Selective Detections Of Epigenetic Modifications In DNA

Beyond the five cationic nucleobases A,T/U,C and G,DNA and RNA are decorated with aboundant modifications,such as 5-methylcytosine(5mC),5-hydromethylcytosine(5hmC),N-6 methyladenine(6mA and m6A).These chemical modifications can dynamically regulate the gene expression and provide mechanistic insights into the occurrence of diseases,and finally generated a new area of research,named as nucleic acid epigenetics.To better understand the exact roles of these modifications in biological process and diseases,in this thesis,we are mainly focused on the establishment of new analytical techiques,aiming at facilitating the accurate detection and mapping of these epigenetic modifications.DNA methylation mainly refers to 5-methylcytosine,which is verified involved in various biological processes.Numberous studies revealed that the aberrant methyaltion is closely associated with the occurrence of cancer,such as global hypomethylation and suppressor gene-specific hypermethylation.Expecially,the aberrant DNA hypermethylation of tumor-suppressor genes is always involved in the evolution of an invasive cancer from precursor lesions,which facilated the early diagnosis of cancer,as well as measuring response of tumors to targeted therapy.Be aware of the drawbacks of traditional methylation-specific PCR(MSP),we specially introduced Fluorescence labelled dGTP and asymmetric PCR into the bisulfite-treated DNA targets.Thus,the issue of methylation detection can be transformed into one of guanine quantification in the complementary DNA strand.Through the PAGE analysis,the methylation status at each CpG site can be monitored by fluorescent signal in a single reaction.Most importantly,this strategy is successfully applied for the determination of methylation status in different cancer cell lines and the cancer cells with 5-aza-2'-deoxycytidine(DAC)treatment.DNA methylation in mammalian genome is dynamically regulated during development.In active DNA demethylation,5mC can be oxidized by TET dioxyenases into 5hmC,5fC and 5caC in a stepwise manner.Compelling evidence suggests that these 5mC oxidative modifications carry additional functions beyond being just demethylation intermediates.Here we develop a new fluorescent strategy for the quantificative detection of 5hmC in genomic DNA based on CCP-FRET combined with KRuO4 oxidization.First,the 5hmC containing DNA is subjected to KRuO4 oxidization and transformed into 5fC with high efficiency.Then the oxidized DNA is labelled with hydroxylamine-BODIPY,followed by the capture of CCP through electrostatic interactions,which can generate significant FRET signal between CCP and BODIPY and enable the quantification of 5hmC.The selective labelling is verified through PAGE,HPLC and MALDI-TOF analysis.This strategy is successfully demonstrated for the 5hmC quantification in mEsc,HeLa and HEK 293T cells,benefiting from light-harvesting,large Stocks shift and optical signal amplification of CCP.Most recently,there has emerged several studies that reveal the presence of 6mA and clues to potential epigenetic functions in higher eukaryotes or even in vertebrates and mammalian embryonic stem cells,beyond bacteria,largly expanding the epigenetic scope of DNA methylation.Interestingly,we found Ag~+ can selectively stabilize the A-C mismatch and efficiently promote primer extension.In contrast,the complex of 6mA-Ag~+-C is instable and therefore cannot be recognized by DNA polymerases,resulting in the termination of primer extension.Based on this finding,we successfully identified and quantified 6mA at the single-base level through the analysis of gel bands of extended primers and fluorescence measurements combined with rolling circle amplification.