Regulation Of G-quadruplex And Studies On Antitumor Activities By Benzimidazole Derivatives And Visual Observation Of G-quadruplex

The G rich strands could be folded up into G-quadruplex by Hoogsteen hydrogen bonds and exist in telomeres and gene promoters, which are genomic regions. Many groups have reported that targeting G-quadruplex nucleic acids with small molecules could be anti-tumor agents since they might block telomerase activities and regulate gene expression by stabilizing G-quadruplex nucleic acids. Therefore, many G-quadruplex binding ligands have been reported as candidates of anti-cancer drugs.In this thesis, the interaction of benzimidazole derivatives with G-quadruplex DNA and duplex DNA have been studied. Their antitumor activities of benzimidazole derivatives were evaluated by MTT assay, growth curve measurement, immunofluorescence, RT-PCR, and western blot. As mentioned above, small organic molecules are able to stabilize the G-quadruplex structure and could provide one stradegy for the discovery of novel anti-cancer agents. For efficient screening of G-quadruplex ligands, it is important that G-quadruplexes can be reliably detected. So, we also established a visual observation of G-quadruplex assay based on the AIE molecule silole. In summary, the main ideas of this thesis were as follows:1. In this thesis, the interaction of benzimidazole derivatives with G-quadruplex DNA and duplex DNA have been studied via a series of biological methods, including CD, exonuclease I hydrolysis assay, TRAP-LIG assay, polymerase stop assay, UV-Vis titration. The results showed that the bisbenzimidazole 1 was dicovered to bind helix DNA, while benzobisimidazole derivatives 2-4 were found to bind and induce different G-quadruplex isomers. The compound 2,3,4 have significant telomerase inhibitory activity, with IC50 values of 10.7,10.2 and 4.7μM respectively. Benzobisimidazole 5 was also a good c-myc G-quadruplex binder. We modified bisbenzimidazole to benzobisimidazole, and then their binding DNA styles changed from binding B-form duplex DNA to recognizing different modes of telomere G-quadruplex. It is concluded that the conformation of aromatic core is the determinate of binding selctivity:the strong and selective binding of compound 2,3 and 4 with intramolecular G-quadruplexes may be attributed to its large ring system, which overlaps completely with the G-tetrad plane; while the twist of dihedral angle between the two benzimidazole rings probably makes compound 1 suitable to interact with helix double-stranded DNA. On the other hand, the G-tetrad arrangement and the orientation of the loops are important factors for the interaction, and the ligand side chains play a significant role in selecting DNA topology.2. The antitumor activities of benzimidazole derivatives were evaluated by MTT assay, growth curve measurement, immunofluorescence, RT-PCR, and western blot. The toxicity of SGC7901 and Hela cell lines were both performed. We found that the compound 2-5, which could induce and stabilize G quadruplex formation, have good toxicity towards the tumor cells, but compound 1 seems less effective in both SGC7901 and Hela cell lines. Immunofluorescence results show that the compound 4 and 5 kill the Hela cell by DNA damage way. More importantly, we found that most of the DNA damage occurs at chromosome telomere. Besides, the G quadruplex ligands also reduce the expression of c-myc oncogene. All the results indicate that the compound 2-5 can inhibit the proliferation of cancer is quadruplex-relevant.3. In this study, AIE molecule silole 1 could be used to detect G-quadruplex formation using an exonuclease I hydrolysis assay. This visual observation of G-quadruplexes has been successfully used in investigating multiple G-quadruplexes, including the one-stranded telomeric, c-myc, c-kit and VEGF G-quadruplexes, and a d(G4T4G4) inter-molecular G-quadruplex. The detection of G-quadruplex can be also applied to G-quadruplex isomers induced by small molecules. Both TMPyP and compound 5 (BBP) were used in this assay. The results are identical to previous reports. This methord might be applied in a rapid-test kit for G-quadruplex ligands screening.