在患有額顳葉失智症 (Frontotemporal Dementia, FTD) 及肌萎縮側索硬化症( Amyotrophic Lateral Sclerosis, ALS ) 之家族性遺傳疾病之患者中發現，大部分患者的大腦內及脊髓中的未解碼區域C9ORF72 (Chromosome 9 open reading frame 72) 基因帶有異常的六核苷酸擴張 (Hexanucleotide expansion, GGGGCC)，經過非ATG起始之轉譯 (non-ATG initiated translation) 產生大量二胜肽重複序列 (Dipeptide Repeats, DPRs)片段。DPRs包含甘胺酸-精胺酸 (poly Glycine-Arginine, (GR)n)、甘胺酸-丙胺酸 (poly Glycine-Alanine, (GA)n)、甘胺酸-脯胺酸 (poly Glycine-Proline, (GP)n)、脯胺酸-精胺酸 (poly Proline- Arginine, (PR)n)、脯胺酸-丙胺酸 (poly Proline-Alanine, (PA)n)，不同序列片段與病灶機制之間關係仍有許多未知的地方。本研究主要使用水溶液小角度X光散射 (Bio-SAXS) 結合分子結構模擬程式探討水溶液中甘胺酸-精胺酸 (poly Glycine-Arginine, (GR)n) 二胜肽在不同序列長度下的結構變化，其中n = 5、10、15、20、25與30，以及 (GR)n 混合以腺嘌呤-胞嘧啶之含氮鹼基 (AC)3 組成的單股DNA (ssDNA) 複合體結構，作為理解 (GR)n 細胞毒性之基礎。第一部份，由我們建立的水溶液小角度X光散射結合分子動力學模擬之數據分析法來解析在溶液下各別 (GR)n 的結構。由結果得知，在n值趨近20時，(GR)n 會形成類似 α螺旋 (α-helix) 結構，但其螺旋間距為6.2 ± 0.1 Å較 螺旋間距5.4 Å寬鬆，故暫稱為軟螺旋結構。此結構特徵歸因於 (GR)n 精胺酸之氫鍵吸引作用及側鏈正電荷排斥的平衡結果。透過圓二色性的分析 (Circular Dichroism) 也發現(GR)n 隨n值而越趨顯著的類似二級結構訊號。此與先前結果相呼應。第二部分，我們結合SAXS、UV-Vis吸收光譜及折射率的同步量測，解析出 (AC)3 與 (GR)25 ，在水溶液中可穩定存在的結合比為1:1;此複合體的結構迴旋半徑Rg = 17.9 ± 0.6 Å，僅略高於 (GR)25 的Rg値16.5 ± 0.2 Å。分子動力學模擬與SAXS數據的綜合分析結果顯示，(AC)3選擇性的吸附與 (GR)25較鬆散之N端 (N-terminal) 形成半穩定 (metastable) 結構。

Segments of abnormal dipeptide repeats (DPRs) are often found, as signature symptom products, from the transcription of an infected gene chromosome 9 open reading frame 72 (C9ORF72) in the brain or spinal cord of the patients of familial frontotemporal dementia and amyotrophic lateral sclerosis. Such dipeptide repeats include poly Glycine-Arginine (GR)n, poly Glycine-Alanine, poly Glycine-Proline, poly Proline-Arginine, and poly Proline-Alanine, which have been associated with cell toxicity of unclarified mechanism. This study aims to provide structural hints on the mechanism of (GR)n cell toxicity on the basis of the solution structures of (GR)n, with n = 5, 10, 15, 20, 25, and 30, and their associations with single strand DNA of selected repeating base pairs in solution, using combined small-angle X-ray scattering (SAXS), UV-vis absorption, and refraction index (RI) measurements, further coupled with molecular structure simulation. In the first part, a combined analysis of SAXS-Molecular structural simulation is developed to reveal the solution structures of neat (GR)n. The result shows that (GR)n forms a loose helical structure of an extended conformation when n is near or above a critical value nc of 20, with a mean helical pitch length of 6.2 Å larger than that (5.2 Å) of the well-known  helix. Consistently, circular dichroism result also suggests a trend of developing secondary structure from random coils in solution with the increase of n value. In the second part of the study, an integrated methodology combining SAXS, UV-vis absorption, and refractive index measurements is developed to resolve the composition and structure of (GR)n complexed with a specific single strand DNA, (AC)n. The (AC)3-(GR)25 complex is found to be stable in solution only at 1:1 association ratio, with a radius of gyration Rg = 17.90.6 Å, slightly larger than 16.5  0.2 Å of the neat the (GR)25. Combined analysis of molecular dynamics simulation and the SAXS data reveals a metastable complex conformation with the (AC)3 selectively binding to the loose N-terminal of (GR)25.

中文摘要 i
Abstract ii
致謝 iii
圖目錄 vi
表目錄 xi
1 緒論 1
1.1 六核苷酸擴張 (GGGGCC expansion) 1
1.2 FTD/ALS 2
1.3 二胜肽重複序列Dipeptide Repeats (DPRs) 3
1.4 研究動機 8
2 研究方法與原理 10
2.1 生物小角度X光散射 (Bio-SAXS) 10
2.2 分子結構模擬結合生物小角度X光散射分析 17
2.2.1 Rosetta分數函數計算與結構自由能最小化程序 19
2.3 紫外光-可見光光譜學 (UV-Visible spectroscopy) 20
2.3.1 定性分析 21
2.3.2 定量分析 22
2.4 折射率 (Refractive Index) 於蛋白質量測 24
2.5 發展UV-Vis/RI 數據分析方法解析GR-DNA複合體之結合比 26
3 實驗方法 27
3.1 實驗樣品製備 27
3.2 水溶液中小角度X光散射量測與流程 28
3.3 Flow-SAXS 量測及數據篩選評估 33
4 結果與討論: 以X光散射觀察(GR)n的水溶液中結構 37
4.1 SAXS結合分子結構模擬分析的數據分析方法 37
4.1.1 (GR)5的熔球結構 38
4.1.2 (GR)10的半熔球結構 41
4.1.3 (GR)15的展開與隨機線圈結構 44
4.1.4 (GR)20螺旋起始點與展開結構 47
4.1.5 (GR)25的軟螺旋結構 50
4.1.6 富含軟螺旋結構的 (GR)30 53
4.2 GR的螺旋結構之增長趨勢 56
4.3 (GR)n二級結構的形成與其穿膜關係 58
4.4 結論 61
5 GR-DNA複合物的水溶液結合比例與其結構方法發展 62
5.1 (GR)25 DPRs-(AC)3 ssDNA複合體UV-Vis/RI的數據分析方法 62
5.2 (GR)10 DPRs與(AC)3 ssDNA 71
5.3 (GR)25 DPRs-(AC)3 ssDNA複合體之分子動力學模擬 72
5.4 複合體結構研究結論 74
6 總結 75
參考文獻 77
附錄 82

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