The Formation, Character And Identification Of G-quadruplex And I-motif Structures In C-myb Proto-oncogene

There exists so many repetitive sequences in human tolemere and oncegene promoters which can fold into special secondary structures. The structure that exist G-rich sequences is called G-quadrulex, and they are proved to be formed under the conditions of physical temperature, pH and ionic strength. The past researches showed that G-quadrupex structure exist in mammalian cells by using appropriate ligands in cell environment. And some ligands binded to G-quadruplex structure have also been used to inhibit telomere and control the expression of oncogenes. Among genomic DNA, the complementary sequences of G-rich sequences are C-rich ones which have the potencial to fold into i-motif structure. In vitro experiments, it was found that stable i-motif structure was formed when pH is below 7 and in the physiological temperature and ionic strength. Even though it remains uncertain that the existence of this structure in cells and the possible intervention of cell biology chemical targets, but what inspires people to research the biological function of i-motif is the protein isolation binding to this structure. So, there has important research significance of i-motif structure.Two G-rich sequences and one C-rich sequences are selected from C-myb oncogene promoter. ESI-MS and CD were utilized to explore the formation, property and recognition of these structure.The main contents and results of this research are as followed:1.The two G-rich sequences S1, S2 and the complementary chain of S1, S3, which are selected from C-myb oncogene promoter. First,it was proved that S1 and S2 could fold into G-qudruplex in NH4OAc solution and the dimer structure of S1 G-quadruplex was formed in high concentration of NH4OAc solution. CD experiment was utilized as the supplementary mean to verify the results of ESI-MS. They formed parallel strand G-quadruplex both in the solution of NH4OAc and KCl. Small molecule brucine was screend out to bind to S1 G-quadruplex with high affinity and good selectivity. Brucine bind to S1 G-quadruplex preferentially even though short chain duplex and genenomic duplex chain coexist in the solution. The binding of brucine to S1 G-quadruplex promote its stability using CD-melting, heating up by ESI-MS, ESI-MS/MS spectrometry.2. The G-rich sequence S4 was selected from C-myb oncogene promoter as the research chain. First,the results of ESI-MS and CD showed S4 fold into G-quadruplex with parallel/anti-parallel hybrid configuration in both NH4OAc and KCl solution. Study of the impact of solution condition showed that content of G-quadruplex structure increased with concentration of cation and configuration varied from parallel to anti-parallel with the increasing of methanol. Protopine was selected as the ligand of S4 G-quadruplex for the sake of its high affinity and good selectivity to this structure. ESI-MS/MS was utilized to prove small molecule could promote the stability of G-quadruplex.3. The two C-rich sequences S5, S6 and the complementary chain of S6, S7 are selected from C-myb oncogene promoter. First, the results of ESI-MS and CD showed S5 and S6 fold into G-quadruplex in the NH4OAc solution with pH4.5. And then changing the condition of solution to study the sturucture, as a result that facilitate the formation of i-motif is pH and crowding enviroment. The acid environment also promote the melting temperature of both i-motif. The binding of molecule fanchinoline to S6 i-motif and the competition experiment with its complemetary chain were also conducted.