探討人類端粒四股結構的結構轉換機制

Elucidation of the Mechanism of Structure Conversion in Human Telomeric G-quadruplexes

王咨富(Zi-Fu Wang)

指導教授 ： 張大釗

國立臺灣大學/理學院/化學研究所/博士(2013年)

https://doi.org/10.6342/NTU.2013.02623

摘要

由於G-四股結構(G-quadruplex)有潛力干擾端粒酶延長端粒的功能，因此設計小分子穩定四股結構抑制端粒酶過度表現的癌細胞，有機會可以成為有效的抗癌藥物。但由於人類端粒四股結構具有多樣性，導致研究四股結構生物上活性或設計藥物增加複雜性。本篇論文主要探討人類端粒四股結構與結構轉換的機制，研究離子與溶劑造成的效應。第一部分探討人類端粒四股結構在鈉、鉀離子交換的結構轉換機制，利用NMR與site-specific isotope (15N) labeling的方法，我們定出了人類端粒序列 d[TAG3(T2AG3)3] (TA-HT21)在鈉離子溶液的四股結構，與文獻中報導的在鉀離子溶液的結構相同，因此光譜快速轉換的主要原因，並非因為大幅的結構改變，而是由於離子交換的結果，我們進而提出了鈉/鉀離子交換的機制。第二部分是觀察PEG (polyethylene glycol)所產生的溶劑效應，利用分子設計，經由四股結構穩定劑BMVC，合成出其衍生物BMVC-8C3O，可以有效轉換人類端粒DNA G-四股結構，從不同的非平行結構轉為平行螺旋型的結構，使複雜的結構歸一化，同時能夠大幅提升端粒DNA G-四股結構的解旋溫度 (melting temperature)。成功的將溶劑效應轉換到區域化的效應，不僅支持了PEG誘導結構轉換並非是經由分子擁擠效應(molecular crowding effect)，而是經由脫水效應(dehydration effect)的結果，此外也提供四股結構周邊化的水分子的環境可以成為分子設計影響端粒四股結構。第三部分利用DSC、CD、 Time-resolved NMR 等方法，探討端粒非平行四股結構經由BMVC-8C3O作用，轉換成平行螺旋型四股結構的轉換機制，發現轉換過程中間存在intermediate，並嘗試建立其動力學機制，進而了解不同四股結構的動力學與熱力學在結構轉換中的關係。

關鍵字

人類端粒四股結構 ； 結構轉換 ； 離子效應 ； 四股結構嵌合劑 ； 去水合效應

並列摘要

Human telomere G-quadruplexes (G4) is a promising target for cancer therapy by telomerase inhibition. Thus, designing and synthesizing of a small molecule that can bind to G4 and inhibit telomerase has been a popular research topic. However, G-quadruplex has a variety of structures and it is not known which of these structures are likely to be present in living cells. Therefore, a rational design of selective ligands to G4 is challenging. Here, we synthesized a novel ligand and apply the technique of ITC, DSC, and NMR with site-specific isotope (15N) labeling to discuss the diversity of G4 structure and to find out the mechanism of structure conversion. The first part of my thesis discusses the mechanism of structure conversion of Human telomere G-quadruplex by sodium- potassium exchange. By solving the structure of Human telomere G-quadrupelx (HT23) under sodium form, we find the structure is the same as the potassium form. Instead of involving large conformation change, the process is mainly through the ion sequential exchange. The second part is applying the idea of solvent effect of PEG (polyethylene glycol) to ligand design where we optimized the fluorescence probe of 3,6-Bis(1-methyl-4-vinylpyridium) carbazole diiodide (BMVC) molecule by substituting a tetraethylene glycol in the N-9 position with a methyl-piperidinium cation to produce a new G4 stabilizer called BMVC-8C3O. The design principle based on solvent effect of PEG can induce structural change from non-parallel to parallel G4 and further stabilize the G4 structure for Human telomere G4 sequences. This provided a basis for the design of novel G4 ligands. The results show the modification of G4 ligand with the oxygen atom plays a crucial role for inducing parallel G4s, and provide evidence of the local dehydration effect from PEG where the local water structure is the key for induce conformational change of human telomere. The final part is applying the ligand to study the G4 strucutre conversion from hybrid to parallel. By NMR result, we discover the intermediate state and provide the kinetic pathway of structure conversion. The mechanism of G4 structure conversion can be further apply to the G4 biological function and G4 ligand design.

並列關鍵字

Human telomere G-quadruplex ； Struture conversion ； Ion effect ； G-quadruplex ligand ； Dehydration effect

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