Studying On The Molecular Recognition Mechanism Of Aptamers And G-quadruplex Conformation In Complex Environment

Nucleic acids are an important class of biological macromolecules involved in every process of life activity.This dissertation aims to study the following two aspects of nucleic acids mainly using nuclear magnetic resonance methods:1,studying on molecular mechanism of artificial Aptamer recognizing certain target with efficiency and specificity;2,conformational study of DNA G-quadruplex under cell-like and living cell environment.Firstly,two DNA Aptamers selected to discriminatory recognize OTA/B were investigated in molecular mechanism study.The results showed that OTA was identified by OBA18 with a ligand-conformation dependent induced-fit mechanism.Combining with molecular simulation method we further clarified that Mg2+ regulated the precise recognition of OBA18 by changing the conformation of ligands to amplify their one atom difference.At the same time,the complex structure of OBA18-OTA was resolved,which help us to expound the mechanism and effect of the recognition process between Aptamer and small molecule toxins at the atomic level,and provide a structural basis for design and synthesis of novel aptamer probes and drugs.Following,in research of recognition mechanism of OBA32 and OTA,the conformation selection mechanism was determined by NMR and CD spectroscopy.And it is found that the joint interference of Mg2+ on both Aptamer and ligand controled the occurrence of the recognition process.Then specific site labeling was used for ambiguous asignment of hydrogen bonds in the complexe.Thereby the G-quadruplex secondary structure’ of OBA32-OTA complex was built,which provides the topology foundation for construction of G-quadruplex ligand three-dimensional structure.G-quadruplexe(G4)structures are believed to exist abundantly in living cells and play essential roles in regulating cellular process,which makes them attractive therapeutic targets.Many in vitro studies have shown that G4 conformation is quite sensitive to environment.So in order to obtaine biological relevant G4 conformation,we firstly characterized G4 conformation under macromolecular crowding and confinement environment.The results showed that crowded environment had little effect on the two kinds of G4 conformations of human telomeric DNA but induced increased thermal stability of them.However,confinement environment promoted K+stablized G4 conformation transformed to Na+ stablized conformation in contrast.Namely,cell-like environment affect the conformation and stability of telomere DNA G-quadruplexe significantly.Therefore,we further quantitatively detected G4 multiple conformation for a defined DNA sequence in Xenopus oocytes and Hela cells.The G4 conformational dependence on cell type demonstrates the importance of studying DNA conformation under natural cellular conditions.Here,we suggest that 19F NMR is a simple and efficacious method for quantitative characterization of nucleic acids conformation under physiological environment.We believe 19F NMR will be a powerful tool for further investigating the conformation,dynamics,and interactions of nucleic acids in cell-like or living cells condition in the future.