- 學位論文
專一性STAT3適體與圈套寡核苷酸之篩選與驗證
Selection and Verification of STAT3-specific Aptamers and Decoy Oligonucleotides
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摘要
STAT3 (Signal Transducers and Activators of Transcription 3, 訊息傳導及轉錄激活蛋白3) 為細胞中重要的轉錄因子蛋白，當STAT3受到細胞外的訊息分子刺激時，位於細胞質的STAT3會磷酸化以及二聚體化，接著轉位至細胞核後，DNA binding結構域會和目標基因的啟動子上的特定序列鍵結，而轉錄活化該目標基因的表現。STAT3異常的持續活化和許多腫瘤形成有關，因此調控STAT3在癌細胞中的表現具有重要的研究價值。在許多調控方法當中，STAT 3-圈套寡核苷酸 (STAT3-decoy oligonucleotide, STAT3-dODN) 為一段模擬目標基因的啟動子上的特定序列，能與STAT3之DNA binding結構域鍵結，進而抑制STAT3下游基因表現。 本研究的實驗中針對STAT家族的六種蛋白質同時進行篩選，發現此方法適合用於判斷哪種蛋白質會在同時篩選中附著最多的DNA，但是可能並不適合針對每一種STAT蛋白質篩選出對其專一性的DNA序列，此外STAT1蛋白質也許並不適合作為STAT3目標蛋白的反向篩選蛋白質。 在本研究中，將以已知的圈套寡核苷酸候選序列作為篩選的資料庫 (library pool)，STAT3作為目標蛋白，透過系統性配位子指數增益演繹技術 (Systematic Evolution of Ligands by Exponential enrichment, SELEX) 篩選出與STAT3有高度專一性與親合力的適體，並且以ELONA (ELISA-mimic enzyme linked oligonucleotide assay) 驗證篩選獲得的適體之專一性與親合力，透過MTT細胞存活率試驗探討篩選得到的序列對於細胞的影響。在本研究中，找到了具有對STAT3高度親合力的適體序列dODN-18與dODN-20，與過去研究的dODN-pi相比有更高的親合力，同時它們也對細胞的存活率有初步的影響。
並列摘要
Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor in the cell. Abnormal activation of STAT3 has been proven to be a factor leading to cancer cell and regulate cell proliferation, differentiation, apoptosis, immune and inflammatory responses. Since over-activated STAT3 is often found in malignant tumor, it has the strong potential to be a biomarker. STAT3-decoy oligonucleotide (STAT3-dODN) is a specific sequence which is simulate to promoter of target gene. It has ability binding to STAT3 DNA binding domain, and also inhibits the gene expression. Aptamers, stable single-stranded nucleotides, which have high binding affinity and specificity to target, are selected through Systematic Evolution of Ligands by Exponential enrichment (SELEX). In the study, it shows that it may not be suitable for simultaneous screening of six STAT proteins. In addition, STAT proteins may be not suitable for being each other counter selection protein. In the present study, we demonstrated micro-bead SELEX with different library pool named as N40 and dODN pool. The result showed that we get candidate aptamers in early round of selection (<5). From the ELONA experiment, we get two aptamers named dODN-18 and dODN-20 which have higher affinity to STAT3 protein then dODN-pi. With MTT assay, we also realize the influence of dODN-18 and dODN-20 to cells that it may cause the cell death. Therefore, there are several applications for diagnosis and treatment in the future.
參考文獻
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