DNA 於脂雙層上擴散行為受離子強度與膜電荷密度影響之研究

Abstract

本研究以控制正電荷脂質濃度與離子強度改變DNA與脂雙層之靜電吸引力，並觀察在不同狀況下DNA在脂雙層上擴散之形態、擴散係數以及纏繞半徑。當帶負電的DNA吸附在鋪設於玻璃基材上的帶正電脂雙層，由於脂雙層本身的流動性讓DNA得以在二維平面上產生擴散行為。有研究觀察到DNA在二維平面上的擴散行為符合勞斯模型(Rouse model, D~1/N)，且纏繞半徑也符合高分子自迴避隨機模型(self avoiding random walk, <R^2>~N^2v)的預測，但至今仍沒有一個研究深入探討DNA在帶正電脂雙層上的擴散機制。 本研究利用螢光顯微鏡來追蹤DNA於二維平面上的擴散狀況以及DNA之纏繞半徑。透過影像分析DNA之質心位置隨時間之變化，我們發現DNA在脂雙層上在長延遲時間下為簡單擴散(simple diffusion)，但短延遲時間中卻為次擴散(sub-diffusion)。在實驗影像中，DNA在脂雙層上有片段受到侷限的情形，我們稱為準黏著點(quasi-sticky point)。DNA片段並不會完全固定於準黏著點上，而是可以在其上滑動，並且有一定的機率掙脫。這些侷限使DNA在短延遲時間時產生次擴散的現象，並且靜電吸引力之上升會增加這些侷限對DNA之影響。同時我們發現DNA之擴散係數與正電荷脂質濃度有高度相關性；除此之外，在高正電荷脂質濃度以及低離子強度的條件下，DNA甚至會出現吸附後無法展開的現象。我們認為這些侷限的出現是因為基材表面在分子層面仍是不平坦的，使脂雙層表面存在著不平整，對DNA來說這些不平坦之凹陷處可視為靜電位能井，而這些位能井的生成可能是因為以下兩點：(1) DNA在凹陷處能夠與正電荷脂質有更強的靜電作用；(2)正電荷脂質(DOTAP+)之親水端較中性脂質(DOPC)之親水端小，因此較易聚集於凹陷處形成位能井。 為了驗證此位能井假設，我們使用另一種親水端較大的正電荷脂質(EPC+)進行實驗；較大的親水端會使脂質分子在凹陷處較不易聚集，使位能井深度下降，因此對DNA之侷限減弱，同時使DNA之擴散係數上升以及延後DNA無法展開之現象。此實驗之結果如同我們所預期，DNA在EPC+/DOPC脂雙層上之擴散係數確實比DOTAP+/DOPC脂雙層上之擴散係數高，DNA無法展開的現象則會出現在更高濃度的狀況。實驗的結果的確支持我們所提出的位能井假設。

In this study, we investigated the behavior of DNA on supported lipid bilayers with different surface charge density and under different ionic strength. When the negatively charged DNA is adsorbed on the positively charged lipid bilayers deposited on the glass substrate, the fluidity of the lipids allows DNA to diffuse in a two-dimensional manner. It has been observed that the diffusion behavior of DNA on a two-dimensional plane is consistent with the Rouse model (D~1/N), and its radius of gyration is also consistent with the prediction given by the self avoiding random walk (<R^2>~N^2v). However, there still lacks indepth study on the diffusion mechanism of DNA on the positively charged lipid membrane. We used fluorescence microscope to track the diffusion of DNA and the radius of gyration on a two-dimensional plane. By image analysis of the position of the centroid of DNA over time, we found that DNA conducts simple diffusion over a long delay time on lipid bilayers, but sub-diffusion over a short delay time. From experimental images, we found that a DNA often has few fragments confined at fixed points, called the sticky points. DNA fragment is not completely fixed on a sticky point, but can slide through and has a certain chance to break free. These sticky points cause sub-diffusion of DNA over a short delay time, and the increase in electrostatic attraction amplifies the impact of these sticky points. At the same time, we found that the diffusion coefficient of DNA is highly correlated with the concentration of positively charged lipids. In addition, under the conditions of high positive charge lipid concentration and low ionic strength, DNA may not be able to expand after adsorption on the bilayers. We believe that these sticky points appear because the surface of the substrate is uneven at the molecular level, which causes unevenness on the surface of the lipid bilayer. For DNA, the concaved places on lipid bilayers can be regarded as electrostatic potential wells. The origin of these energy wells may be due to the following two reasons: (1) DNA located at concaved places can have a stronger electrostatic interaction with positively charged lipids due to the shape of the surface. (2) The size of hydrophilic end of positively charged lipid (DOTAP+) is smaller than that of neutral lipid (DOPC). Therefore, positively charged lipids are prone to gather in the concaved places to form potential energy wells. In order to verify this potential well hypothesis, we experimented with another positively charged lipid with a larger hydrophilic end (EPC+). The larger hydrophilic end makes EPC+ less likely to aggregate in the concaved places, reducing the depth of energy wells. Therefore, the effect of sticky points to DNA is expected to be weakened. As a result, the diffusion coefficient of DNA is expected to increase and the phenomenon that DNA cannot be unfolded is expected to happen only at higher EPC+ concentration. In omparison with the experimental results, the diffusion coefficient of DNA on the EPC+/DOPC lipid bilayer is indeed higher than that on the DOTAP+/DOPC lipid bilayer, and the phenomenon that DNA cannot be unfolded occurs at a higher concentration. The agreement between our expectation and experiments supports the potential well hypothesis.