DNA 適體於免疫治療及微生物檢測上之應用研究

Abstract

適體 (Aptamer) 泛指一種DNA 或RNA 的核酸分子結構，可因內含序列的不同而形成多樣的立體結構並結合相對應之目標分子。 適體分子可利用PCR 技術進行分子數之放大，而利用反覆與目標物結合，分離及放大的適體篩選技術(SELEX， Systematic evolution of ligands by exponential enrichment)，專一性的適體分子可以完全以試管實驗 (In vitro)的步驟篩選出來，相對於單株抗體需要動物或大量的融合瘤細胞來製造，適體的篩選明顯方便許多。 因此自從90 年代初期發現適體具有類似抗體的特性後，許多針對不同目標物的適體分子便接連被分離且發表，其中某些適體分子更具有調節蛋白質或細胞活性之功能。 除此之外，許多報告更將適體應用於化學分子，蛋白質，或癌細胞檢測上。本論文研究主要分成兩個部分，第一部分的研究專注於將適體分子應用於免疫系統的調節上。 研究過程中，成功的建立了一個名為IP-SELEX 技術，此技術成功的將傳統SELEX 的步驟簡化並縮短篩選的時間至兩個星期內。利用IP-SELEX，成功的篩選出專一性結合TLR2 接受器 (為一調控先天免疫反應的接 受器) 的適體分子，而其中某些適體分子更具有調節TLR2 相關免疫反應之功能。此外本論文也應用基因微陣列技術(DNA microarray)於快速分析與TLR2 結合之適體分子序列上。此部分的研究完整的包含了篩選技術的建立、TLR2 適體分子的篩選、適體分子的分析及鑑定、一直到適體分子的應用。第二部份的研究主要針對如何將適體分子應用於高敏感度的細菌檢測上。利用全細胞篩選技術 (Cell-based SELEX)，專一性結合金黃色葡萄球菌的適體分子被成功的分離出來，並進行專一性測試。 本研究進一步將所分離的適體分子與奈米金粒子結合，並利用奈米金粒子對光產生的強共振散射特性，建立了一個平台技術，此技術可在1.5 小時內檢測微量金黃色葡萄菌的存在，並且達到接近PCR 技術的偵測敏感度。 同時也利用這個檢測平台，成功的將抗生素檢定的時間由傳統的16 小時以上縮短至3 小時內。綜合而言，本論文研究成功地將適體分子應用於免疫系統的調節及高敏感度病原體檢測上，而這個研究也說明了適體分子不論在學術研究或臨床應用上都具有極高的潛力。

Aptamers are DNA or RNA molecules capable of folding into a variety of structures based on their sequence compositions. Like antibody, aptamers can specifically bind to their target molecules with nano- to pico-molar range of dissociation constant. However, aptamers possess several advantages over monoclonal antibodies. For examples, aptamers are stable in a wide range of temperatures and pH values, and they can be easily amplified by PCR or produced by chemical synthesis. Moreover, aptamers for a particular protein are identified with a technology called SELEX (Systematic Evolution of Ligands by EXponential enrichment) operated totally in vitro and the process can be automated. For these reasons, since the discovery of aptamers in early 1990’s, numerous aptamer sequences have been identified to interact with a wide range of targets from as small as antibiotics to as large as mammalian cells. Some of the aptamers even exhibit capability to regulate the biochemical function of their targets. On the other hand, aptamers had also been applied in detection of small chemical compounds, proteins, or cancer cells. This dissertation is composed of two parts. The first part focuses on identifying the immune-regulatory aptamers. Toll-like receptor 2 was selected as a model, for its important role in regulating innate immune system. In this study, an advanced selection method called IP-SELEX was developed, which simplified the traditional SELEX protocols and reduced the time for aptamer selection to within two weeks. Moreover, high affinity TLR2 aptamers were isolated and characterized. The antagonistic aptamers were identified by using large-scale functional screening and analyzed for its binding domain by using high throughput DNA microarray. This dissertation is a complete report including development of IP-SELEX method, isolation and characterization of TLR2 aptamers, and application of the isolated aptamers. The second part of the dissertation focuses on applying DNA aptamer in ultrasensitive pathogen detection. In this study, aptamers specifically targeted Staphylococcus aureus were isolated with cell-based SELEX. The isolated aptamers were conjugated with gold nanoparticles for S. aureus detection. The instrument that detects resonance light scattering signal of gold nanoparticles is constructed. The system can reach PCR-like sensitivity within 1.5 hr. Moreover, the technology is also applied in antimicrobial susceptibility testing to reduce the required time span from 16 hr to 3 hr.