A Combination Of Multiple Methods Is Used To Analyze The Interaction Between Human C-myb Oncogene G-quadruplex DNA And Natural Products

The G-quadruplex in promoter region of human oncogene plays a crucial role in the transcription and replication of gene,and has become a key part of genomics,clinincal medicine and drug design research.In recent years,the experimental methods for analyzing the structures and specificity of G-quadruplex have developed rapidly.Because the special structure was dynamic variable structure,many methods need to label DNA,including surface plasmon resonance(SPR),fluorescence energy resonance transfer(FERT)and molecular beacon,and may affect the formation of G-quadruplex.At the same time,there is the equilibrium between the strucutres of DNA double helix,G-quadruplex and i-motif due to the existence of complementary sequences in life.Therefore,it is necessary to design and establish an analytical method that can efficiently identify the structural of DNA and the mechanism of binding ligandsBased on the above of thinking,the specific contents and results of this subject are as follows1.A section of G-rich sequence S1,which is downstream of the 17th base of the human C-myb proto-oncogene transcription initiation site,was selected.First,natural products(Chrysin,Solasonin,Solamargine and Solasodine)were screend out to bind to S1 G-quadruplex with obvious affinity by the electrospray ionization mass spectrometry(ESI-MS).Then,the interaction of S1 with ligands(Chrysin,Solasonin,Solamargine and Solasodine)was studied by UV experiments and CD titration.The results showed that the order of binding constants was Solasodine>Chrysin>Solasonin?Solamargine.The results of CD-melting experiments showed that the greater affinity of alkaloids ligands to G-quadruplex was stronger for the stability of the G-quadruplex structure,and the stability of the G-quadruplex would deteriorate due to flavonoids Chrysin bingding to the structure.The experiments of fluorescence confirmed that the four natural products may be bind to the groove or surface of G-quadruplex.2.In order to establish capillary electrophoresis(CE)for analyzing interaction accurately,the interaction between cyclodextrin of the naphthalene derivatives was used as the model for quantitative analysis,and theoretical calculation was used to evaluate the results of CE.The selected naphthalene derivatives were different in the positions of sulfonates and carboxylates,and affinity capillary electrophoresis(ACE)based on viscosity correction analyzed the interaction between cyclodextrin of the naphthalene derivatives and obtained the binding constants.In addition,the relative binding energy was obtained by molecular simulation and proved the accuracy of ACE results.3.The interaction of S1 G-quadruplex and alkaloid was studied by ESI-MS and frontier analysis of capillary electrophoresis(CE-FA).Fisrtly,the three natural molecules that could bind to the G-quadruplex was screened out by ESI-MS,and the sequence of affinity was Pseudolaric acid B>Scopolamine N-butyl bromide>Nuciferin.Then,the specificity of interaction between three small molecules and S1 in liquid phase was studied by the CE-FA.The results of CE-FA were as follows:Pseudolaric acid B was not interacting with S1.The reason may be that Pseudolaric acid B and G-quadruplex carry negative charge and repel each other in CE-FA.In the process of electrospray ionization,negatively charged Pseudolaric acid B improved the ionization efficiency of DNA and induced false positive of ESI-MS.Beside,Nuciferin interacted with S1 G-quadruplex,but was non-specific binding-Scopolamine N-butyl bromide interacted with S1 G-quadruplex specificly,and the binding ratio was 1:1 and the binding constant was 1.18×105M-1.