Liquid Chromatography-mass Spectrometry For The Study Of RNA Hydroxylmethylation

RNA modifications,especially N⁶-methyladenine?N⁶-methyladenosine,m⁶A?,participate in the regulation of many physiological processes,including the mRNA splicing,the mRNA transport and the mRNA stability,which has important effect on immune tolerance.Therefore,m6A is an important epigenetic modification.The latest research suggests that m6A modification is reversible.The FTO and ALKBH5 protease catalyze it to produce hydroxylmethylation form of hm6A?N⁶ hydroxymethyladenosine?which is an unstable intermediate.The process is closely related to many human diseases and cell growth.Similarly,5mC in DNA is catalyzed by Tet protein?Ten to Eleven Translocation?,relying on alpha glutaric acid and Fe^2?catalytic oxidation to form 5-hydroxymethylcytosine?5hmC?,5hmC can also be catalyzed by Tet protein and further oxidizes to 5-aldehydecytosine?5fC?and 5-carboxylcytosine?5caC?.Such methylation phenomenon in DNA and RNA might exist similar mechanisms,namely methylation modification may be catalyzed by enzyme to form hydroxyl methylation modification as the intermediate,and then revert to normal nucleosides.We hypothesized that there might be hm²G,hm⁴C,hm⁵CM hm⁶Am in the organism,and they were produced by m²G,m⁴C,m⁵Cm,and m6Am under the action of the enzyme.Based on previous researches,we know that hydroxylmethylation of hm6A is unstable.It is difficult to accurately analyse hm6A by the conventional enzyme solution method,and because of the lack of standards comparison,we are difficult to determine whether new hydroxymethyl modified nucleosides exist in RNA.In the research of this paper,we developed a new analytical method for hydroxymethyl modification on the above two problems.The main contents of this work are as follows:We set up a new enzyme digestion method,choosing phosphodiesterase and alkaline phosphatase as the main enzyme,and carried out in moderate neutral pH to avoid the conventional digestion process of high temperature heat denaturation and acid or alkali environment.Through enzymatic hydrolysis efficiency investigation,we found that at 37 ?,under the condition of pH 7.0,4 h can make 15 ug RNA completely digest,and hydroxymethyl content of modified nucleosides reached the highest.Meanwhile,we synthesise hm4C,hm2G,hm6A hm5Cm,hm4Cm,hm6Am as standard comparison,confirmed by secondary mass spectrometry and high resolution mass spectrometry,greatly increased the qualitative accuracy of the hydroxymethyl modified nucleosides in actual samples.Based on the sensitive analysis of high performance liquid chromatography-tandam mass spectrometry,we detected six hydroxyl methylation modification in 293T cells,HeLa cells and thyroid tissues,reducing the false positive in the testing process.In addition,compared with normal people,we found that the m2G in the tissue of thyroid cancer significantly increased,illustrates the RNA modification may have a regulating effect on cancer,which laid a good foundation for the pathology and physiology research.The discovery of these new hydroxymethyl modifications further proves that the demethylation mechanism of the methylation modification we suspect is a general rule.