Site-specific Quantification Of 5-carboxylcytosine In DNA By Chemical Conversion Coupled With Enzyme Assisted Ligation

5-Methylcytosine(5mC)is an important epigenetic modification in mammals.The active DNA demethylation could be achieved through the ten-eleven translocation(TET)protein-mediated oxidization of 5m C with the generation of 5-hydroxymethylcytosine(5hm C),5-formylcytosine(5f C)and 5-carboxylcytosine(5caC).It has been known that5 m C,5hm C and 5f C play critical roles in modulating gene expression.However,unlike the 5m C,5hm C,and 5f C,the functions of 5caC are still underexplored.Investigation of the functions of 5caC relies on the accurate quantification and localization analysis of5 caC in DNA.At present,the quantitative analysis of 5caC mainly relies on liquid chromatographymass spectrometry(LC-MS)technology,and the quantitative information of 5caC is obtained.The high-throughput sequencing of 5caC in DNA is mainly based on the traditional bisulfite sequencing method,which can obtain the single-base resolution information of 5caC in genome.However,it is expensive and time-consuming.Meanwhile,the harsh reaction condition of bisulfite treatment will lead to the significant degradation of DNA.Besides,the site-specific quantitative information of 5caC could not to be obtained through the quantitative analysis and high-throughput sequencing of 5caC.The existence of modifications in DNA or RNA may affect the formation of hydrogen bond between the modified nucleobases with complementary nucleobases,and DNA ligase has an ability to identify the single-base mismatch and weakened base pairing.Therefore,in the current study,we developed a method by chemical conversion in conjugation with enzyme assisted DNA ligation-real-time quantitative PCR(q PCR)for the site-specific quantification of 5caC in DNA.This method depends on the selective conversion of 5caC to form dihydrouracil(DHU)by pyridine borane treatment.DHU behaves like thymine and pairs with adenine(DHU-A).Thus,the chemical conversion by pyridine borane leads to the transformation of base paring from 5caC-G to DHU-A,the unaffected C will mismatch with A.The method is utilized to achieve the site-specific detection and quantification of 5caC in DNA.As a proof-of-concept,the developed method was successfully applied to distinguish the synthesized DNA template 5caC-DNA from C-DNA,and the 5caC level in DNA mixture was quantitatively evaluated.Otherwise,as low as 125 f M of 5caC-DNA could be identified from the blank.Besides,the method was successfully applied in the site-specific quantification of 5caC in synthesized DNA spiked in complex biological samples,and the recovery was between88.0%-91.1%.The method is rapid,straightforward and cost-effective,and shows promising in promoting the investigation of the functional roles of 5caC in future study.