3,6-bis(4-methyl-2-vinylpyrazinium) carbazole diiodide與DNA四股結構及雙股結構作用之共振拉曼光譜研究

Investigation of the Interactions Between 3,6-Bis(4-Methyl-2-VinylPyrazinium) Carbazole Diiodide and G-Quadruplex/Duplex DNA by Resonance Raman Spectroscopy

蘇信逢(Shin-Feng Su)

指導教授 ： 楊子萱

國立中正大學/理學院/化學暨生物化學系/碩士(2013年)

摘要

在DNA染色體末端有一段長約一萬五千個鹼基對所組成的序列稱之為端粒。端粒的存在是為了保護染色體及避免DNA在複製時造成結構熔合(fusion)。隨著每一次的細胞分裂，端粒會減少50-200個鹼基對。當端粒的長度減少到一定程度後，細胞會進入衰老階段(senescent stage)並停止分裂。近年來對於端粒的研究也拓展到癌症上；研究指出85%以上的癌細胞能夠活化用來延長端粒長度的端粒酶(telomerase)，使癌細胞的端粒長度不會隨著每一次的細胞分裂而減少。
人類端粒的最末端是由一連串重複的TTAGGG單股鹼基序列所組成。因富含鳥糞嘌呤(guanine)能以Hoogsteen氫鍵形成穩定的鳥糞嘌呤四股結構(G-quadruplex)。端粒酶無法延長形成鳥糞嘌呤四股結構的端粒，因此如何穩定端粒的鳥糞嘌呤四股結構成為研究癌症治療的一個方向。
咔唑衍生物3,6-bis(4-methyl-2-vinylpyrazinium) carbazole diiodide (BMVC4)已發現能夠有效穩定鳥糞嘌呤四股結構。但對於BMVC4和鳥糞嘌呤四股結構的作用情形並不清楚。由於BMVC4不具螢光性質，因此本研究利用共振拉曼光譜來探討BMVC4和寡核苷酸鳥糞嘌呤四股結構d[TAG3(T2AG3)3] (Hum23)以及線性雙股螺旋結構d[(GC)2A2T2(GC)2)] (LD12)的作用情形。藉由光譜訊號的改變並搭配理論計算來瞭解BMVC4與DNA四股結構及雙股結構的作用形式。此研究有助於藥物設計上增加藥物對四股結構的選擇性。
實驗結果顯示BMVC4在和Hum23作用後，其吸收光譜譜峰產生24~35 nm的紅位移且有額外的譜峰顯現，顯示BMVC4和Hum23之間的作用力影響到BMVC4電子能階的能量。同樣BMVC4在和LD12作用後吸收譜峰也產生13~27 nm的紅位移，但位移量較BMVC4與Hum23作用的小，也沒有額外的譜峰顯現。透過理論計算可以得知使譜峰位移的作用力與BMVC4的吡嗪有關，推論是吡嗪上的氮因接上甲基後所產生的正電與DNA磷酸骨架的負電產生庫倫作用力導致BMVC4吸收譜峰的激發態軌域能量降低，最終造成譜峰紅位移。
比較BMVC4和BMVC4/Hum23共振拉曼光譜，兩者的光譜形態並沒有太大差異，顯示BMVC4和Hum23作用後其電子位能曲面沒有受到太大影響。透過兩者的差異光譜與訊號擬和來進行譜峰的綜合比較後歸納出三個可能的作用形式：(1) 咔唑會受到作用力的影響。作用力有可能是與DNA鹼基的π-π stacking；(2) 吡嗪與磷酸骨架之間有庫倫作用力；(3) 乙烯基氫不共平面的彎曲振動受到作用力影響而消失，顯示BMVC4受到Hum23空間的局限。
接著比較BMVC4和BMVC4/LD12共振拉曼光譜。兩者的光譜形態差異較BMVC4和BMVC4/Hum23的差異更小。差異光譜顯示兩者之間的作用力較微弱，但仍然有吡嗪與磷酸骨架間的庫倫作用力。
最後以推論出的作用力來模擬BMVC4與DNA之間可能的作用位置。BMVC4可能會與Hum23結合在TTA環(T12、A14)與G-quartet (G5-G11-G15-G23)之間，透過咔唑與G-quartet的π-π stacking以及吡嗪與磷酸骨架的庫倫作用力使鳥糞嘌呤四股結構不易被解開。BMVC4可能會與LD12以吡嗪與磷酸骨架的庫倫作用力作用在同一條序列並橫跨四個磷酸骨架，兩者之間的作用可能很容易受環境擾動而分離。

關鍵字

咔唑衍生物 ； 鳥糞嘌呤四股結構 ； 端粒 ； 共振拉曼光譜

並列摘要

The DNA sequence at the end of a chromosome is called telomere. It stabilizes the DNA structure and prevents chromosomal fusion. It had been confirmed that cancer cells can activate telomerase to extend the length of telomere, keeping carcinoma alive. In the solution with Na+/K+ ions, the single strand overhang of the telomere folds into a G-quadruplex structure so that telomerase cannot prolong the length of telomere, which causes telomere shortening and induces cell apotosis. Thus, stabilizing the G-quadruplex structure of telomere becomes an active research topic in anti-cancer therapeutic treatment.
The carbazole derivative compounds have been found to be able to stabilize human DNA telomeric G-quadruplex in vitro. Nevertheless the detailed interactions between carbazole derivatives and the G-quadruplex are still unknown. Since 3,6-bis(4-methyl-2-vinyl pyrazinium) carbazole diiodide (BMVC4), one of carbazole derivatives, is lack of fluorescence, we used resonance Raman spectroscopy to investigate the interactions of BMVC4 with the G-quadruplex d[TAG3(T2AG3)3] (Hum23) and the double helix d[(GC)2A2T2(GC)2)] (LD12) to identify interacting moieties of BMVC4 with these DNA sequences.
Upon mixing with Hum23, the UV-Vis spectrum of BMVC4 shows a large bathochromic shift of 24~35 nm, suggesting that BMVC4 indeed strongly interacts with Hum23. The bathochromic shift is also observed in the absorption spectrum of the BMVC4 mixed with LD12, but is smaller in magnitude (13~27 nm) than the case of BMVC4 mixed with Hum23. Through the theoretical calculations, we conclude that the shift is caused by the Coulombic interaction between pyrazinium ion of BMVC4 and DNA phosphate backbone. It lowers the excited state energy and causes the red shift of absorption peaks.
Comparing resonance Raman spectra of BMVC4 and BMVC4/ Hum23, we propose that there are three types of interactions between BMVC4 and Hum23: (1) Interaction of carbazole moiety with DNA, probably due to π-πstacking; (2) Interaction of pyrazinium with negatively charged phosphate backbone of Hum23 by Coulombic attraction; (3) The restriction of the out-of-plane bending of vinyl hydrogen. This band vanishes when BMVC4 interacts with Hum23, indicating that BMVC4 is constrained in Hum23 cavity.
Through the difference spectrum of BMVC4 and BMVC4/LD12, we proposed that the interaction between BMVC4 and LD12 is mainly the weak Coulombic interaction between pyrazinium moieties and phosphate groups of DNA.
Finally, we propose a possible interaction model of BMVC4 with quadruplex and duplex DNA. BMVC4 may intercalate into Hum23 between the second TTA loop (T12-A14) and the third G-quartet (G5-G11-G15-G23) plane by π-π stacking and interact with the DNA backbone through Coulombic attraction. BMVC4 may interact with LD12 by Coulombic attraction of pyrazinium ions and the phosphate backbone of one strand.

並列關鍵字

BMVC4 ； G-quadruplex ； Telomere ； Resonance Raman spectroscopy

參考文獻

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