Quantitative Analysis Of 6-methyladenine In RNA Based On Ligation-polymerase Chain Reaction

N6-methyladenosine(m~6A)is the methylation of the sixth nitrogen atom in the RNA's adenine A,and it is the most common and abundant post-transcriptional modification in eukaryotic messenger RNA(mRNA).Recent studies have shown that m~6A presents not only in eukaryotic mRNA such as yeast,fruit flies,plants,mammals,but in viral mRNA.Besides,m~6A presents in other RNAs in the eukaryon,such as long non-coding RNA(lncRNA),ribosomal RNA(rRNA),transfer RNA(tRNA),and small nuclear RNA(snRNA).With the development of the enzymology,the discovery of methyltransferase(METTL 3,METTL 14,WATP)and demethylase(FTO,ALKBH5)for m~6A suggests that m~6A modification is revisable and dynamically regulated,and further indicates that m~6A modification is associated with gene regulation,including mRNA translation efficiency,stability and splicing.What's more,m~6A is related to many serious disease,such as the cancer and leukaemia.Therefore,the detection of m~6A in RNAs has gteat significance for the basic biological study and the diagnosis of disease.In this dissertation,we first find that T3 DNA ligase has a strong selectivity for m~6A,and then establish a new method to accurately recognize and quantify m~6A in RNA with polymerase chain reaction(PCR)based on the ligation reaction.Firstly,we choose the fragment containing the 2577th site of MALAT1 LncRNA(Metastasis-associated lung adenocarcinoma transcript 1,which has been reported to contain m~6A modification at the 2577th site)as the RNA target.Next,for detection of the RNA target,the DNA probe L1(left probe)and probe R1(right probe)are designed.Each probe contains two parts.One part is a universal primer-specific sequence used for PCR amplification and the other part is a target-specific sequence,which is respectively complementary to the RNA target.Besides,Probe L1 is modified with a phosphate group at its 5'-terminus and probe R1 is modified with two ribonucleotides at its 3'-terminus.When the RNA target's 2577th site is adenine A,Probe L1 and Probe R1 will adjacently hybridize with the RNA around the 2577th A site,and thus they can be ligated with the catalysis of the T3 DNA ligase.Then the ligation products can be subsequently amplified by PCR using the universal forward primer and reverse primer.During the amplification reaction,SYBR Green I is utilized as the fluorescent dye for real-time detection of PCR products.However,when the adenine A in the RNA target's 2577th site turn into m~6A after the methylation,the m~6A at the 2577th site can significantly hinder the ligation of probe L1 and probe R1.Then the ligated products and final PCR products will be greatly reduced.Finally,we can calculate the content of the m~6A in the 2577th site,according to the reduction of the PCR amplification products.With the proposed assay,the selectivity is up to 54.1-fold to discriminate m~6A against the adenosine A.What's more as low as 4 fM RNA-containing m~6A target can be detected and the sensitivity has been improved about 106-fold compared with the existing methods.This assay has been successfully applied to m~6A detection in the RNA extracted from the Hela and HEK293T cells.