Differential Antisense Hybridization to Screen G-Quadruplex Stabilizers by Using Isothermal Titration Calorimeter

端粒位於真核細胞的染色體末端，其DNA 由富含鳥糞嘌呤的重複核苷酸序列接續而成。當適當大小的單價陽離子存在時，富含鳥糞嘌呤的核苷酸序列可纏繞成穩定的鳥糞嘌呤四股結構(G-quadruplex)。G-quadruplex 二級結構，使得端粒酶的RNA 模版不易與之鍵結，阻斷端粒酶作用而無法延長端粒長度。因此，科學家研發有機小分子穩定端粒四股結構，希望藉由加強G-quadruplex 的穩定性以抑制癌細胞中的端粒酶作用，使癌細胞失去無限制增生的能力，有效的穩定劑即成為頗具潛力的抗癌藥物。 傳統上，藉由比較相同序列DNA 添加有機小分子前後的解旋溫度差異來推斷有機小分子穩定DNA 的能力。然而，通常解旋溫度都高於人體的生理溫度許多，在高溫的鉀鹽溶液環境中，藥物可能促使DNA 結構轉換，但此一轉換在正常的生理條件下卻不易發生；其次，人體中DNA 存在的環境是高濃度鉀鹽溶液，但文獻中以大幅解旋溫度差異來宣稱該小分子擁有良好穩定效力的卻是在鈉鹽或低濃度鉀鹽等非生理狀態的溶液的實驗結果，但不同的溶液條件可能會造成不同的結果；再者，若欲加入相關的蛋白質一併做測試也不適合進行高溫實驗。基於以上三大理由，我開始反思以「解旋溫度」當作篩選藥物的標準是否適當。因此，我從H22 開始，以其互補股d(CCCTAACCCTAACCCTAACCCT，C22)作為解旋的驅動力，搭配使用實驗室「超微量恆溫滴定量熱儀」(ITC)來做測定。 從ITC 實驗結果中，H22 和C22 的配位比值(N 值)大小可比較出藥物穩定G-quadruplex的效力：在未添加有機小分子時，H22 與C22 的N 值約等於1。當有機小分子愈能穩定G-quadruplex 時，則H22 的四股結構愈不容易解開，相對地和C22 互補的情況降低，因此所測得之N 值下降。簡言之，穩定力愈好，所測得的N 值愈小。例如，加入本實驗室合成的有機小分子 BMVC 的N 值約為0.62；加入市售有機小分子TMPyP4 的N 值則約為0.8；同時，我以相同配製條件的樣品做解旋溫度的測試，各有機小分子間並無明顯的差異，突顯傳統方法的囿限。另外，我們可以從實驗結果中得到H22 和C22 雜交的平衡常數，利用赫斯定律求得該有機小分子對G-quadruplex 及雜交形成的雙股之選擇性。選擇能力由大到小分別為：8C3O > Braco19 > BMVC-4 > BMVC > BMVC-5 > TMPyP4。實驗結果與文獻中的選擇性結果相符，可證明本研究透過互補股競爭法搭配使用超微量恆溫滴定量熱儀有其重要價值。 本研究建立在恆溫、生理狀態的鉀離子溶液且四股與雙股DNA 共存的篩選方法，期望透過模擬人體生理環境的方法來篩選鳥糞嘌呤四股結構穩定劑，可從中篩選出合適的藥物結果，以提供科學家未來藥物設計合成的正確方向。This work intended to establish a novel method to screen G-quadruplex ligands that stabilize the G-quadruplex structures formed by Hoogsteen Hydrogen bond of guanine-rich sequences. Telomere, in the end of chromosomes, has guanine-rich sequences, these sequences are prone to adopt G-quadruplexes in vivo. These unusual structures might play an important role in biological processes, especially inhibiting the elongation of telomerase, an enzyme essential for immortalization of tumor cell. Thus, G-quadruplexes are likely as the promising target for anticancer drug design. Since a number of small molecules were reported, a novel method is requested to examine if they really have the ability to stabilize G-quadruplex structure. In general, a thermal melting analysis is a popular method, in which the unfolding of G-quadruplex is monitored by changes in spectroscopic signal. One can screen the G-quadruplex stabilizer by comparing melting temperature (Tm) before and after adding small molecules to G-quadruplexes. However, Tm of human telomeres is normally much higher than the physiological temperature. At high temperature, some compounds would induce structural change of G-quadruplexes in 150 mM K+ buffer condition, but this conversion would not happen at the physiological temperature. Moreover, melting analysis cannot be applied to G-quadruplexes associated with the related proteins because of the tolerance of the proteins. In the work, we established an isothermal method to screen G-quadruplex stabilizers based on antisense hybridization. We used isothermal titration calorimetry (ITC) to investigate the effects of each G-quadruplex ligand on duplex formation by dAG3(T2AG3)3 and its complementary strand d (C3TA2)3C3T. It is believed the better the G-quadruplex stabilizer, the less the duplex formation. Therefore, the binding stoichiometry ratio (N) for the duplex formation is small. Accordingly, we have successfully distinguished those G-quadruplex stabilizers in 150 mM K+ buffer condition, in which those stabilizers could not be distinguished by CD melting analysis. We found that the order of stabilizing ability is 8C3O > Braco19 > BMVC-4 > BMVC > BMVC-5 > TMPyP4. In addition, we can combine equilibrium constant of hybridization from this method and Hess’s law to derive the stabilizer selectivity to G-quadruplex from duplex. The results are in agreement with those K ratio obtained by fluorescent titration. Among these stabilizers, 8C3O has best selectivity owing to its hydrophobic property. In summary, we developed a mimic physiological method to screen G-quadruplex stabilizers. We believe that this method can provide more reliable information for drug design