二胜肽重複序列 (Dipeptide Repeats, DPRs) 源自於 GGGGCC 六核苷酸擴張 (G4C2 expansion) 經重覆關連之非 ATG 起始之轉譯而成，由正義股 (Sense sequence) 產生甘胺酸-精胺酸 (poly Glycine-Arginine)、甘胺酸-丙胺酸 (poly Glycine-Alanine) 及甘胺酸-脯胺酸 (poly Glycine-Proline)；反義股 (Antisense sequence) 產生脯胺酸-精胺酸 (poly Proline- Arginine) 、脯胺酸-丙胺酸 (poly Proline-Alanine) 及甘胺酸-脯胺酸 (poly Glycine-Proline) 。先前研究指出二胜肽重複序列是額顳葉失智症 (Frontotemporal Dementia, FTD) 及肌萎縮側索硬化症 (Amyotrophic Lateral Sclerosis, ALS) 之發病原因之一，目前證據顯示其細胞毒性或許與其在水溶液中的結構相關，但機制仍未確定。本實驗室先前已成功解析出poly Glycine-Arginine (GR)n (n = 5、10、15、20、25、30) 之結構，發現當 n ≥ 20 時，會開始形成軟螺旋結構，但並非為 α-螺旋。本研究主要利用先前發展的水溶液小角度 X 光散射 (SAXS) 結合分子結構模擬的方法探討水溶液中進一步探討poly Proline- Arginine (PR)10、(PR)30、(GR)25-1 及 (GR)25-2結構的差異性並以此理解與細胞毒性之相關性。由結果得知 (PR)30 比先前已解析出來之 (GR)30 的軟螺旋結構更延伸，但無明顯之螺旋結構；(PR)10 與先前已解析出的 (GR)10皆無螺旋結構產生，但 (PR)10 的結構較為鬆散。造成結構較為鬆散的之原因推測為較大的脯胺酸 (Proline) 主鏈結構無法如甘胺酸 (Glycine) 主鏈配合精胺酸 (Arginine) 之帶電側鏈螺旋排列以有效降低自由能之故。為證實此以推測，我們再在 (GR)25 中在不同位置選擇性置換一至兩個 GR 為 GP 序列，並觀測其相對應的結構改變。結果顯示， (GR)12-GP-(GR)12， (GR)25-1 及 (GR)8-GP-(GR)7-GP-(GR)8， (GR)25-2，會因為脯胺酸之序列重複長度改變而減少其螺旋結構，歸因於中間有 (GP) 序列阻擋螺旋結構的形成，由圓二色性分析 (CD) 觀察結果發現也有類似二級結構減少的結果。這些結構的特徵與差異性或許對詮釋 (GR)n、(PR)n 兩者在細胞毒性的不同機制表現上有所啟發。本研究中也嘗試結合靜態光散射儀 (MALS)、場流分離系統 (AF4) 及折射儀 (RI)，進行 (GR)25 與短單股DNA 序列 (AC)3 之複合結構分子量測量，以確定兩者之間的結合比列狀況。雖然我們成功地組合此系統，並能進行牛血清蛋白及細胞色素 C 的分子量量測達 1-5 % 準確度，然而由於 (GR)25-(AC)3 之複合物體積小且樣品濃度不足，並未能量測到足夠的光散射訊號導致無法決定此複合體分子量。

Dipeptide Repeats (DPRs) are derived 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 (FTD) and Amyotrophic Lateral Sclerosis (ALS). The sense sequence translates into poly Glycine-Arginine (GR)n, poly Glycine-Alanine (GA)n and poly Glycine-Proline (GP)n ; antisense sequence translates into poly Proline-Arginine (PR)n, poly Proline-Alanine (PA)n and poly Glycine-Proline (GP)n. Current research shows that different DPRs have different cytotoxicity in aqueous solutions. Previous research in our research group resolved the structure of (GR)n (n = 5, 10, 15, 20, 25, 30) and found helical structure formation when n ≥ 20; which is less compact as that of α-helix. This study aims to provide the structure of (PR)10 and (PR)30 using small-angle X-ray scattering combined with the protocol of molecular structure simulation, Rosetta-SAXS. The result shows that (PR)30 is more extended than (GR)30 resolved previously, but with less helix structural features compared to the latter. (PR)10 has a relatively extend structure with no helix features, compared to the globular (GR)10 of also no helical structure. The reason for the loose structure is attributed to steric effects of the fifth member-ring of Proline, which frustrates formation of helix structures of (PR)n. To examine the proposed mechanism, we have selectively mutated 1 or 2 GR-DPR with GP-DPR for (GR)12-GP-(GR)12, (GR)25-1, and (GR)8-GP-(GR)7-GP-(GR)8, (GR)25-2, to suppress formation of helical structure. The result indeed shows that there is largely reduced helical structure in (GR)25-2, which result supports the steric effects proposed. The structural differences revealed in this study might provide important insights in the interpretation of the different behaviors of the two DPRs of (GR)n and (PR)n in cell toxicity. We have also integrated measurements of multi-angle light scattering, asymmetric flow field-flow fractionation, and the combined spectroscopy of UV-vis adsorption and refraction index, in an integrated system, in an attempt to determine the molecular weight of the complex of (GR)25 and single-strand DNA (AC)3, and deduce their binding ratio. Although, we have successfully intergraded the system and determined the molecular masses of bovine serum albumin (66.5 kDa) and cytochrome C (12.3 kDa) to a mass resolution of 1-5%. The system, however, could not determine the molecular weight of (GR)25-(AC)3, due presumably to low intensity of light scattering with the insufficient sample concentration used.

中文摘要--------i
Abstract--------iii
致謝--------v
圖目錄--------viii
表目錄--------xiii
一、 緒論--------1
1.1 二胜肽重複序列 (Dipeptide Repeats, DPRs)--------1
1.2 以 SAXS 結合分子結構模擬研究生物分子的結構--------3
1.3 GR 系列的結構變化--------5
1.4 GR 與單股 DNA (ssDNA) 的結合狀況--------9
1.5 研究動機--------10
二、 研究方法與原理--------12
2.1 生物小角度 X 光散射 (Bio-SAXS)--------12
2.2 小角度 X 光散射結合分子結構模擬分析--------17
2.3 紫外光-可見光光譜學 (UV / Visible spectroscopy)--------18
2.4 折射率 (Refractive Index) 於蛋白質量測--------23
2.5 場流分離系統 (Field-Flow Fractionation, FFF)--------25
2.6 光散射的量測 (Light Scattering , LS)--------28
2.6.1 靜態光散射 (Stastic Light Scattering, SLS)--------28
2.6.2 動態光散射 (Dynamic Light Scattering, DLS)--------30
三、 實驗方法--------32
3.1 實驗樣品--------32
3.2 生物小角度 X 光散射量測與流程--------33
3.3 場流分離系統及光散射測量流程--------38
四、 結果與討論：利用小角度X光散射結合分子結構模擬分析二胜肽重複序列水溶液結構--------40
4.0 回顧： (GR)25 之結果--------40
4.1 (PR)10--------41
4.2 (PR)30--------44
4.3 (PR)10 與 (PR)30 之結構比較--------48
4.4 (GR) 與 (PR) 之結構比較--------49
4.4.1 (PR)10 與 (GR)10--------49
4.4.2 (PR)30 與 (GR)30--------50
4.5 (GR)25-1--------54
4.6 (GR)25-2--------57
4.7 (GR)25、(GR)25-1、(GR)25-2 之結構關係--------60
五、 總結--------62
參考文獻--------63
附錄一、SAXS 數據選用說明--------67
附錄二、(GR)25-(AC)3 DPR-ssDNA水溶液之複合物分離及分子量計算--------68
附 2.1 場流分離系統與光散射量測--------68
附 2.1.1 標準品校正--------68
附 2.1.2 (GR)30-(AC)6 複合物之量測--------69
附 2.2 利用 HPLC 之Bypass 模式搭配光散射量測 (GR)25-(AC)3 複合物分子量--------73
附 2.3 複合體結合狀況研究結論--------76
附錄三、(GR)25 及複合物之圓二色性分析--------77
附錄四、多肽與雙層磷脂酸膜之關係--------79
附 4.1 多肽與奈米圓盤之結構分析--------79
附 4.2 多肽與磷脂酸膜厚度之關係--------80

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