利用拉曼探針分辨不同去氧核糖核酸結構

Abstract

80年代末期，學者了解到真核生物的染色體尾端，端粒，為一段單股富含「鳥糞嘌呤鹼基(G)」的單股DNA序列，並且會形成特殊的「鳥糞嘌呤四股結構」，而端粒的G-四股結構與癌症研究有密切關係。近年來又陸續在非「端粒」的位置，發現到四股結構的存在，特別是原致癌基因啟動子的地方，因此更加引發學者對於四股結構研究的高度興趣。另外有些學者致力於研發能夠穩定四股結構的小分子，進而可能成為抗癌藥物。 本實驗的目的是探討能夠穩定四股結構的小分子之作用機制，進而發展出可以分辨不同DNA結構的探針分子。首先利用分子的吸收、螢光、旋光光譜、及膠體電泳實驗，比較咔唑衍生物BMVC-4及二苯胺衍生物BMVPA-4與四股結構的作用能力，發現剛性的咔唑結構比不具剛性的二苯胺結構更能有效地穩定四股DNA的結構。 由於咔唑衍生物BMVC-4及二苯胺衍生物BMVPA-4的螢光強度都很微弱，可以經由共振拉曼光譜，根據拉曼譜線所對應到的分子振動態，鑑別它們與不同DNA結構作用的主要官能基。根據譜線的位移顯示BMVC-4對於四股結構的作用較大於雙股結構，但BMVPA-4則恰好相反。因此在分辨雙股或四股DNA結構時，這兩個分子是可以相互補。特別的是，由於BMVPA-4分子較不具剛性結構，對於DNA結構具有較高的靈敏度，可用來辨識同為DNA四股結構的bcl2mid與bcl2mid-M（經替換兩鹼基），在結構上存在差異。此外，我們的實驗結果也顯示，BMVPA-4主要針對雙股DNA的AT鹼基對作嵌入。因此，我們認為BMVPA-4較BMVC-4更適合作為拉曼探針分子。 藉由本實驗的討論，我們不僅提高拉曼探針分子的應用性，甚至將來在設計穩定DNA結構的藥物分子時，也提供了重要的資訊。

At the end of 80s, scientists realized that the end of eukaryotic chromosomes contain G-rich single-stranded DNA, telomere. These telomeres could adopt novel secondary structures, termed G-quadruplexes. Also, quadruplex-folded telomeric DNA has been found related to cancer activity. Recently, G-quadruplexes at non-telomeric sites were also detected, especially in promoter regions of proto-oncogene. These findings inspired scientists to get more involved in G-quadruplexes research. Some scientists aim to discover small molecules for stabilization of G-quadruplex structure that could potentially lead to cancer therapeutic drugs. In this work, we study the binding modes of G-quadruplex stabilizers and develop molecular probes that can recognize different DNA structures. First, we compare the binding affinity, between carbazole derivative (BMVC-4) and diphenylamine derivative (BMVPA-4) with G-quadruplex by using absorption, fluorescence, circular dichroism spectra and gel electrophoresis experiments. We found the rigid carbazole structure could stabilize G-quadruplex DNA more effectively than flexible diphenylamine structure. Furthermore, owing to the low intrinsic fluorescence intensity of BMVC-4 and BMVPA-4, we could obtain the Raman spectra through resonance Raman, and do the vibrational mode assignments to identify the functional group which is mainly used to interact with DNA. The shifts of Raman lines imply the interaction of BMVC-4 to G-quadruplex is larger than that to duplex DNA while BMVPA-4 show the opposite interaction. So, BMVC-4 and BMVPA-4 could cooperate in DNA structure recognition. In addition, since BMVPA-4 is more flexible than BMVC-4, it is more sensitive to DNA structure differences, especially in investigating the variations of bcl2mid and bcl2mid-M (two bases mutant of bcl2mid). Also, our results implicate AT loci as the primary sites of BMVPA-4 intercalation. Therefore, we suggest that BMVPA-4 is a better Raman probe for DNA structure recognition. Based on these works, we could not only extend the application of Raman probes, but also provide information to drug design of DNA stabilizers in the future.