我們以光學鉗平台研究人類端粒序列 H24 ( (TTAGGG)4 ) 中的鳥嘌呤-四股結構 G-quadruplex (G4) 的摺疊與解開機制。首先為了達成量測此結構轉變所出現的微小距離變化，針對光學鉗平台的操作模式作改進，目標在達成定力量測的模式。為了提高系統中雷射的穩定性，將聲光調變器加入光學系統中，藉由記錄雷射強度後，對應調整外加電壓成功達成穩定雷射功率，在二十分鐘內標準差小於 0.01 mW。關於 G4 實驗，我們選擇以 H24 序列作為研究標的，並以 150 mM 高濃度 Na+ 離子溶液使序列透過 Hoogsteen basepairing 形成 G4 的二級結構，且摺疊形式出現較為單純的反平行籃子型 (anti-parallel basket type)。首先以受力與伸長量模式 (force-extension assay) 量測發現 G4 結構在逐漸增加施力 (unfolding) 過程及降低施力 (refolding) 過程中均出現一到兩個轉折事件，不過在加入穩定劑 BMVC (3,6-bis(1-methyl-4-vinylpyridinium) carbazole) 後G4結構在 unfolding 過程中轉折事件上升至兩到三個，暗示著中間態的存在。接著轉換到定力量測 (force-clamp assay) 模式下觀察 G4 解構機制，由單獨只有 H24 序列存在所得到的時程圖，可以觀察到除了全展開及全摺疊的長度之外，出現了第三種長度分布，並且在加入 BMVC 與 H24 序列共同培養之下，我們觀察到第四個分布的出現。由 H24 序列在兩種光學鉗的量測模式下所得到的結果，確實的發現 G4 結構的解構機制在有 BMVC 的處理下會有不同於其單獨存在的情形。

We used an optical tweezers platform to study the folding and unfolding pathway of individual molecules containing single-stranded DNA human telomeric G-quadruplex (G4) sequence, (TTAGGG)4. We home-built an optical tweezers platform with force-clamp capability, which held a DNA tether at a constant force, to determine the DNA length in high spatro-temperal resolution. We included an acousto-optic modulator (AOM) to maintain the laser stability and madulate laser output intensity. By modulating the applied voltage of AOM, feedbacked from the laser power measurement, we achieved to maintain the laser power with standard deviation less than 0.01mW over 20 minutes. For G4 experiments, these G4 containing DNA molecules are found to form the G-quadruplex structure based on Hoogsteen basepairing in 150 mM Na+ solution. When forces were applied to unfold the G4-containing DNA molecules, most of the unfolding traces showed one or two transitions, suggesting the existence of one stable intermediate state. The total unfolding distance was consistent with the expected value of unfolded G4 structure. However, when the DNA molecules were pre-incubated with a G4 stabilizing ligand BMVC, 3,6-bis(1-methyl-4-vinylpyridinium) carbazole, most DNA molecules showed three unfolding transitions, suggesting of two stable unfolding intermediate states. Using the force-clamp assay, we found that three extension states exist in the traces of G4 DNA. When G4 was pre-incubated with BMVC, the number of extension states also increases to four. Both force-extension and force-clamp results suggest that BMVC-bound G4 structures are able to withstand higher force than the ligand-free G4 structure, thus revealing more intermediate states.

目 錄……III
圖目錄………VI
表目錄………VII
中文摘要……VIII
英文摘要……IX

第一章 前 言

1.1 端粒、端粒&;#37238;與鳥嘌呤-四股結構之間關係............................. 1
1.1.1 端粒……………………….… 1
1.1.2 端粒&;#37238;………………………………………. 2
1.1.3 鳥嘌呤-四股結構…………………………..…. 3
1.2 鳥嘌呤-四股結構的多樣性………………..…….. 4
1.3 鳥嘌呤-四股結構穩定劑………………………… 5
1.4 單分子雷射光學鉗系統………..………………. 7
1.5 對於鳥嘌呤-四股結構構型及解構機制之先前研究….… 8
第二章 實驗架設
2.1 實驗核酸基質的製備…………..……………….. 13
2.1.1 雙股核酸長柄製備………….…………… 13
2.1.2 製造雙股核酸長柄的黏狀末端 (sticky end) 16
2.1.3 利用黏合反應組合反應所需最終產物………. 17
2.1.4 實際進行核酸製備過程中階段性所得結果…… 19
2.2 玻片之表面修飾………………..………… 21 IV
2.2.1 製備反應所需的反應槽 (Flow chamber)……… 21
2.2.2 製備表面修飾卵白素 (streptavidin) 的 1040 奈米聚苯乙烯球(polystyrene bead)………….22
2.2.3 實驗前玻片表面處理與核酸基質之固定………. 23
2.2.4 實驗所用之溶液條件………………….. 24
第三章 光學鉗兩種量測模式的建立

3.1 光鉗平台架設………………………. 25
3.1.1 光學系統設計概念…….…………………… 26
3.1.2 光學系統架設步驟……………….…………… 27
3.1.3 檢驗光路平行…………….……………………… 28
3.1.4 光位移感測器 (QPD) 訊號與 1040 奈米球相對雷射中心距離轉換常數的量測………………31
3.1.5 由光位移感測器訊號量測光鉗彈簧係數 (Force constant)….. 32
3.1.6 受力與伸長量 (Force-extension) 的量測…………………………….. 33
3.1.7 定力下伸長量 (Force-clamp) 的量測………………………………... 35
3.1.8 建立定力量測施力與距離補償時使用的校正曲線……………..…… 35
3.1.9 利用聲光調變器 (AOM) 及光強度感應器 (Power meter) 建立雷射強度回饋系統……37
3.1.10 實際架設雷射光強度的訊號回饋系統…………………….. 38
3.1.11 利用 2100 奈米球建立顯微鏡聚焦高度回饋系統…………….…... 41
3.2 測試光學鉗定力量測模式…………………………. 43
3.2.1 測試髮夾序列在定力模式下解構情形……….. 44
第四章 結果與討論
4.1 實際實驗中拉扯核酸基質幾何位向……..……………….……………….. 46
4.2 人體端粒序列在受力與延長量量測結果 (force-extension assay)……… 46 V

4.2.1 人體端粒序列單獨存在下不同事件次數的施力與伸長量曲線…… 48
4.2.2 人體端粒序列與 BMVC 共同存在下不同事件次數的施力與伸長
量曲線……………….………………………………………………..

50
4.2.3 人體端粒序列在有無 BMVC 存在下事件發生次數統計圖……… 52
4.3 定力下延長量量測 (force-clamp assay) 結果……………………………. 54
4.3.1 人體端粒序列單獨存在下長度改變的時程路徑圖 (time course)….. 54
4.3.2 人體端粒序列與 BMVC 共同存在下長度改變的時程路徑圖 (time
course)…………….…………………………………………………...

57
4.3.3 人體端粒序列在有無 BMVC 存在時程路徑圖的長度分布統計圖.. 60
4.4 BMVC 如何穩定人體端粒序列的解構過程模型…………..…………….. 62
第五章 總結與未來展望

5.1 總結……………………………………….…… 65
5.2 未來展望………………………………………….… 66

參考文獻..……………………………………………… 68
附錄 數據列表
1. 人類鳥嘌呤序列受力與伸長量圖…………………………... 71
1.1 小範圍伸長量區間內無 BMVC 的表現…………………….71
1.2 小範圍伸長量區間內有 BMVC 的表現………………………. 75
1.3 大範圍伸長量區間內無 BMVC 的表現………………………………. 80
1.4 大範圍伸長量區間內有 BMVC 的表現………………………………. 84
2. 人類鳥嘌呤序列定力下伸長量圖…………………………………………... 88
2.1 在溶液中無 BMVC 定力下伸長量改變………………………………. 88
2.2 在溶液中有 BMVC 定力下伸長量改變………………………………. 96
VI

圖目錄
圖 1-1 端粒序列示意圖…………………………………… 1
圖 1-2 核酸複製反應的進行架構圖………………………… 2
圖 1-3 端粒序列形成二級結構中平面結構圖………………… 3
圖 1-4 過往研究中解出的鳥嘌呤-四股結構………… 5
圖 1-5 鳥嘌呤-四股結構穩定劑 BMVC………………….. 6
圖 1-6 先前對於鳥嘌呤-四股結構解構機制所提出的模型………….. 12
圖 2-1 核酸序列目標產物示意圖…………………… 13
圖 2-2 各部分的核酸及最終核酸基質電泳圖………………. 20
圖 2-3 玻片反應槽設計示意圖……………………………. 21
圖 3-1 光學鉗實驗整體概念示意圖…………………. 25
圖 3-2 光學鉗實驗光路系統示意圖………………………. 27
圖 3-3 不同情況的 1.04 μm 球在 QPD 的總和訊號所呈現的圖譜………... 30
圖 3-4 1.04 μm stuck bead 在雷射兩軸掃描所得訊號反應…………………. 32
圖 3-5 QPD 訊號標準差反應球在雷射下受力大小…………... 33
圖 3-6 受力與伸長量量測模式……………….……………… 34
圖 3-7 建立 QPD 訊號與球在雷射中位置的校正曲線…………… 36
圖 3-8 AOM 驅動雷射強度改變及穩定長時間強度成效…………………... 39
圖 3-9 AOM 電壓給入與光強度讀取之間等待時間決定…………….…….. 40
圖 3-10 根據 2.1 μm bead 建立顯微鏡聚焦高度回饋系統………….………. 42
圖 3-11 定力量測的 stuck bead 及 DNA tether 控制組…………………...... 43
圖 3-12 髮夾序列在定力量測下的結果…………………….. 45
圖 4-1 核酸基質架設光學鉗平台下的位向………………… 46
圖 4-2 受力與伸長量曲線中轉折的辨認……………………… 47
圖 4-3 H24 序列單獨存在下的 force-extension curve………… 49 圖 4-4 BMVC 與 H24 序列共同培養後所得的 force-extension curve……. 51
圖 4-5 H24 序列在單獨及添加 BMVC 所得受力與伸長量曲線中的轉折次數…………………………53
圖 4-6 H24 序列單獨存在下長度改變的時程路徑圖……… 56
圖 4-7 H24 序列與 BMVC 共同存在下長度改變的時程路徑圖……. 59
圖 4-8 人體端粒序列在有無 BMVC 存在的時程路徑的長度分布統計圖…. 61
圖 4-9 人類端粒序列解構過程模型與 BMVC 參與步驟………………. 64

表目錄
表 2-1 DNA handle 所使用的引子對序列………………. 14
表 2-2 PCR 反應使用之 buffer 條件………………… 14
表 2-3 PCR 反應使用之 program 流程…………… 15
表 2-4 限制&;#37238;截切反應配對組合………………... 16
表 2-5 限制&;#37238;截切反應之 buffer 條件………………. 17
表 2-6 Ligation 反應中使用的單股 DNA 序列……… 18
表 2-7 Ligation 反應之 buffer 條件與步驟……. 19

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