Chemical Strategies To Study RNA Demethylation And To Detect 5-Formylcytosine

Nucleic acids carry a wide range of chemical modifications and exert different influences in a variety of cellular processes in eukaryotic organisms.Among these modifications,5-methylcytosine and N6-methyadenosine are the most abundant epigenetic modifications in DNA and RNA respectively which play crucial roles in gene expression and various biological processes.The discovery of demethlation pathway and oxidized intermediates suggest broader epigenetic regulation roles.In this thesis,we mainly studied two parts:N6-methyladenosine chemical demethylation study and detection of 5-formylcytosine based on chemical labeling method.N6-methyIadenosine is the most abundant epigenetic modification that ubiquitously occurs in the mRNAs of animals,plants and viruses,and play important roles in regulating mRNA splicing,nuclear export,translation,stability and miRNA synthesis.The discovery of FTO and ALKBH5 enzymes revealed the enzymatic demethylation mechanism of N6-methyladenosine,which implicate a dynamic and reversible role of N6-methyladenosine.N6-hydroxymethyladenosine and N6-formyladenosine intermediates identified in the demethylation process may substantially modulate RNA-protein interactions and dynamically affect the gene expression.These findings indicate that the RNA demethylation exhibit function roles in the physiological process.Here we find that the demethylation of N6-methyladenosine also ocurred in the presence of hydrogen peroxide and ammonium bicarbonate,which produces three oxidative intermediates during the reaction process:N6-hydroxymethyladenosine,N6-formyladenosine and N6-peroxyethyladenosine.The structures of the intermediates were identified by HPLC,NMR,mass spectrometry and fluorescence detection.The study showed that newly discovered N6-peroxymethyladenosine was the key intermediate in the demethylation process and the demethlation was mediated by hydroxyl radical mechanism.We also find the demethylation occurred in RNA oligos and RNA samples extracted from Hela cells.5-formylcytosine is an oxidation intermediate generated from iterative oxidation of 5-methylcytosine catalyzed by TET dioxygenases.Emerging evidence suggests that 5-formylcytosine is a stable epigenetic modification involved in many biological processes such as transcription and chromatin regulation.For further functional studies,there is still a need to develop alternative methods for 5-formylcytosine detection.We focused here on the use of Pictet-Spengler ligation for selective labeling of 5-formylcytosine using alkoxyamine derivatives and the combination with bisulfite sequencing method to detect DNA 5-formylcytosine sites.First,through HPLC,LC-MS,DNA mass spectrometry and gel electrophoresis experiments,we found the compounds quantitatively labeled 5-formylcytosine in single-stranded DNA and double-stranded DNA.From NMR and single-nucleotide primer extension assay,we confirmed that treatment of labeling 5-formmylcytosine with bisulfite resulted in a T?C mutation during PCR.Then the presence and number of 5-formylcytosine at defined sites was successfully confirmed through bisulfite sequencing method.Finally,we added a biotin group to the compound to enrich 5fC-containing DNA,this procedure further improved the detection sensitivity.