DNA Methylation Detection And Small Molecule-mediated G-quadruplex Imaging And DNA Damage Repair

Gene regulation is an important mechanism for controlling gene expression in organisms and it is the basis of cell differentiation,morphogenesis and ontogeny in organisms.The influencing factors of gene regulation include histone modifications,DNA methyl ati on and other epigenetic modifications,secondary structure of nucleic acids(DNA/RNA)and chromatin conformational changes,etc.The study of the influencing factors of gene regulation helps people to explore the complex regulatory relationship and regulation between genes,which has important biological and biomedical significance.Epigenetics plays an important role in gene regulation.DNA methylation is the most widely studied epigenetic modification,which can regulate gene expression,transcription and splicing by changing the physicochemical properties of DNA sequences and the state of chromatin.Some genetic diseases and malignant tumors are often closely related to abnormal DNA methylation.Methylation detection has become one of the most important tests in early cancer diagnosis.In addition to epigenetic modifications,nucleic acid secondary structure is also crucial in gene regulation.Among the secondary structures of nucleic acids,G-quadruplex(G4)has attracted much attention due to its biological functions.Studies have shown that G4 plays a vital role in regulating gene expression and transcription,protein translation and proteolysis,DNA repair,maintaining the stability of chromosome ends and epigenetic regulation.Given the important roles of DNA methylation and G4 in gene regulation,we have proposed a single-molecule strategy for the direct identification of 5mC by the combination of DNA fiber FISH and AFM and imaged mitochondrial DNA G4s in living cells by small molecule fluorescent probes.In addition,in order to further explore how G4 structure regulates genes and affects the cell fate,this paper also studied the mechanism of G4 ligandinduced DNA damage and its repair.The specific research contents of the full text are as follows:1.Single-molecule analysis of genome-wide DNA methylation by fiber FISH coupled with AFM.In this chapter,DNA fiber-FISH and AFM technology were used to realize the visualization of 5mC in whole genomes of several cell lines at single-molecule level,the characterization of anti-5mCDNA complexes and detailed spacing distribution of two neighboring 5mC sites on single DNA molecule.Furthermore,this approach can be applied for reporting other epigenetic modifications,not limited to 5mC or one single epigenetic modification.It can be anticipated to contribute to the development of clinical diagnosis of epigenetic-related diseases.2.Imaging of mitochondrial DNA G-quadruplexes in living cells.The folding and unwinding of mitochondrial DNA G4(mtDNA G4)is associated with mitochondrial DNA replication,transcription and mitochondrial genomic stability.In this chapter,we specifically lighted mtDNA G4 structures by the two-photon fluorescent molecule CQ.In addition,due to the excellent photoblinking and high photostability,CQ has been used for super-resolution imaging of mitochondrial DNA G4.This research will provide important reference value for the design and application of fluorescent dyes for biological imaging and super-resolution G4 imaging.At the same time,it can also provide a favorable visualization tool for the study of the role of mitochondrial DNA G4 in gene regulation.3.G-quadruplex ligand induced DNA damage.In this chapter,we confirmed that G4 ligand PyBI could induce and stabilize G4 in vitro and in cells via a series of experiments.PyBI can induce replication fork arrest,activate cellular checkpoints and cause cell cycle arrest,thereby cause cytotoxic DNA double-strand breaks(DSBs).DSB-seq data showed that the position of DSBs caused by PyBI highly overlapped with the formed G4s,which provides direct evidence for the theory that G4 induced by G4 ligands can lead to DNA breaks.In addition,the data also showed that G4 ligand was involved in the biological process,molecular function and cellular component,which will promote the study of various regulatory roles of G4 in cells.4.G4 DNA damage repair mechanism based on synthetic lethality.In this chapter,we found that PyBI had an ideal synthetic lethality in combination with DNA repair pathway inhibitors.According to the results of DSB-seq and inhibitor screening,we selected HR and NHEJ pathway inhibitors of DSB repair for further research.The combined strategies can significantly inhibit the proliferation of cells in long-term medication.These studies indicate that PyBI is expected to be used in tumor therapy,especially for tumors with DNA damage repair gene defects,which will open up new avenues for precise tumor therapy.In addition,PyBI can be used as a scaffold,which has the potential for modifications,and this has important reference value for the design of more optimized G4-specific ligands and anti-cancer drugs.