pH value, stabilizing this G4 structure can inhibit gene transcription and potentially suppress the cancer cell formation. Previous studies have shown that this 39nt GC-rich region might fold into two different G4 conformations, among them, Bcl-2midG4 sequence fold into hybrid conformation and Bcl-2Pu30 sequence form parallel conformation. Interestingly, an interconversion between these two structures was proposed but no direct experimental evidence was shown. These two different interchangeable G4s in Pu39 may be important for the regulation of transcription, as each G4 is likely to be recognized by different proteins leading to different gene modulation. We applied single-molecule fluorescence resonance energy transfer (smFRET) spectroscopy to directly reveal the interconversion dynamics between Pu39 G4 states. We found that the interconversion between these two conformational states undergoes a stepwise mechanism through one unidentified intermediated state. Interestingly, the 6th tandem G-sequence, which was usually believed to be unimportant and truncated in the previous studies, modulate the mechanism of interconversion. Moreover, an additional G4 state was also found in the Pu39 configuration.

    致謝 i 摘要 ii Abstract iii 目錄 iv 圖目錄 vi 表目錄 ix 第一章 緒論 1 1-1 前言 1 1-2 癌症成因 3 1-3 B 細胞淋巴瘤基因 2 5 1-4 G-四股結構 8 1-5 研究動機 18 第二章 實驗內容 19 2-1 實驗方法 19 2-1.1 單分子技術 19 2-1.2 螢光共振能量轉移 21 2-1.3 全內反射式螢光顯微鏡 23 2-1.4 圓二色光譜儀 24 2-2 實驗樣品製備 25 2-2.1 實驗序列設計 25 2-2.2 螢光染料分子標記 28 2-2.3 寡核苷酸黏合反應 30 2-2.4 樣品槽製備與組裝 31 2-3.5 固定樣品於樣品槽 35 2-2.6 影像緩衝溶液 36 2-3 數據處理與分析 39 2-3.1 數據處理 39 2-3.2 擬合分析 43 第三章 實驗結果與討論 45 3-1 實驗設計 45 3-2 原版序列的鑑定 46 3-3 點突變序列的鑑定 52 3-4 第六組連續 G 片段的影響 57 3-5 非 G4 結構的鑑定 59 3-6 加入 PDS 穩定 G4 結構 61 3-7 未知狀態的結構鑑定 63 第四章 結論 69 第五章 未來展望 71 參考文獻 72

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