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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48796

Title: 利用質譜儀鑑定適體核酸與目標蛋白複合物之結構
Examination of Aptamer-Protein Complex Structure by Mass Spectrometry
Authors: Hung, Guo-Ming
洪國明
Contributors: 國立臺灣海洋大學:生命科學暨生物科技學系
Keywords: 質譜儀;適體核酸;化學交聯鍵結
mass spectrometry;aptamer;chemical cross-linking
Date: 2017
Issue Date: 2018-08-22T06:31:24Z
Abstract: 適體核酸因其具有特定結構,能專一性辨認目標分子,如:蛋白質、藥物、小分子,適體核酸具有良好的化學穩定性且能夠透過簡單的化學合成方式製造,因此在生物科技應用、臨床診斷及治療上被視為是替代抗體的分子之一。配體指數增強系統進化技術(Systematic Evolution of Ligands by Exponential Enrichment)是篩選適體核酸之主要方式,能透過小分子、蛋白質、癌細胞做為目標,用以篩選出適體核酸,然而,後續用以決定適體核酸與其辨識分子在空間上之結合位仍須依靠耗時的方法,如:核磁共振以及X射線結晶繞射學。因此我們發展以質譜儀鑑定適體核酸與蛋白質複合體結合位結構的方法。預先透過化學交聯鍵結將空間中具有交互作用的蛋白質序列及適體核酸的核苷酸以共價鍵進行固定,再利用液相層析串聯式質譜儀進行分析。我們以細胞色素C作為模式蛋白,作為方法開發之材料,以甲醛作為交聯試劑以共價鍵方式固定細胞色素C與其適體核酸形成之複合物,再藉由質譜儀進行分析,得到兩段可能適體核酸結合位之胜肽序列,以細胞色素C的三維結構分析胜肽資訊,發現其位於環狀結構上,且與人類凝血酶蛋白與其適體核酸形成複合物之文獻作為比較,於文獻中,適體核酸與人類凝血酶蛋白交互作用位置亦為環狀結構區域,藉此驗證此實驗結果,推測適體核酸與蛋白質的環狀結構具有交互作用的可能性較高。期望此方法的建立能應用於適體核酸與其目標分子結合位之分析。
The oligonucleotide aptamers can specifically recognize target molecules such as proteins, drugs, and small molecules due to their special oligonucleotide sequences with the formation of unique spatial structures. Aptamers have good chemical stability and can be easily prepared by chemical synthesis; therefore, they have high potential to be the substituent for antibody in biotechnology application, clinical diagnosis, and therapeutic usage. The systematic evolution of ligands by exponential enrichment (SELEX) method was developed for screening proper aptamers to a variety of target ligands, including small molecules, proteins and tumor cells. However, to determine the spatial interaction between aptamer and its target ligand still depends on time- consuming methods such as x-ray crystallography or NMR techniques. We hereby developed a mass spectrometry based method to investigate the aptamer-protein complex structure. The spatial correlation of aptamer-protein complex was fixed by chemical cross-linking reaction and further investigated by MS analysis. We used cytochrome c as a model protein to develop the new methods and used formaldehyde as a cross-linker to induce crosslinking and fix cytochrome c-aptamer complex. After protein digestion and mass spectrometry identification, we found two possible candidate peptides which the aptamer interaction region. We also compared our results with known data, the thrombin-aptamer complex structure. We found both our results and thrombin-aptamer complex showing the aptamer interaction region in loop structure of protein. Through this method can identify target proteins for SELEX selected aptamer as well as to determine their interaction region on molecule.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G001043B010.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48796
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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