以單分子光學鉗系統探討鳥嘌呤-四股結構穩定劑對人體端粒酶解構機制之影響

Abstract

我們以光學鉗平台研究人類端粒序列 H24 ( (TTAGGG)4 ) 中的鳥嘌呤-四股結構 G-quadruplex (G4) 的摺疊與解開機制。首先為了達成量測此結構轉變所出現的微小距離變化，針對光學鉗平台的操作模式作改進，目標在達成定力量測的模式。為了提高系統中雷射的穩定性，將聲光調變器加入光學系統中，藉由記錄雷射強度後，對應調整外加電壓成功達成穩定雷射功率，在二十分鐘內標準差小於 0.01 mW。關於 G4 實驗，我們選擇以 H24 序列作為研究標的，並以 150 mM 高濃度 Na+ 離子溶液使序列透過 Hoogsteen basepairing 形成 G4 的二級結構，且摺疊形式出現較為單純的反平行籃子型 (anti-parallel basket type)。首先以受力與伸長量模式 (force-extension assay) 量測發現 G4 結構在逐漸增加施力 (unfolding) 過程及降低施力 (refolding) 過程中均出現一到兩個轉折事件，不過在加入穩定劑 BMVC (3,6-bis(1-methyl-4-vinylpyridinium) carbazole) 後G4結構在 unfolding 過程中轉折事件上升至兩到三個，暗示著中間態的存在。接著轉換到定力量測 (force-clamp assay) 模式下觀察 G4 解構機制，由單獨只有 H24 序列存在所得到的時程圖，可以觀察到除了全展開及全摺疊的長度之外，出現了第三種長度分布，並且在加入 BMVC 與 H24 序列共同培養之下，我們觀察到第四個分布的出現。由 H24 序列在兩種光學鉗的量測模式下所得到的結果，確實的發現 G4 結構的解構機制在有 BMVC 的處理下會有不同於其單獨存在的情形。

We used an optical tweezers platform to study the folding and unfolding pathway of individual molecules containing single-stranded DNA human telomeric G-quadruplex (G4) sequence, (TTAGGG)4. We home-built an optical tweezers platform with force-clamp capability, which held a DNA tether at a constant force, to determine the DNA length in high spatro-temperal resolution. We included an acousto-optic modulator (AOM) to maintain the laser stability and madulate laser output intensity. By modulating the applied voltage of AOM, feedbacked from the laser power measurement, we achieved to maintain the laser power with standard deviation less than 0.01mW over 20 minutes. For G4 experiments, these G4 containing DNA molecules are found to form the G-quadruplex structure based on Hoogsteen basepairing in 150 mM Na+ solution. When forces were applied to unfold the G4-containing DNA molecules, most of the unfolding traces showed one or two transitions, suggesting the existence of one stable intermediate state. The total unfolding distance was consistent with the expected value of unfolded G4 structure. However, when the DNA molecules were pre-incubated with a G4 stabilizing ligand BMVC, 3,6-bis(1-methyl-4-vinylpyridinium) carbazole, most DNA molecules showed three unfolding transitions, suggesting of two stable unfolding intermediate states. Using the force-clamp assay, we found that three extension states exist in the traces of G4 DNA. When G4 was pre-incubated with BMVC, the number of extension states also increases to four. Both force-extension and force-clamp results suggest that BMVC-bound G4 structures are able to withstand higher force than the ligand-free G4 structure, thus revealing more intermediate states.