Liquid Chromatography-mass Spectrometry For The Study Of DNA/RNA Modifications

DNA cytosine methylation plays regulation roles in growth and development as well as various biological processes. In addition to DNA methylation, recent studies demonstrated that RNA modification constitutes another important layer of epigenetic modification. RNA adenine methylation (N6-methyladenosine) has been considered to be dynamic and reversible and closely related to many physiological processes.In recent years, DNA/RNA modifications were studied from different aspects, including quantitative analysis of modifications, inveistigatioin of methylation and demethylation mechanism, exploration of new modifications, and functional correlation with multiple diseases, etc. However, there are still many intriguing points to be further investigated.Different from the widespread existence of m6A in RNA of organisms from all three kingdoms, m6 was mainly reported to exist in DNA of prokaryotes. The existence of m6A in eukaryotes, especially higher eukaryotes, remains to be further investigated. Besides, the demethylation of 5-methylcytosine (5-mC) in DNA has been extensively studied, but the demethylation mechanism of 5-mC in RNA remains unclear. Although many studies about m A have been reported, the role of m A in the formation and development of many disease is still unclear. In addition, present studies on DNA/RNA modifications mainly focus on analysis with a large amount of samples, while analysis of several cells has not been reported. Owing to the high sensitivity and selectivity of liquid chromatography coupled with mass spectrometry (LC-ESI-MS/MS), in this study, we established highly sensitive methods based on LC-ESI-MS/MS for the study of DNA/RNA modifications and further explored the functions of DNA/RNA modification.1. Study of adenine methylation in DNA. Due to the important functions of DNA adenine methylation (m6dA) in prokaryotes as well as the widespread existence of m6A in RNA, we employed high-resolution mass spectrometry to investigate the m6dA modification status in eukaryotic DNA. We established a highly sensiitive method based on LC-ESI-MS/MS for the analysis of m6dA in DNA. We found the widespread existence of m6dA in genomic DNA of higher eukaryotes including human cells, rat tissues, and plants, with the contents varying in different cell types.2. Study of adenine methylation and cytosine methylation in RNA.(1) By using chemical labeling coupled with LC-ESI-MS/MS analysis, we were able to simultaneously determined 5-mC,5-hydroxylmethylcytosine (5-hmC), 5-formylcytosine (5-foC) and 5-carboxylcytosine (5-caC) in RNA with high sensitivity. By using 2-bromo-l-(4-diethylamino-phenyl)-ethanone as the labeling reagent, the detection sensitivity of the four cytosine modifications increased by 70 to 313 folds. Using this method, we discovered 5-caC from mammalian RNA, which implys that RNA may undergo the same cytosine oxidative demethylation as that in DNA. In addition, we successfully quantified 5-mC,5-hmC,5-foC and 5-caC in RNA from tissues of liver cancer and colorectal cancer patients. The results showed a significant depletion of 5-hmC in tumor tissues compared to tumor adjacent normal tissues, suggesting 5-hmC in RNA may have profound effects on epigenetic regulation in development and formation of cancers.(2) We developed a quantitative method based on LC-ESI-MS/MS for the determination of m6A in RNA. Through the analysis of 180 RNA samples derived from human peripheral blood of T2DM patients and healthy controls, we found the m6A contents in the RNA from T2DM patients were significantly lower compared with the control groups. The results demonstrated that m6A content in RNA was highly related to the incidence of T2DM. m6A could be used as a novel potential biomarker of T2DM.(3) We established a highly sensitive method for the determination of DNA/RNA modifications in circulating tumor cells (CTCs) using an effective CTCs capture system coupled with a combined strategy of sample preparation for the captured CTCs lysis, nucleic acids digestion, and nucleosides extraction in one tube. With the developed method, we were able to detect DNA and RNA methylation (5-mdC, m6A, 5-mC) in a small amount of cells. We then further successfully determined DNA and RNA methylation in CTCs from lung cancer patients and found a significant difference between CTCs and the corresponding whole blood cells.In all, our results and findings set the basis for further understanding of the biological functions of DNA/RNA modifications.