阮文滔黃太煌Wen-Tau JuanTai-Huang Huang許又仁You-Ren Hsu2019-09-052013-8-232019-09-052011http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=id=%22GN0697410133%22.&%22.id.&http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/102723SARS-CoV核殼鞘蛋白(nucleocapsid protein)與其病毒RNA結合形成核殼體(ribonucleocapsid)是包裹形成病毒顆粒必要的過程。近來SARS-CoV核殼鞘蛋白的研究顯示其蛋白質的結構靠近N端與C端個別有一個具有結構的區塊(NTD與CTD)，且兩者皆能與RNA與DNA結合。在這個過程涵蓋了複雜的蛋白質-核酸交互作用，以及蛋白質之間彼此的交互作用。在傳統的生物化學技術如Electrophoretic Mobility Shift Assay (EMSA)應用於研究蛋白質-核酸交互作用有其技術性的限制，難以直接性的區別CTD與NTD與核酸結合過程中所扮演的腳色的差異。 這篇論文的目標是應用單分子技術構建一個可直接觀察SARS-CoV CTD與NTD個別與單一核酸分子產生交互作用過程的實驗系統。我們透過螢光染劑YOYO-1和DNA結合並接著在經聚乙二醇(PEG)改質的玻璃上，並透過Alexa594和蛋白質結合，構築螢光影像。使得實驗過程可透過兩種螢光的受激發後放光的波長不同，在不同的濾鏡頻道下分別觀察DNA與蛋白質產生交互作用時的影像。 實驗的結果意外的發現SARS-CoV CTD在低濃度的環境下，SARS-CoV CTD會產生聚合(Aggregation)的現象，使得少量SARS-CoV CTD以Oligomer形式存在；而SARS-CoV NTD在相同的濃度條件卻並不產生聚合的現象。而在蛋白質與核酸交互作用的實驗當中，我們觀測到SARS-CoV CTD與DNA之間的親和力(affinity)大於SARS-CoV NTD與DNA之間的親和力與相關文獻的EMSA結果一致。透過此實驗系統的建構，可進一步應用於了解核殼鞘蛋白(NP)-RNA交互作用與SARS-CoV RNA的包裹機制。SARS-CoV nucleocapsid protein (N protein) binds to SARS-CoV RNA to form the viral ribonucleocapsid which is essential for packaging of the virus particle. Recently, SARS-CoV N protein has been shown to compose of two structured domains, the N-terminal domain (NTD) and the C-terminal domain (CTD). Both domains have been shown to be able to bind RNA and DNA. Formation of the nucleocapsid involves complex protein-nucleic acid and protein-protein interactions. The traditional biochemical technique used to study protein-nucleic acid interaction such as Electrophoretic Mobility Shift Assay (EMSA) has its limitation and is hard to differentiate the roles of the two domains of SARS-CoV N protein in the N protein-nucleic acids binding process. The goal of this thesis is to develop a single molecule technique to directly observe the interaction between individual nucleic acid and SARS-CoV CTD and NTD of N proteins. We have prepared Alexa 594-labeled NTD and CTD, as well as YOYO-labeled DNA. The DNA is immobilized on a chip coated with polyethylene glycol (PEG). Protein and DNA signals can be observed in different channels of a fluorescent microscope. Protocols for observing and analyzing the interaction of the labeled protein with labeled DNA have been developed. Surprisingly, we observed protein aggregates in low concentration of CTD, but not NTD. Thus, we concluded that a small fraction of the CTD exists in oligomer state. We can also observe the binding of CTD and NTD to DNA and found, in consistent with other biochemical and molecular biology studies, that CTD binds to DNA with higher affinity. Further investigation should leads to better understanding of the NP-RNA interaction and SARS-CoV RNA packaging.單分子技術蛋白質核酸交互作用Single Molecule TechniqueSARSNucleocapsidProteinDNAInteraction