Study On The Modification And Metabolic Labeling Of Nucleic Acids Based On Oxidative Amination Of Thionucleosides

Thionucleosides are a class of sulfur-containing modified nucleosides formed by replacing the oxygen atom directly attached to the pyrimidine(or purine)ring with a sulfur atom and possessing a wealth of chemically reactive properties.Natural thionucleosides exist in the transport ribonucleic acid(t RNA)of various organisms and serve to stabilize the structure and function of t RNAs,while non-natural thionucleosides can be incorporated into nucleic acids by chemical synthesis or cellular metabolism.Chemically synthesized thionucleoside-containing oligonucleotide probes are usually used to study the interaction between nucleic acids and other biomolecules in vitro,while metabolic labeling of nucleic acids with thionucleosides are utilized to track the dynamic processes of nucleic acid in cells.Therefore,it is important to develop the methods of analysis and modification of thionucleotides in nucleic acids for studying the biological functions of natural thionucleotides,constructing functionalized oligonucleotide probes,and revealing the molecular mechanisms of nucleic acid dynamics.In this dissertation,we explored the oxidative amination reaction of thionucleosides in nucleic acids and applied it to the analysis and modification of thionucleosides in nucleic acids,involving quantitative analysis of natural thionucleosides,post-synthetic modification of thionucleoside-containing oligonucleotides,and sequencing analysis of cellular genomic DNA metabolic labeling with thionucleosides.First,we designed a fluorescence derivatization and quantification method of 4-thiouridine in nucleic acids based on oxidative amination reaction.The high sensitivity and selective fluorescence quantification of 4-thiouridine in E.coli t RNA were achieved with the detection limit of 1.0 n M owing to the fluorescence enhancement of aminofluorescein after selective derivatization of 4-thiouridine by oxidative amination reaction.The changes of 4-thiouridine levels in t RNAs from E.coli under the UVA radiation was also quantified by this method.Then we presented a general method for post-synthetic modification of oligonucleotides based on oxidative amination reaction of 4-thio-2'-deoxyuridine.Oligonucleotides with certain functions were prepared by reacting 4-thio-2'-deoxyuridine in oligonucleotides with alkyl amines.Fluorophore-arylated amines were also attached to form fluorophore-arylated deoxycytidine in oligonucleotides,and the constructed fluorescent oligonucleotides have potential applications in biosensing.Finally,we established a DNA metabolic labeling analysis method based on oxidative aminolysis reaction of 4-thiothymidine combined with sequencing.The low cytotoxic 4-thiothymidine was used for metabolic labeling of nascent DNA,and the 4-thiothymidine incorporated in DNA was converted into 5-methyl-2'-deoxycytidine through oxidative aminolysis reaction.The method is well compatible with high-fidelity DNA polymerase and allows identification of labeled sites according to the mutation in sequencing read after the reaction,therefore,it enables enrichment-free and single-base resolution assay of metabolic labels in DNA and assessing DNA dynamics at the genomic level.