| 摘要: | 本論文以核酸四股結構 i-Motif 作為研究目標。構成i-Motif 之四股核酸鏈，其各股序列需含有兩個或兩個以上的胞嘧啶；該結構可由四條核酸鏈組成，亦可由二條或單條核酸鏈自我折疊而成。i-Motif結構是一種鑲嵌型的獨特核酸四股結構，係由胞嘧啶形成鹼基對C•C+ 構成四股結構 i-Motif，故 i-Motif 結構在酸性溶液中較容易形成。我們設計一個富含胞嘧啶的寡核酸鏈5’-d (CCTTCCTCTCCTCC) (C2T2) 運用紫外光分光光譜儀與圓二色光譜儀觀測 i-Motif 在 pH 6.0、不同的緩衝溶液 ( Phosphate、Cacodylate、MOPSO、PIPES 與 Bis-Tris) 中的結構與穩定度。
運用紫外光分光光譜儀在波長 265 nm 與 285 nm 下觀測寡核酸鏈 C2T2，在定波長 265 nm 的熔點曲線呈現增色效應，而波長 285 nm呈現減色效應。在波長 265 nm 觀測所獲熔點溫度為：25°C (MOPSO) > 22°C (PIPES) > 21°C (phosphate) > 19°C (cacodylate) ≈ 19°C (Bis-Tris)，i-Motif 在 MOPSO 緩衝溶液中是最穩定的，與在 Bis-Tris 緩衝溶液中所生成之結構 (相較下結構穩定度最低)，兩者熔點溫度差值達 6 °C，由此可知緩衝溶液的性質對 i-Motif 結構穩定度有顯著的影響性。
我們進一步研究在波長 285 nm 觀測 i-Motif 的熔點曲線，在 Phosphate、Cacodylate 與 PIPES 緩衝溶液中，寡核酸鏈 C2T2 的熔點曲線呈現一個漂亮的 S 型，然而在波長 285 nm 觀測熔點溫度，MOPSO 與 Bis-Tris 緩衝溶液的吸收度變過小 (< 4%) 導致熔點溫度的不準確性。在波長 285 nm 觀測所獲的熔點溫度，在 PIPES、Phosphate 與 Cacodylate 緩衝溶液樣品之熔點溫度與 265 nm 量測結果相差值在 1°C內；而在 MOPSO 與 Bis-Tris 緩衝溶液之樣品因吸收度值變化過小，無法準確計算熔點溫度。
為了進一步了解 i-Motif 在不同緩衝溶液中的構型變化，運用圓二色光譜儀在 5°C、pH 6.0 觀測寡核酸鏈 C2T2 在MOPSO、PIPES、Phosphate、Cacodylate 與 Bis-Tris 緩衝溶液中的圓二色光譜圖。根據圓二色光譜圖我們發現圓二色光譜的整體波形是相似的，但是波峰與波谷的位置與振幅強度略有不同。表明緩衝溶液的性質對 i-Motif 構形與穩定度是有明顯的影響性，也暗示 i-Motif 在不同緩衝溶液中可能存在不完全相同的構形。
此外，我們運用紫外光分光光譜儀在不同溫度下用波長範圍 200 nm-320 nm 觀測 i-Motif 結構在不同波長下的吸收度變化；在全波長實驗中，將高溫 (80°C) 的吸收度值減去低溫 (5°C) 的吸收度值後，將吸收度變化量對波長作圖。藉由此實驗結果，我們可以了解在紫外光熔點實驗中波長的選擇是很重要的;由前述紫外光熔點實驗，在波長 265 nm 與 285 nm 所得實驗結果，可印證全波長實驗之重要性，用以預測紫外光熔點實驗之待測波長。
The i-motif, which is formed by DNA stretches of two or more cytidines, is the only known nucleic acid structure involving systematic base intercalation. The base pairing of hemiprotonated cytosine and cytosine forms C•C+ duplex structures, and two parallel stranded C•C+ duplexes with an antiparallel orientation form i-motif, a four stranded structure. An understanding of both its structural stability and solution properties is essential for a rational approach to the development of sensors and drug design. We investigated one C-rich strand of 5’-d(CCTTCCTCTCCTTCC) (C2T2). A combination of UV and CD spectroscopy techniques were used to determine the spectral characteristics and structural stability of i-motif in a variety of solutions (e.g. phosphate, cacodylate, MOPSO, PIPES and Bis-Tris) of pH 6.0.
The melting curves were characterized by a hyperchromic effect at 265 nm and a hypochromic effect at 285 nm upon i-motif dissociation. The melting temperatures (Tm) derived from the curves at 265 nm showed the order: 25°C (MOPSO) > 22°C (PIPES) > 21°C (phosphate) > 19°C (cacodylate) ≈ 19°C (Bis-Tris). There was a span of 6°C in Tm from the most stable situation in the MOPSO to the least stable in the Bis-Tris. In consequence, the nature of buffer species had significant influence on the structural stability of the i-motif.
We further investigated whether the formation of the i-motif structure could be monitored at 285 nm. The nice sigmoid transitions were observed clearly in buffers of phosphate, cacodylate and PIPES containing the C2T2 strand. The Tm’s measured at 285 nm were the same as those at 265 nm in the same conditions. However, a small variation (< 4%) of absorbance was recorded at 285 nm in the MOPSO or Bis-Tris buffer. Thus, the recordings at 285 nm in the MOPSO and Bis-Tris buffers would lead to a much larger uncertainty in the measurements of the Tm values.
The CD spectra of the C2T2 strand were further carried out in the MOPSO, PIPES, phosphate, cacodylate, and Bis-Tris buffers of pH 6.0 at 5°C. The overall shape of CD spectra is similar, but the magnitude and location of the positive and negative bands varies with the nature of buffer species. It indicates that changes in the buffer environment critically affect the detection of i-motif dissociation, implicating that the i-motif in different buffers might exist in different topologies.
Then use the observation wavelength UV spectrometer 200 nm-320 nm absorbance change at different temperatures, the full wavelength experiment cryogenic temperature minus the absorbance absorbance change amount of the absorbance is plotted versus wavelength, with the result we can understand the melting point of the experiment in the UV wavelength selection is important. |
