- 學位論文
以WT22為例探討DNA和RNA從髮夾彎結構到G-四股結構之結構轉換
Investigation of conformational transition of DNA and RNA from a hairpin to G-quadruplex in WT22 sequence
摘要
富含鳥糞嘌呤(Guanine，G)的單股DNA及RNA序列能夠透過Hoogsteen 氫鍵鍵結形成G-四股結構(G-quadruplex)，進而影響基因的轉錄及調控。先前，Shanker的團隊在一段他們所設計的RNA序列上發現藉由Mg2+和K+的濃度可以去調控RNA二級結構間的轉變，並認為此轉換機制對於調控基因表現扮演著重要的角色。我們實驗室也發現位於WNT1啟動子區域中一段富含鳥糞嘌呤的DNA序列(WT22:5’-GGGCCACCGGGCAGGGGGCGGG-3’)，在加入鉀離子後會由髮夾彎(hairpin)結構轉成G-四股結構。 本篇論文以WT22為例，來探討DNA序列及其對應的RNA序列從髮夾彎結構到G-四股結構的轉換速率是否有差異，若有差異則進一步討論其差異所造成的原因為何。首先，利用圓二色光譜(CD)、核磁共振光譜(NMR)搭配膠體電泳(gel electrophoresis)和分析級超高速離心(AUC)確認WT22 RNA能如同WT22 DNA一樣在加入鉀離子後能從髮夾彎轉成G-四股結構，且觀察到WT22 RNA結構轉換所需的時間少於WT22 DNA。接著利用軟體Mfold，發現WT22 RNA的髮夾彎結構較WT22 DNA所形成的穩定。且進一步去計算從髮夾彎轉變成G-四股結構所需跨過的能量屏障(energy barrier)，發現造成WT22 RNA轉換速率快於WT22 DNA的關鍵在於WT22 RNA的髮夾彎結構打開所需要的能量屏障較低。由於序列相同的WT22 DNA及WT22 RNA，兩者的不同在於RNA五碳醣上2號碳所接的是羥基(-OH)而非氫(-H)，因而推測這個相異之處可能讓RNA的環境處在脫水狀態使得結構轉換速率加快。因此，藉由加入聚乙二醇(PEG)和乙腈(ACN)等能造成脫水效應(dehydration effect)的分子使DNA處在和RNA一樣的脫水環境下，結果顯示WT22 DNA在脫水環境下結構轉換速率加快許多，也驗證了脫水效應可能是造成WT22 RNA結構轉換速率快於WT22 DNA的主要原因。
並列摘要
Single-stranded G-rich DNA and RNA sequences can form G-quadruplex through Hoogsteen hydrogen bonds, and they are important in gene transcription and regulation. Previously, Balasubramanian’s group has reported an artificial RNA sequence shifts the hairpin/G-quadruplex structural equilibrium controlled by the concentration of Mg2+ and K+, and suggested that the structural conversion plays a crucial role in regulating gene expression. We have shown that a native G-rich DNA sequence (WT22: 5'-GGGCCACCGGGCAGGGGGCGGG-3') in the WNT1 promoter region can undergo conformational transition from a hairpin structure to G-quadruplex structure upon addition of K+. Taking WT22 as an example, we found that WT22 RNA is capable of undergoing conformational change from hairpin to G-quadruplex, confirmed by CD, NMR, gel electrophoresis and AUC. In addition, the transition rate of structural conversion from a hairpin to G-quadruplex of WT22 RNA is faster than that of WT22 DNA. The simulated results of Mfold showed that the hairpin structure of WT22 RNA is more stable than WT22 DNA. Moreover, the energy barriers required to unfold hairpin structure are calculated, and the results indicate that the conversion rate of WT22 RNA is faster than WT22 DNA since the energy barrier of WT22 RNA is much lower. Finally, the dehydration reagents like polyethylene glycol (PEG) and acetonitrile (ACN) are used to verify whether dehydration effect is the main reason to accelerate the conversion rate of WT22 RNA. This is because the only difference between WT22 RNA and WT22 DNA is the -OH and -H attached to the second carbon in pentose sugar.