利用結合位特異性SELEX法篩選具抑制流感凝集素功能之DNA適體

Abstract

如何篩選具有功能性之核酸適體是目前相關領域研究中一個重要的課題，本研究即提出一種新穎的核酸適體篩選方法，利用競爭標的蛋白之功能性結合位方式篩選核酸適體，以此獲得具功能性之核酸適體，故此法稱為結合位特異性系統配位子指數增益演繹程序(ES-SELEX)。本研究以人類流感(A/New Caledonia/20/99)血液凝集素做為標的蛋白，並利用前述方法篩選具有抑制血液凝集素功能之核酸適體。經過九個回合篩選後，可獲得對血液凝集素高度親和性之核酸適體，其平均解離常數可達1.2 nM，其中編號CP9P536之核酸適體被確認具有血液凝集素抑制功能，該核酸適體於62.5至1000 nM濃度中可完全抑制血液凝集素活性。除此之外，在免疫沉澱試驗中亦驗證該核酸適體能於人類血清及流感抗原中辨識流感血液凝集素，顯現該核酸適體除具高度親和力外並具專一性。在核酸適體結構上，利用圓二色性分析推測CP9P536核酸適體的結構屬於平行鳥糞嘌呤四面體之複合結構，同時該核酸適體在攝氏4度至37度間結構相當穩定。由實驗結果可以發現，本研究所提出之結合位特異性核酸適體篩選方法，不但可獲得高度專一性之核酸適體，更可以直接且快速獲得具有預期功能之核酸適體，並且由本方法所篩選出之核酸適體具有高度結構穩定之特性，可供未來進一步發展相關流感抑制藥物之應用。

Antagonistic aptamer selection is an important issue in the field of aptamer study. In this thesis, a novel methodology of aptamer selection is designed and proposed. This method is called epitope-specific SELEX (systematic evolution of ligands by exponential enrichment), in which antagonistic aptamers are obtained by the SELEX with competition for the receptor-binding epitope on a target protein. Hemagglutinin of human influenza A/New Caledonia/20/99 is chosen as a model to demonstrate this strategy. After nine SELEX rounds, anti-hemagglutinin aptamers with high affinity are obtained, and the mean dissociation constant of the aptamer pool is as low as 1.2 nM. In the aptamer pool, aptamer CP9P536 shows high antagonistic activity against hemagglutinin. It can inhibit the agglutination activity of hemagglutinin with the concentration range from 62.5 to 1000 nM. Furthermore, the specificity of this aptamer is identified by the immunoprecipitation assay, which shows that the aptamer can recognize hemagglutinin in human serum and viral antigen. Also, the structure of CP9P536 is characterized by circular dichroism. It is supposed that the structure of CP9P536 is a parallel G-quadruplex structure. Moreover, CP9P536 is structurally stable between 4 to 37°C. As a result, it is considered that ES-SELEX can effectively screen the aptamer with high affinity and desired functionality. Moreover, the aptamer identified and characterized in this study is structurally stable for clinical applications. It has potential for DNA-based anti-influenza drug development.