2005-11-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/711897摘要：本計畫擬發展光適性體微陣列晶片技術並應用此技術解析蛋白質表現差異。藉由「系統性配位子指數增益演繹程序」（SELEX）所演繹出的單股DNA適性體，對蛋白質標的物具有高度專一性及親和性，其解離常數可達nM甚至pM的範疇。此外DNA分子穩定性亦遠優於胺基酸所組成的抗體，且可直接利用聚合脢連鎖反應或核酸合成法大量複製。因此適性體晶片兼具抗體晶片的功能及基因晶片的穩定性，且可與DNA晶片製程相容，使得實用性大幅地增加。為進一步提升檢測靈敏度與專一性，本研究擬採用光適性體作為微陣列晶片上的探針。光適性體上的鹵素鹼基可藉UV光活化使其與蛋白質標的物進行光交聯反應形成穩定的共價鍵結，於是晶片便能藉由高度嚴苛甚至失活性的洗滌條件大幅地降低背景干擾。然而光適性體的演繹過程PhotoSELEX遠較SELEX複雜且目前已知的適性體幾乎都不具有光交聯能力，所以如何使一般的適性體微陣列晶片「升級」為光適性體晶片並利用升級後的光適性體晶片解析蛋白質表現差異便成為本計畫的研究目的。研究初期，我們將以凝血蛋白及bFGF之適性體建立升級標準程序與找尋最適化實驗條件；之後以大腸桿菌K12之蛋白質表現差異評估升級程序與光適性<br> Abstract: This project aims at development of photoaptamer microarrays and use of the microarrays for differential protein expression profiling. Single-strand DNA aptamers, evolved from the SELEX process (Systematic Evolution of Ligands by EXponential enrichment), feature both high specificity and affinity to the target proteins. The typical dissociation constant for an aptamer-protein complex can reach nanomolar, even picomolar, range. Moreover, DNA aptamers are more structurally stable than antibodies composed of amino acids, and they can be prepared and scaled up through polymerase chain reaction (PCR) or oligonucleotide synthesis. Thus, aptamer chips are not only like antibody chips in function but also as stable as gene chips. Also, the chip fabrication is compatible with the other DNA chip process. Consequently, applications of such an antibody-mimic chip are visible. To further enhance detection sensitivity and specificity, we will array photoaptamer probes on slides in stead of aptamers. The halogen bases inside a photoaptamer molecule can be activated by UV irradiation and then form a stable covalent bond with the target protein. This process is called photocrosslinking. After photocrosslinking, chips can stand for highly stringent, even denatured, wash conditions and thus greatly reduce background contamination. However, the screening process of photoaptamers, namely PhotoSELEX, is much more complicated than a typical SELEX process. Besides, almost all aptamers discovered to date are lack of the ability of photocrosslinking. As a result, our objectives are finding an appropriate way to “upgrade” aptamer chips to photoaptamer ones and utilizing the upgraded chips for differential protein expression profiling. At the beginning, we will employ both aptamers of thrombin and basic fibroblast growth factor (bFGF) to establish the standard upgrading protocol and to optimize assay conditions. Afterward, we will evaluate the upgrading protocol and the performance of photoaptamer chips through differential protein expression profiling of E. coli K12. The relevant techniques developed in this project can be applied to essential biotech aspects, such as disease diagnosis and functional proteomic research.微陣列晶片光適性體蛋白質表現microarrayphotoaptamersprotein expression