臺灣博碩士論文加值系統

目前有許多關於DNA吸附在脂雙層上的行為的實驗，但受限於實驗上觀察手段的限制，許多現象的背後機制難以驗證，只能利用模擬來加以解析。本研究利用布朗動態法(Browian dynamics,BD) 模擬DNA吸附於帶正電荷的脂雙層上之行為，並探討兩種實驗觀察的成因:(1)DNA為何能在具有正曲率的脂雙層上自發性展開，(2)脂雙層上若出現物理性障礙是否會導致DNA的次擴散及蜷縮行為。
本實驗室先前利用DNA在脂雙層上自發性延展的行為開發出新型DNA基因圖譜分析平台，其原理為在具有週期性的溝槽之基材上鋪設帶正電脂雙層，DNA會先吸附在脂雙層上並藉由擴散至溝槽，並於溝槽具有正曲率處自發性延伸呈一直線，使我們能夠標定DNA上特定序列並產生基因圖譜。對於DNA沿著溝槽彎曲處自發性延伸機制，推測是因為在溝槽彎曲處存在一靜電位能井，而產生位能井的原因有兩個可能: (a)截圓錐型的帶正電脂質傾向聚集在溝槽彎曲處(立體效應)，(b)DNA在溝槽彎曲處能感應到較多正電荷(幾何效應)。我們的模擬結果顯示，當DNA尚未吸附於脂雙層上時，脂質在彎曲處的分佈確實會受到其構型及基材曲率的影響，截圓錐型之脂質傾向聚集具有正曲率的彎曲處，而構形為倒截圓錐型則相反。當基材曲率越高時，脂質構型對於基材彎曲處的脂質分佈影響會越明顯。而關於幾何效應，我們的模擬結果則顯示帶電粒子在彎曲處所具有的靜電位能的確較在平面處低，而當基材彎曲處曲率越高時，其平面處與彎曲處的靜電位能差會越大，幾何效應越大。
接著我們改變基材曲率和脂質構型來觀察對DNA伸展率的影響。DNA的伸展率明顯隨基材曲率上升而增加。但脂質構型的改變，在不同曲率下，均對DNA伸展率影響不大。因此我們得知基材形狀的幾何效應較脂質構型的立體效應對DNA自發性展開的現象具有較強的影響力。而模擬中也發現不論是改變基材曲率或脂質構型皆會造成DNA在表面彎曲處濃度分佈的變化，因此可以佐證DNA自發性伸展機制的確是由幾何效應與立體效應所造成。
而當DNA於平面脂雙層上擴散時，實驗上觀察到DNA於短延遲時間下會出現次擴散(sub-diffusion)行為，當基材表面粗糙度增加時，不但次擴散現象更加嚴重，甚至連DNA型態也會改變。為了瞭解基材表面的突起結構是否阻擋DNA的擴散並影響DNA的型態，我們也運用模擬來解析物理性的障礙對吸附在脂雙層上DNA的行為影響。我們在二維脂雙層模型中加入物理性障礙，當系統中有物理性障礙時，DNA擴散行為會在短延遲時間下出現次擴散(sub-diffusion)的行為，而長延遲時間下回到正規擴散( normal diffusion )，但DNA的型態並沒有太大變化，因此推斷物理性障礙的確會造成DNA在短延遲時間下造成次擴散行為，而在實驗上所觀察到的DNA構型變化，應該來自其他原因，如前述由表面曲率所造成的位能井。

There are many experiments investigating the behavior of DNA adsorbed on lipid bilayers. Although many intriguing phenomena have been reported, the underlying mechanisms are difficult to understand and can only be verify by simulation. In this study, we used Brownian dynamics (BD) to simulate the behavior of DNA adsorbed on a positively charged lipid bilayer, and explored the causes of two experimental observations: (1) why DNA can spontaneously extend along the region with positive curvature, (2) whether physical obstacles on the lipid bilayer can cause the sub-diffusion and the collapse of DNA.
Our lab has previously developed a DNA gene mapping analysis platform utilizing the phenomenon that DNA can spontaneously extend along the grooves on glass surface covered with cationic lipid bilayers. More precisely, DNA can spontaneously extend along the place where the curvature is positive. The mechanism for the spontaneous extension of DNA is presumed to be due to the presence of an electrostatic potential energy well for DNA at the bend of the grooves, and there are two possible sources of the electrostatic potential well: (a) the positively charged lipids with truncated cone shape tend to accumulate at place with positive curvature (steric effect), (b) DNA can interact with more positively charged lipids at the bend of the groove (geometric effect). Our simulation results show that both the curvature of the substrate and the shape of lipids indeed affect the lipid distribution in the lipid bilayer. Lipids with truncated cone shape tend to concentrate at bends with positive curvatures while lipids with inverted truncated cone shape tend to become more dilute. The higher the curvature of the substrate, the more obvious the effect of lipid shape on the lipid distribution at the bend of the substrate. Regarding to the geometric effects, our simulation results show that the electrostatic potential of a negatively charged particle at the bend is indeed lower than that at the plane and the electrostatic potential well becomes more prominent as the curvature increases.
We also examined how lipid shape and substrate curvature affect the degree of extension of DNA. We found that the degree of extension of DNA increases with the curvature of the substrate. However, changes in lipid shape have little effect on DNA extension. Therefore, our results suggest that the geometric effect of the substrate shape has stronger influence on the spontaneous extension of DNA than the steric effect of lipid shape. We also found that the probability distribution of DNA segment at the bend will be affected by both lipid shape and substrate curvature, another evidence supporting that the spontaneous extension of DNA is indeed caused by geometric effect and steric effect.
When DNA diffuses on a lipid bilayer set on a planar substrate, it is observed that DNA exhibits sub-diffusion behavior in a short delay time. As the surface roughness of the substrate increases, the sub-diffusion behavior also becomes more prominent. Moreover, DNA conformation may also change from an unraveled one to a collapsed one. In order to understand whether the protrusion structure on the surface of the substrate can hinder the diffusion of DNA and affect the conformation of DNA, we used our simulation to investigate the effect of physical obstacles on the behavior of DNA. It is found that the physical obstacles indeed induce DNA sub-diffusion at short delay time while the normal diffusion is recovered at long delay time. However, the physical obstacles show no effects on DNA conformation. Therefore, we conclude that DNA conformation change observed in experiments is more likely due to other reasons, such as the aforementioned electrostatic potential well caused by surface curvature.

致謝 i
摘要 ii
Abstract iv
目錄 vii
圖目錄 x
表目錄 xviii
符號表 xix
第1章 緒論 1
1.1 前言 1
1.2 研究動機與目的 2
第2章 文獻回顧 4
2.1 DNA的物理性質 4
2.1.1 去氧核糖核苷酸(DNA) 4
2.1.2 堅韌長度(Persistence length) 4
2.1.3 輪廓長度(Contour length, Lc) 5
2.1.4 擴散係數(Diffusivity) 5
2.1.5 迴轉鬆弛時間(Rotational relaxation time , τR) 6
2.2 DNA模型 7
2.2.1 Bead-spring model 9
2.3 脂質 12
2.3.1 脂質結構 12
2.3.2 脂質自組裝 13
2.3.3 脂質於彎曲表面排序(Lipid Sorting) 15
2.3.4 DNA在脂雙層上的行為 17
2.4 脂質模型 20
2.4.1 改變親水頭基有效大小達到不同脂質構型 20
2.4.2 脂質疏水尾基之間作用力 22
2.5 直線線性分析法(Direct Linear Analysis,DLA) 25
2.6 DNA自發性延伸於脂雙層上 26
2.7 本實驗室研究現況 28
2.7.1 DNA自發性延伸平台 28
2.7.2 位能井 32
2.7.3 DNA在脂雙層表面上的擴散行為及環動半徑變化 34
2.7.4 模擬DNA在脂雙層上的擴散行為 35
2.7.5 以AFM量測基材表面 38
2.8 本研究模擬策略之設計 39
第3章 模擬方法 40
3.1 布朗動態法(BD) 40
3.2 本研究脂質模型 43
3.2.1 Lipid本身的交互作用力 44
3.3 脂質與基材間的作用力 49
3.4 本研究DNA模型 49
3.5 週期性邊界條件(Period boundary conditions) 49
3.6 模擬參數 50
3.7 基材設計 51
第4章 結果與討論 53
4.1 影響脂質在彎曲處(正曲率)分佈因素 53
4.1.1 脂質分佈受脂質密度影響 54
4.1.2 脂質分佈受脂質構型影響 57
4.1.3 脂質分佈受靜電作用力影響 58
4.1.4 脂質分佈受到基材曲率影響 60
4.1.5 對於脂質分佈總結 61
4.2 幾何效應 62
4.3 研究DNA吸附在脂質後其伸展率及脂質濃度變化 66
4.3.1 DNA於平面上的伸展率 67
4.3.2 DNA 初始位置 68
4.3.3 基材彎曲處曲率為0.2時DNA伸展率及脂質濃度變化 69
4.3.4 基材彎曲處曲率為0.1時DNA伸展率及脂質濃度變化 72
4.3.5 基材彎曲處曲率為0.05時DNA伸展率及脂質濃度變化 75
4.3.6 DNA侷限行為: 78
4.3.7 比較實驗上與模擬上DNA伸展率 80
4.4 物理性障礙對DNA擴散係數的影響 82
4.4.1 物理性障礙有序排列對於DNA運動之影響 84
4.4.2 物理性障礙隨機排列對於DNA運動之影響 87
4.4.3 比較兩種物理性障礙排列方式 90
4.4.4 與實驗結果做比較 91
第5章 結論 92
第6章 參考文獻 94

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