利用布朗動態法研究基材形貌對DNA在脂雙層上擴散及伸展行為之影響

Abstract

本實驗室先前開發出新型的DNA基因圖譜分析平台，其原理是在具有週期性溝槽結構之基材上鋪設帶正電脂雙層後，將帶負電的DNA吸附於脂雙層上，利用DNA會聚集在基材溝槽的正曲率處進行自發性伸展的特性，使DNA上的特定序列能被精確標定，從而產生基因圖譜。為了將本平台應用在醫療檢測上，我們以優化此檢測平台為目標，然而受限於實驗上觀察方法的限制，許多現象的背後機制難以驗證，因此本研究利用布朗動態法(Browian dynamics,BD)來模擬並進一步解析DNA於脂雙層上之特殊行為背後的基本原理。本研究之重點有二：(1)探討基材表面的正曲率處所引發的靜電位能井對DNA的擴散行為及型態之影響，(2)重現DNA吸附於脂雙層後擴散至正曲率面進行自發性伸展的過程。 在先前的實驗研究中發現，當DNA吸附於脂雙層上，會在短延遲時間下出現次擴散行為，代表DNA的運動行為因為某種不明原因而被侷限；而我們的研究團隊已經證明在基材溝槽的正曲率處具有一靜電位能井，使DNA可以感受到更低的靜電位能而聚集。故我們推測實驗上所使用的基材表面並不平整且具有局部的正曲率，使DNA鏈段可能會被侷限於上述靜電位能井中並造成次擴散現象。 為了驗證我們的假設，我們分別建立了凸結構和凹結構這兩種基材結構來模擬實驗上具有突起和凹陷的基材表面形貌。我們的模擬結果顯示，當DNA吸附於脂雙層後，其鏈段的確會被侷限於具有靜電位能井的正曲率處，使DNA的運動行為在短延遲時間下呈現次擴散，並在長延遲時間下回到正規擴散。為了進一步確認靜電位能井對DNA擴散行為的影響，我們使用了三個參數來增加靜電位能井的深度，也就是增加基材曲率、降低離子強度和增加脂雙層正電荷濃度。模擬結果顯示，當靜電位能井的深度較深時，不僅DNA的環動半徑較小，次擴散行為也更嚴重，與實驗上觀察到的趨勢一致，證明DNA的次擴散現象確實是由位能井所引起的。 對於DNA在溝槽的正曲率處進行自發性伸展的現象，先前我們的研究團隊已經分別透過實驗和模擬探討了其背後的基本原理，而在本研究中我們改良了原先的模擬系統，重現了實驗上DNA吸附於脂雙層後擴散至具有正曲率的彎曲面進行自發性伸展的過程，並探討了彎曲面的曲率大小對DNA伸展率的影響，模擬結果顯示當DNA達到高度伸展態時(平均伸展率大於80%)，繼續增加基材曲率對DNA的平均伸展率的影響不大。

Our lab has previously developed a new DNA gene mapping platform utilizing the phenomenon that negatively charged DNA can adsorb on and spontaneously extend along the grooves on a glass substrate covered with cationic lipid bilayers. In order to apply this platform to practical medical diagnostics, we aim at optimizing this mapping platform. However, it is difficult to understand the mechanism behind the unexpected DNA behaviors due to the limitations of experimental observation methods. Therefore, we use Brownian dynamics (BD) to simulate and investigate the unexpected behavior of DNA adsorbed on lipid bilayers. This research focuses on two main topics: (1) To investigate how the diffusion behavior and conformation of DNA affected by the electrostatic potential well induced by the positive curvature of the substrate . (2) To reproduce the phenomenon that DNA adsorb on lipid bilayers and spontaneously extend along the places with positive curvature. Recent studies have reported that DNA adsorbed on cationic lipid bilayers exhibit an unexpected sub-diffusion behavior, but the cause of the phenomenon remains unclear. Previously our lab has also found that DNA adsorb on cationic lipid bilayers and aggregate spontaneously at the places with positive curvature where exists an electrostatic potential wells. Therefore, we speculated that these topology-induced potential wells are also the origin of the DNA sub-diffusion. To verify our speculation, we constructed in our simulations a substrate consisting of cavity and protrusion structures to represent the topology observed in experiments. The simulation results showed that those places with positive curvature do restrict the motion of DNA and induce sub-diffusion behavior at short delay time while the normal diffusion is recovered at long delay time. In addition, under the conditions of deeper potential wells: higher substrate curvature, lower ionic strength, and higher cationic lipid concentration, the radius of gyration of DNA decreases and the sub-diffusion behavior becomes more significant, also in accordance with our observation in experiments. Therefore, the simulation results support our speculation that those topology-induced potential wells do cause DNA sub-diffusion. In the second part of this study, we improved our simulation model with a substrate of more realistic topology and successfully reproduced the process of DNA adsorption on lipid bilayers and its spontaneous extension along the places with positive curvature. Furthermore, we also simulated the relationship between the substrate curvature and the degree of DNA extension. We found that when DNA reaches a highly stretched state (the average degree of extension is greater than 80%), increasing the substrate curvature has weak effect on DNA extension.