李以仁Lee, I-Ren吳佳諭Wu, Jia-Yu2019-09-042017-08-032019-09-042016http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=id=%22G060342078S%22.&%22.id.&http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/100084人類B細胞淋巴癌基因（B-cell Lymphoma 2，BCL2）是一致癌基因，基因的過表達已被顯示出跟多數癌症息息相關，而在此基因啟動子區域所形成的G-四股結構已被研究顯示出可以抑制啟動子的活性，調節癌基因的轉錄，進而抑制癌蛋白的形成。先前，已有使用圓二色光譜及核磁共振光譜檢測出BCL2基因可以形成至少三種以上的G-四股結構構形。在此，我們使用全內反射式螢光顯微光譜結合單分子螢光共振能量轉移技術來探討BCL2序列在不同鹽類濃度條件下所形成的G-四股結構構形以及其含量變化。本實驗主要研究的目標為完整的BCL2序列（39個鹼基對）以及其縮短版序列（27個鹼基對）（Full length BCL2和BCL2MidG4），由實驗結果觀察到當低鉀離子濃度時，縮短版序列的G-四股結構構形有快速變動的現象，隨著鉀離子濃度提升，漸漸趨向於一穩定的構形。完整的BCL2序列觀察到其構形之間除了有快速變動的現象之外，還觀察到相對較慢的不同構型間的相互轉換，這些狀態之間的轉換不需要經過打開的構形，與文獻報導的人類端粒酶序列的G-四股結構構形之間的轉換不同，且觀察到有循序折疊的現象。而當溶液中鉀離子濃度提升，其構形也出現停留在某一特定狀態的現象。由於穩定G-四股結構的構形對於致癌基因的表達調節有相當的重要性，近年來已有相關研究發展可穩定G-四股結構的小分子藥物，然而這些小分子藥物對於G-四股結構可能有選擇性的結合，因此了解G-四股結構的多樣性以及其之間的轉換有助於藥物的發展及合成。B-cell lymphoma 2 (Bcl-2) is an oncoprotein that involves in the regulation of programmed cell death or apoptosis. The overexpression of BCL2 gene has been observed in a wide range of human cancers. It has been reported that the P1 promoter of BCL2 gene contains a 39-nucleotide(nt) GC-rich region which can form multiple G-quadruplex structures that inhibit gene transcription, and the most stable one is formed by internal four G-tracts. Previous studies have shown that this 39nt GC-rich region might fold into at least three different G-quadruplex conformations. Here, we apply single molecule fluorescence resonance energy transfer (smFRET) spectroscopy using total internal reflection fluorescence (TIRF) microscope to investigate the G-quadruplex conformations in the BCL2 gene promoter and further analyze the structural dynamics of these conformational transitions. We found that the conformations formed in the BCL2MidG4 sequence at low potassium concentration interconverts quickly, while at high salt concentration, it tends to maintain at a high FRET steady state. For Full length BCL2 sequence, the fast conformational interconversion is also observed. Besides the fast change, BCL2 G-quadruplex undergoes a relatively slow interconversion between conformational states. Unlike the conformational transitions of human telomere G-quadruplex, this interconversion does not go through an open conformation, suggesting a direct conformational transition. In addition, a sequential folding is observed. At higher potassium concentration, similar to the BCL2MidG4 sequence case, majority of the molecules remain in a steady state. Because the stability of G-quadruplex is highly correlated to oncogene transcription, great efforts have been made to develop small molecule drugs of binding and stabilizing the G-quadruplex. Understanding the interchange of the conformations and the structural dynamics further provides valuable information on the development of small molecule drugs.人類B細胞淋巴癌基因G-四股結構全內反射式螢光顯微鏡單分子螢光共振能量轉移B-cell Lymphoma 2G-quadruplextotal internal reflection fluorescence microscopesingle molecule fluorescence resonance energy transfer