Application Of C5-Derived Uracils In Post Synthetic Modification Research Of DNA

As the carrier of genetic information,DNA has been widely concerned.Single nucleotide polymorphism(SNP)refers to a single nucleotide(A,T,G,and C)variation causing the DNA sequence polymorphism at the genome level.SNP research has be-come a focus of common concern scientific researchers around the world.So that to design sensitive nucleic acid probe for detection of single-base variation through chem-ical approach is of great significance.DNA synthesis in the cell is a basic core process of biological cell proliferation.Quantitative monitoring DNA synthesis is often used to study the biochemistry of the cell cycle,organization development and regeneration,efficacy evaluation and many other important biological processes.In this thesis,research is based on the nucleotide modification in DNA,the main work is focus on using modified uracil to detect DNA bases variations and to monitor replication process in the cell.The research content is divided into two parts,the first part is the detecting SNP in vitro,the second part is the monitoring of DNA replication in the cells.In the first part,C-5 modified deoxyuridine-dTetU was the designed and synthe=sized,then its homologous phosphoramidite monomer for the solid-phase synthesis was synthesized,and through the solid phase synthesis dTetU was successfully inserted the oligonucleotide.The oligonucleotide containing dTetU itself has no fluorescence emis-sion,but irradiated under the light of 305 nm for 1 minutes,an intramolecular cycliza-tion reaction quickly happened with a molecular nitrogen release and the fluorescent emission was increased at the same time.This process can be verified by fluorescence spectrum and matrix assisted laser desorption ionization time of flight mass spectrom-etry(MALD-TOF-MS).Based on the fluorescence spectrometric,the reaction in oligo-nucleotide level apparent rate constant was calculated.We found that before UV irra-diation there was not any response to the change of base,but after ultraviolet activating the probe can distinguish different bases including AP site in fluorescence.In the second part,a C-5 vinyl modified deoxyuridine-dvinU and a bioorthogonally active and fluorescence quenched before tetrazine ligation reaction compound-Tz-DAF were designed and synthesized.In vitro test,we found that the reaction between vinyl modified nucleoside and Tz-DAF was completed after 1 hour,after that there appeared more than 10-fold of fluorescence enhancement,and the product was confirmed by electrospray ionization mass spectrometry(ESI-MS).By MTT assay to determine cy-totoxicity of dvinU,we found its IC50 value concentration is 164 ?M(less than the com-monly used alkynyl deoxyuridine DNA nucleoside EdU).And when the cell incubation,adding dvinU incubation cell cultures of 1 day with Tz-DAF staining for 1 hour,found strong green fluorescence in the nucleus of cells via confocal microscopy.In addition,high performance liquid chromatography(HPLC)and agarose gel electrophoresis for the cells' DNA extract were also analyzed.