四重摺疊結構之核酸適體抑制登革熱病毒研究

DNA aptamer forms a G-quadruplex can neutralize all four serotypes of dengue viruses

蕭雯心(Wen-Hsin Hsiao)

指導教授 ： 程家維

國立清華大學/生命科學院/生物科技研究所/碩士(2011年)

摘要

Flavivirus family includes Yellow Fever virus (YFV), Dengue virus (DENV), Japanese Encephalitis virus (JEV), Tick-borne Encephalitis (TBEV), and West Nile virus (WNV) that are are very important arthropod-born virus(mosquital or tick) associated with human diseases. Dengue fever is caused by dengue virus (DENV), which is primarily transmitted by Aedes albopictus and Aedes aegypt. There are four serotypes of virus, termed DENV-1, DENV-2, DENV3, DENV-4. World Health Organization estimated that over 50-100 million DENV infections occur globally each year. And there is still no specific drug and vaccine available for treatment and prevention. Previous studies suggested that envelope proteins are present on viral surface and are the dominant antigen, particularly domain III. Domain III plays the most important role in viral attachment and fusion with the target cell. Therefore, many studies have used domain III as a target for drug development. Our lab has used SELEX technology to create an aptamer (S15) forms a quadruplex structure and targets an unique epitope of four subtypes of DENV. In this study, we created a new aptamer (S15G3), which has similar structure as S15. Using Circular Dichroism and Fluorescence Spectrometer, we confirmed that S15G3 still have affinity to domain III. Furthermore, we identified the critical region for the binding to domain III by the deletion of nucleotides at the terminal and loop. Our results provided a shorter sequence (S15G3L123A) to make it easier for structure calculation, and it would have more potential to be a therapeutic drug than its parental aptamer.

關鍵字

登革熱病毒

並列摘要

黃熱病毒家族包含黃熱病毒(YFV)，登革熱病毒(DENV)，日本腦炎病毒 (JEV)，蜱媒腦炎病毒(TBEV)，西尼羅病毒(WNV)，都是非常重要的節肢動物傳 染病毒，黃熱病毒被視為造成人類致病率與死亡率的重要病原體。其中登革熱是 由登革熱病毒(DENV)所引起，藉由埃及斑紋以及白線斑蚊作為傳播媒介，依病 毒血清型不同可分為四型 : DENV-1, DENV-2, DENV-3, DENV-4。世界衛生組織 (The World Health Organization)估計每年約會出現五千萬到一億個登革熱(Dengue fever)感染病例，目前沒有特殊的藥物治療和疫苗可用。許多研究認為登革熱病 毒外層覆蓋的封套蛋白(Envelope protein)是一主要抗原，特別是封套蛋白中的第三 區域(domain III)，在病毒辨識宿主受體與發生細胞融合過程中扮演重要的角色。 因此，許多研究利用domainIII作為標的物進行藥物的開發。我們實驗室利用了 SELEX技術篩選找到能與DENV-2 ED3親和性鍵結的核酸適體(S15)，結構為鳥嘌 呤-四重摺疊結構(G-quadruplex structure)，並且，發現S15與domainIII鍵結的區域在 四種不同血清型登革熱病毒中，有著高度相似性。於此次研究中，我們設計了一 個衍生自S15的新適體(S15G3)，S15G3與S15同樣為鳥嘌呤-四重摺疊結構，也能親 和性鍵結DENV-2 ED3。接著，進而研究S15G3序列中的Terminal與loops分別對於 親和性鍵結的影響，目的是想盡可能縮短適體(aptamer)的序列長度，降低合成所 需成本。研究結果顯示，terminal會影響親合性鍵結，而loops則不會影響鍵結，所以，我們將S15G3序列中的loops都縮短為A(Adnine, 腺嘌呤)，命名為: S15G3L123A，此較短序列的適體除了能有利於結構的分析之外，也更具潛力成 為抑制登革熱病毒感染的有效藥物。

並列關鍵字

Dengue virus

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