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標題: | 基於表面電漿子共振生物感測器之適體結構最佳化工程應用於偵測轉移性癌症胞泌體整合素蛋白 Development of Metastatic Exosome Sensing Strategy via Surface Plasmon Resonance based Aptamer Structural Rationalization |
作者: | Hui-Wen Liu 劉惠文 |
指導教授: | 林啟萬(Chii-Wann Lin) |
關鍵字: | 表面電漿子共振,適體,胞泌體,整合素, Surface Plasmon Resonance,Aptamer,Exosome,Integrin, |
出版年 : | 2020 |
學位: | 碩士 |
摘要: | 近年來,癌症胞泌體(Exosome)被發現與癌症細胞轉移(Metastasis)有密切相關,其癌細胞在從原位癌轉移至其他器官之前,會先分泌帶有靶向性蛋白之胞泌體至欲轉移之器官建立腫瘤微環境(pre-metastatic niche),因此若是能在癌細胞轉移前早期發現,可望為更有效地癌症治療帶來一線生機。因此本研究欲利用結構最佳化的核酸適體(Aptamer)作為探針,結合高靈敏度且免標記的相位式表面電漿子共振感測器(Phase-sensitive Surface Plasmon Resonance Biosensor, pSPR biosensor)進行肺臟靶向性之乳癌細胞(4175-LuT)之胞泌體膜蛋白α6β4整合素蛋白(Integrin)之檢測。 一般核酸適體是經由SELEX(Systematic evolution of ligands by exponential enrichment)所選殖而得。然而經由SELEX選出的核酸適體存在著潛在的缺點:包含前後引子可能參與目標分子的結合反應或PCR步驟傾向排除二級結構導致擴增反應的偏誤等,導致該適體結構並非為最佳設計。另一方面,若是可以精簡化SELEX篩選出來的適體序列,即可根據目標情境的需求不同,進一步結合其他核酸適體序列或是加入人工序列,設計具有複合功能之適體。 為使適體結構最佳化,我們使用核酸二級結構模擬工具及序列相似度分析工具,推測出合理之適體共識區域後進行裁切或是延長數個鹼基以維持重要二級結構,設計出21個鹼基的α6β4適體IDA3t,並經由SPR感測器即時檢測核酸適體與目標分子的結合情形,同時在掃描式電子顯微鏡底下觀察其形貌及進行顆粒數統計。另外,我們進一步設計了一個含有一段人工序列的適體IDAχ,並透過實驗證明其仍保有對目標分子的專一性。此人工適體預期可以在捕捉目標分子之後進一步釋放與觸發核酸雜合連鎖反應(Hybridized Chain Reaction, HCR)有關的序列,達到放大訊號的功能,以期未來可以達到在血液樣品當中專一性檢測的目標。 Recently, tumor exosome is discovered to play a vital role in cancer metastasis process, that the integrins on the surface of tumor exosome would determine organtropic metastasis. Tumor-derived exosome would be uptaken by organ-specific cells and prepare the pre-metastatic niche before metastasis happened. If we can detect these exosomes in advance, it’s expected to bring other possible approaches to cancer treatment. In this work, we demonstrate the development of metastatic exosome sensing strategies using Phase-sensitive Surface Plasmon Resonance (pSPR) as a platform, which is highly sensitive and label-free, for aptamer structural rationalization. 4175 lung-tropic tumor cell derive exosome (4175-LuT) is studied with surface integrin α6β4 as targeted surface marker. Traditionally, aptamers are made by SELEX(Systematic evolution of ligands by exponential enrichment). However, there’s some sequence-structural bias with it. For instance, the primers might also attend in the binding with target molecules, or PCR (Polymerase chain reaction) is preferred to amplify the DNA sequence that is lack of secondary structure, while aptamers usually bind with target molecules via it. On the other hand, we can design a bifunctional aptamer based on structural rationalization to meet the need of sensing different target molecules. In order to optimize the structure of α6β4 aptamer made by SELEX, we use secondary structure simulation tools (NUPACK, Mfold) and similarity analysis tool (Lalign) to analyze the structure of aptamer, the optimized α6β4 aptamer named IDA3t. We confirm the binding affinity between optimized aptamer and target molecule by pSPR biosensor. Also, we observe the exosome morphology through Scanning Electron Microscopy (SEM), do particle size analysis and particle number statistics through the images. Furthermore, we design an artificial aptamer here, named IDAχ, which is combined with 21 mer IDA3t and 22 mer designed sequence, and we demonstrate that it can capture 4175-LuT exosome still. This “IDAχ” aptamer has tailor made hairpin secondary structure that can be developed into more elaborated molecular machinery such as hybridization chain reaction, for further application of detecting target molecules with clinical samples. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17232 |
DOI: | 10.6342/NTU202002559 |
全文授權: | 未授權 |
顯示於系所單位: | 醫學工程學研究所 |
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