Interaction Study Of Compound3d With HSA And Screening Aptamer For MCF7Cell

Human serum albumin (HSA) has been developed to be a model protein to studydrug-protein interaction. In the present work, the interaction between our synthesizedflavonoid derivative3d (possessing potent antitumor activity against HepG2cells) andHSA was investigated using fluorescence spectroscopy, circular dichroism spectroscopy,UV-vis spectroscopy and molecular modeling approach. Fluorescence spectroscopyshowed that the fluorescence of HSA can be quenched remarkably by3d underphysiological condition with a slight shift of maximum fluorescence emission bandsfrom360nm to363nm. Calculated results from Stern-volmere equation and modifiedStern-volmere equation indicated that the fluorescence was quenched by staticquenching processing with association constant5.26±0.04×104L mol-1at298K.After comprehensive consideration of the free energy change ΔG, enthalpy change ΔHand entropy change ΔS, electrostatic interactions were confirmed as the main factorparticipate in stabilizing the3d-HSA complex. Both dichroism spectroscopy andUV-vis spectroscopy indicated conformational change of HSA after binding to3d.Moreover, the structure of HSA was loosened and the percentage of α-helix decreasedwith the increasing concentration of3d. Molecular modeling results demonstrated that3d could bind to HSA well into subdomain IIA, which is related to its capability ofdeposition and delivery. Three cation-π interactions and three hydrogen bonds occurredbetween3d and amino acid residuals ARG218, ARG222and LYS199. In conclusion,flavonoid derivative3d can bind to HSA with noncovalent bond in a relatively stableway, so it can be delivered by HSA in circulatory system.In this dissertation, another work was conducted to screen aptamer for MCF7cellusing SELEX method combined with RFD model. In this research, culture medium ofMCF7cell was set as the object of the study and we hope to identify MCF7cell throughits excretion. After24rounds of screening and enrichment, an oligonucleotide librarywas obtained and straight after that was cloning and sequencing. Some of the sequenceschosen from the identified group were found that they could interact with culturemedium of MCF7and generate obvious fluorescence signal. Investigation of theirspecificity of reactions showed that these sequences could not only recgnise MCF7cell but also two kind of liver cells (HepG2and HL7702), as well as MCF10A cell. Theseinformation indicated that secretion of these cell lines might all contain one kind ofmolecules and it happens to be the target molecule of the sequences. Sensetivityresearch demonstrated that the sequence ABE2-1could detect MCF7with its limitationof103cell/mL. At last, we used ultrafiltration membrane with their cut-off molecular of30kD,50kD and100kD to explore the molecular weigh of the target. From the resultswe confirmed that the molecular weigh of target is between30kD and50kD.