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研究生:蔡松樺
研究生(外文):Sung-Hua Tsai
論文名稱:利用震盪式光鉗之不同震盪頻率探討去氧核醣核酸(DNA)與RecA蛋白之交互作用
論文名稱(外文):Probing DNA and RecA Protein Interactions via Different Oscillation Frequency of Oscillatory Optical Tweezers
指導教授:邱爾德
指導教授(外文):Arthur Chiou
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生醫光電工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:75
中文關鍵詞:光鉗
外文關鍵詞:Optical tweezersDNARecA
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去氧核醣核酸 (Deoxyribonucleic acid, DNA) 為生物體的遺傳物質,經過高度聚集、堆疊、纏繞成為染色體並存在於細胞核中。當細胞進行轉錄、分裂或修補時,DNA會與不同的功能蛋白作用並執行任務,延伸細胞的功能。在DNA的重組及修復中RecA蛋白扮演重要的角色,在三磷酸腺苷協助催化下可與DNA形成核絲,將雙股DNA解旋,改變其結構並完成重組。
本研究使用震盪式光鉗分別以固定拉力施與單分子DNA量測其彈力常數,此外也探討高濃度DNA溶液的黏彈性質對光鉗震盪頻率的變化。主要目的是利用上述兩種實驗平台,配合相關的物理模式,探討DNA與RecA蛋白質的交互作用。
在單分子實驗中,我們將雙股DNA (~ 10 kilo base-pair) 視為一條非線性彈簧,利用震盪式光鉗,量測固定施力 (25.5 pN) 下DNA的彈力常數 (~164.5 ± 16.4 pN/μm),發現其彈力常數不因震盪頻率 (1~1000 Hz) 上升而變化,我們加入RecA 蛋白與之作用後,在低頻 (1~100 Hz) 時彈力常數維持在約154.8 ± 8.5 pN/μm,因為結構改變當震盪頻率超過100 Hz (100 ~ 1000 Hz) 時DNA與RecA蛋白結合的分子彈力常數隨著頻率而上升。
其次,DNA溶液可視為一種高分子聚合溶液。我們測量DNA溶液在加入RecA蛋白之前,與加入RecA蛋白之後的黏彈性模數隨著光鉗震盪頻率的變化。彈性與黏性模數分析顯示作用後的DNA溶液偏向彈性溶液。我們量測與比較不同溶液: (a) DNA、(b) DNA與RecA蛋白、(c) DNA與RecA蛋白並含有ATPγs、(d) DNA與ATPγs、(e) DNA與變異RecA蛋白 (I26C) 並含有ATPγs等之實驗數據,經由彈性模數的冪次 (Power Law) 分析判別發現在 (c) 情況中的結果與其他結果有明顯差異,顯示唯有在ATPγs的媒介協助下RecA蛋白始能與DNA結合,並造成RecA-DNA溶液硬度上升。
Deoxyribonucleic acid (DNA) plays an important role in genetic coding in hereditary materials; it is compactly folded and packed in chromosome inside the cell nucleus. Among a wide variety of DNA-binding proteins, RecA protein plays a critical role in homologous recombination and in DNA sequence repair. In the presence of appropriate enzymes such as ATP or ATPγs, RecA can bind directly to dsDNA to form RecA-DNA nucleoprotein filaments.
In this research, we used oscillatory optical tweezers to study the interaction of DNA and RecA protein under two different conditions: (1) a single DNA segment stretched at constant stretching force by optical tweezers, and (2) an aggregate of DNA molecules in appropriate buffer solution.
In the single molecule study, the differential elastic constant of a segment of DNA (~ 10 kilo base-pair), stretched by oscillatory optical tweezers at a constant stretching force of 25.5 pN was measured as a function of oscillation frequency before and after its interaction with RecA. In the case of naked DNA (without RecA conjugation), the differential elastic constant was approximately ~164.5 ± 16.4 pN/μm, independent of the oscillation frequency, in the range of 1 to 1000 Hz. After interacting with RecA, the differential elastic constant of the RecA-DNA filament, measured under identical condition, increased from 154.8 ± 8.5 pN/μm at low oscillation frequency (~ 1 to 100 Hz), to 350 pN/μm at high oscillation frequency (100 ~ 1000 Hz).
In solution, DNA molecules become entangled and form a complex polymer matrix. In this case, we measured with oscillatory optical tweezers, the elastic modulus and viscous modulus as a function of the oscillation frequency, in the range of 1 to 1000 Hz, for the following cases: (a) DNA only, (b) DNA with RecA, (c) DNA with RecA and ATPγs, (d) DNA with ATPγs, (e) DNA with RecA mutant (I26C) and ATPγs. The results vary significant for case (c) compared with all the other cases. A simplified theoretical model based on power law analysis indicates that the stiffness of the solution in case (c) is much higher than that of all the other cases, implying the conjugation of RecA with DNA in the presence of ATPγs.
致謝 i
摘要 ii
目錄 vi
第一章 序論 - 1 -
1-1 微流變學 - 1 -
1-2 DNA的結構及其功能 - 3 -
1-3 RecA蛋白的結構與功能 - 4 -
1-3.1 RecA蛋白的功能 - 4 -
1-3.2 RecA蛋白的結構 - 5 -
1-3.3 RecA蛋白與DNA結合研究 - 6 -
1-4 光鉗簡介 - 6 -
1-4.1 光鉗的歷史 - 6 -
1-4.2 光鉗的原理 - 7 -
1-4.3 光鉗的力學量測 - 8 -
第二章 實驗架設、材料與方法 - 9 -
2-1 實驗架設 - 9 -
2-2 系統解析度 - 10 -
2-2.1 光耦合元件影像解析 - 10 -
2-2.2 壓電致動鏡移動解析 - 11 -
2-3 四象限位置感測器的基本原理 - 11 -
2-4 光鉗彈力係數測量 - 12 -
2-5 壓電致動反射鏡的響應頻率測量 - 13 -
第三章 實驗步驟 - 15 -
3-1震盪光鉗下光鉗彈力係數的測量 - 15 -
3-2 製備DNA黏接聚苯乙烯球 - 16 -
3-3 震盪光鉗下DNA分子彈力係數的測量 - 16 -
3-4 震盪光鉗下DNA與RecA作用之彈力常數的測量 - 18 -
3-5 DNA溶液彈性與黏滯模數隨著頻率變化測量 - 18 -
3-6 DNA與RecA蛋白作用之彈性與黏滯模數變化測量 - 20 -
第四章 實驗結果 - 21 -
4-1 震盪光鉗下光鉗彈力係數的測量比較 - 21 -
4-2 以震盪式光鉗測量DNA的彈力常數 - 21 -
4-3 DNA與RecA蛋白作用之彈力常數隨著頻率的變化 - 22 -
4-4 DNA溶液彈性與黏滯模數隨著頻率的變化 - 23 -
4-5 DNA溶液與RecA蛋白彈性與黏滯模數隨著頻率的變化 - 23 -
第五章 討論、結論與未來工作 - 25 -
5-1 單分子層級DNA震盪實驗 - 25 -
5-2 DNA溶液震盪實驗 - 25 -
5-3 結論 - 27 -
5-4 未來工作 - 27 -
第六章 參考文獻 - 29 -
附圖 - 33 -
圖1. 布朗運動示意圖 - 33 -
圖2. 雙股DNA的鹼基配對 - 34 -
圖3. RecA蛋白將單股DNA與雙股做序列交換 - 35 -
圖4. RecA-DNA核絲結構 - 36 -
圖5. 雷射通過粒子的受力圖 - 37 -
圖6. 光鉗彈簧示意圖 - 38 -
圖7. 光學架設圖 - 39 -
圖8. 倫奇刻線 - 40 -
圖9. 壓電致動鏡接收電壓對應光點移動距離 - 41 -
圖10. 四象限位置感測器結構圖 - 42 -
圖11. 小球位移與四象限位置感測器接收電壓關係圖 - 43 -
圖12. 藉由小球布朗運動得到光鉗的彈力 - 44 -
圖13. 改變雷射光功率對應光鉗彈性系數的變化。 - 45 -
圖14. 壓電致動鏡的頻率響應 - 46 -
圖15. 實驗樣本槽 - 47 -
圖16. 雙股DNA黏接聚苯乙烯大小球示意圖 - 48 -
圖17. 測量DNA彈性係數模型 - 49 -
圖18. 振幅與相位差的曲線配適 - 50 -
圖19. 不同方法測得光鉗彈力常數的比較 - 51 -
圖20. DNA受力與相對伸長關係圖 - 52 -
圖21. 不同震盪頻率下DNA彈力常數變化 - 53 -
圖22. Rec-DNA分子受力與相對伸長量的關係圖 - 54 -
圖23. 不同震盪頻率下RecA-DNA分子彈力常數變化 - 55 -
圖24. 不同DNA濃度下震盪頻率對應的小球振幅 - 56 -
圖25. 不同DNA濃度下震盪頻率對應的小球的相位差 - 57 -
圖26. 震盪頻率對應不同濃度DNA溶液彈性模數的變化 - 58 -
圖27. 震盪頻率對應不同濃度DNA溶液黏滯模數的變化 - 59 -
圖28. RecA-DNA溶液下震盪頻率對應的小球振幅 - 60 -
圖29. RecA-DNA溶液下震盪頻率對應的小球相位差 - 61 -
圖30. Rec-DNA溶液下震盪頻率對應的小球彈性模數的變化 - 62 -
圖31. Rec-DNA溶液下震盪頻率對應的小球黏滯模數的變化 - 63 -
圖32. RecA-DNA彈性模數的冪次分析 - 64 -
圖33. 蠕蟲鏈模型 - 65 -
圖34. 不同濃度DNA黏彈模數對應頻率的變化 - 66 -
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