以分子嵌合軟體輔助H1N1流感病毒DNA適體篩選後微小化之研究

經由系統配位子指數增益演繹程序(SELEX)篩選而得的適體長度通常介於60至120個鹼基之間，其中對標的物具有辨識能力的片段只占15至40個鹼基，此片段稱為微小適體。這些微小適體相較於全長適體除了具備更好的結合力和更快速的腎臟清除率之外，也大大降低了生產成本。本研究利用電腦模擬方法預測標的蛋白與其人工適體所形成之複合物構型，並進一步分析可能的微小適體序列。此微小適體預測程序應用於五條經實驗驗證的序列上，預測準確率可達0.92，顯示此程序在微小化適體研究上具有相當的潛力。本研究也利用相同程序針對自行篩選而得的H1N1流感病毒人工適體進行微小化，H1N1病毒中和試驗結果顯示，微小化適體之病毒中和能力相較於全長適體有顯著的提升，平均可達2.1倍，其中人工適體BR92在微小化後可使細胞存活率提高至98%，約為全長適體的3.4 倍。此外，分子嵌合結果顯示，利用唾液酸進行競爭式篩選所得的人工適體傾向結合於血球凝集素之第一結構域(HA1 domain)，酵素結合適體分析(enzyme-linked aptamer assay, ELAA)也證實其對血球凝集素重組蛋白的專一性。本研究所提出之電腦模擬微小化適體方法可用於預測可能的微小適體參考序列，未來透過更多適體資料的蒐集可進一步進行程序優化，提高預測準確度。

關鍵字

核酸適體；微小化；電腦模擬；分子嵌合； H1N1流感病毒

並列摘要

Aptamers screened out from SELEX (systematic evolution of ligands by exponential enrichment) are generally 60 to 120 bases in length, and only a fragment, called mini-aptamer, containing 15 to 40 bases contributes to the specific binding activity. In this study, structures of the aptamer-protein complexes were predicted, and mini-aptamers were identified from the predicted structures of complexes. The accuracy of prediction was as high as 0.92 when applying the pipeline to five verified aptamers. The result suggests that this pipeline is a potential tool for minimization of aptamers. The same pipeline was used to predict mini-aptamers from H1N1 aptamers obtained by viral SELEX. Compared to the full-length aptamers, mini-aptamers showed 2.1-fold improvement in viral neutralization test in average. Among these aptamers, viral neutralization test using truncated BR92 resulted in 98% of cell viability, which is 3.4-fold higher than full-length BR92. Furthermore, the docking results provided evidences that aptamers screened out by sialyllactose competitive selection tended to bind to the HA1 domain on hemagglutinin, and enzyme-linked aptamer assay (ELAA) also proved that these aptamers do specifically bind to hemagglutinin recombinant protein. In silico aptamer minimization pipeline proposed in this thesis provides a novel method to predict possible mini-aptamer sequences. Optimization of the pipeline can be done to enhance the prediction performer by introducing more aptamer database in the future.

並列關鍵字

aptamer ； minimization ； in silico ； molecule docking ； H1N1 virus

參考文獻

Amina, R., Cheng, Y., Akhtar, H., Jean-Louis, M., 2013. Aptamers: a promising tool for ochratoxin A detection in food analysis. Toxins 2013.

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以分子嵌合軟體輔助H1N1流感病毒DNA適體篩選後微小化之研究

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