DNA 於脂雙層上自發伸展機制及侷限行為之研究

Abstract

我們開發出具有低成本、操作容易等優勢的新型DNA基因圖譜分析平台，其原理為在具週期性的溝槽結構之基材上鋪設帶正電之脂雙層，使帶負電的DNA吸附於脂雙層上，並於自發性地於溝槽夾角之根部伸長呈一直線，使我們能夠直接標定DNA上的特定序列。我們已經在此平台上成功地使用缺口標定法產生基因圖譜。 為了能進一步提升對此系統之瞭解與掌控，我們希望能了解DNA沿著溝槽夾角根部自發拉伸之機制。DNA能自發拉伸顯示在夾角處存在一位能井，此位能井來自於DNA和正電脂質之靜電作用力，其可能成因有二: (1)正電脂質因立體效應而在夾角處聚集；(2)幾何效應的影響，即DNA在彎曲處能感應到較多正電荷。為了探討此一機制，我們使用三組不同脂質進行拉伸實驗: (A)DOPC/DOTAP，(B)DOPC/EPC，(C)DOPE/EPC。實驗結果顯示，立體效應與幾何效應的影響同時存在，在(A)、(B)組中帶正電脂質與電中性脂質親水端差距不大的情形下，幾何效應使DNA在帶有低正電比率的脂雙層上即可得到高DNA拉伸率；但在(C)組親水端差距較大的情形下，立體效應的影響使得即使在高正電比率的脂雙層上，DNA拉伸率仍然不高。 由於幾何效應的影響在文獻中未被提到，因此我們以Possion equation推導DNA在側壁夾角處的靜電位能，發現由幾何效應所產生的電位能差足夠提供DNA拉伸時的商損失。另外，我們也從理論上估計在(A)(B)(C)中正電脂質在側壁夾角處與平面處之分布情形，發現由脂質形狀所引發的濃度差異雖不大，但結合幾何效應的影響後，已足夠左右DNA的拉伸型態，這也解釋了我們在實驗中觀察到的現象。 最後我們希望以原子力顯微鏡量測DNA在側壁夾角拉伸的侷限寬度，並與de Gennes 和Odijk的理論比較。然而疏水端含雙鍵的脂雙層在液相時橫向擾動太快，使DNA無法以一般頻率的探針測得，對此我們希望藉由更換脂質，使脂雙層在常溫下為膠凝相，並能固定住DNA，此實驗仍有待進一步之研究。

We have recently developed a DNA optical gene mapping platform. The working principle of the platform relies on the phenomenon that DNA can be adsorbed and spontaneously extended along a groove covered with cationic lipid bilayers. The physical gene map can then be readily obtained using nick-translating method. This new platform has great potential because of its low cost and easy operation; however, the reason why DNA can spontaneously extend along the grooves is not clear and it has to be investigated in order to optimize the platform. Since DNA extends only along the grooves, it suggests there exists an electrostatic energy well for DNA. Two possible sources of this well are postulated: (1) the steric effect drives cationic lipids to aggregate on curved surface, (2) the geometry effect allows DNA to interact with more cationic lipids on the curved surface. In order to examine our postulations, we observed DNA behavior on three sets of lipid bilayers (A) DOPC/DOTAP, (B) DOPC/EPC and (C) DOPE/EPC, in which the ratio of the area of the headgroup to that of the tail varies gradually from larger than one to smaller than one. The experimental results show both the streic and the geometry effect exist. However, the geometry effect is always in favor of DNA extension while the steric effect can either enhance or undermine the phenomenon. We have also investigated the effect of the concentration of the positively charged lipids and found higher concentration always help DNA extend. In order to quantify the geometry effect, we have derived the electrostatic potential of DNA on curved surface and found the theoretical prediction is in quantitative agreement with the experimental results. We have also calculated lipid distribution due to steric effect and found that a modest variation of lipid concentration in conjunction with the geometry effect can lead to drastic change on DNA extension. Last but not least, we want to measure the width of the DNA confinement using atomic force microscopy and compare our results with the prediction between DNA extension and the confinement width proposed by de Gennes and Odijk. However, since the lateral diffusion of the lipid bilayer is too fast, we cannot find DNA on lipid bilayer using normal AFM probes. We plan to solve this problem in the future by employing lipids with transition temperatures higher than the room temperature so that lipid bilayer can remain in gel phase during experiments.