Research On The Formation, Identification And Quantitative Analysis Of The Quadruplex Nucleic Acid In The Promoter Region Of The Oncogene Bcl-2

Bcl-2 oncongene protein(B-cell CLL/lymphoma-2)is relevant to some important life processes,such as gene replication,cell information transmission,and the occurrence of diseases.The over-expression of Bcl-2 protein can lead to a series of cancers,just like bladder cancer,pancreatic cancer,breast cancer,thyroid cancer and other cancers.Researches have shown that the oncogene Bcl-2 promoter has a large number of guanine-rich(G-rich),and cytosine-rich(C-rich)regins,and they can be potential targets of quadruplex,which control the gene transcription and expression.In Recent years,the technologys and methods about the quadruplexs identifition,properties and its specificity recognition have a rapid development,so the DNA secondary structures gradually become the targets on genomics research and clinical medicine,drug design.Capillary electrophoresis.(CE)has a short analysis time,high resolution,small sample consumption,and is widely accepted in the field of biomolecular determination and drug screening.This thesis focuses on the capillary electrophoresis technology,through the establishment of the interaction between ligands and biological molecules,and aimed to find a rapid,accurate,and efficient quantitative analysis method of the specificity interactions.Thus provide theoretical basis and analysis strategy on cell recognition,information transmission,immune response,DNA transcription replication,and gene expression.Therefore,this thesis focuses on the quantitative analysis method to interactions between quadruplex and natural products,implement effective identify to the specificity interaction.Based on these ideas,I carried out the following three research contents:1.Select ibuprofen as the ligand molecule and its affinity with different cyclodextrins is used to demonstrate the method.First of all,Electrospray ionization mass spectrometry(ESI-MS)and CE-FA methods are respresent different affinity binding constants in gaseous phase and aqueous phase,respectively.Than,with the help of two different computer simulations,calculate the lowest stable energy in gaseous phase and aqueous phase,and a brief discussion is made to explain the order of different sizes of cyclodextrins successfully.We find that ESI-MS analysis method is the most powerful technique for measuring interaction,and CE is a good complement in free solution.2.We choose the oncogene Bcl-2 G-quadruplex as the target,establish the combination method of ESI-MS and circular dichroism,and explore the formation of G-quadruplex from G-rich sequence.Natural products with high affinity were select as the liginds to study the specificity interaction.In this sction,a combination analysis method is established to determine the binding constants and the ratio.The method first used ESI-MS to rapidly screen the small molecule candidates,and then confirmed the affinity of the binding pair with capillary electrophoresis frontal analysis(CE-FA).In addition,a new equation capable of extracting specific binding constants in the presence of non-specific binding without the need of reference compounds was proposed and tested.Using this reference-free method based on the CE-FA data,jatorrhizine and palmatine were found to bind specifically to the Bcl-2 promoter G-quadruplex.3.The oncogene Bcl-2 promoter i-motif structure was selected as the target to further verify the efficient and simple analysis method built in the previous system.First,the formation of i-motif structure was studied systematically.S2 sequence can spontaneously formed into the intramolecular i-motif structure in the acidic condition.Secondly,the effects of pH value and molecular crowding on the stability of i-motif structure were studied.Then,natural products were quickly screened by ESI-MS and flavonoids were found have high affinity to the i-motif structure.Finally,CE-FA was used to determine the interaction in solution,which can quantitatively calculate the binding constant of each step and ratio.We conclude that in all natural products that fisetin and luteo showed good affinity to Bcl-2 oncogene in all natural products.