Study Of 5-methylcytosine Detecting Method Based On A DNA Strand Exchange Reaction

5-methylcytosine(5mC)is an epigenetic modification that frequently appears in CpG islands.It has been reported that the global hypomethylation of genomic DNA and frequently methylated in CpG islands within gene promoters could result in the activation of oncogenes and the high rate of occurrence of various diseases.Given the strong correlation of this genetic modification with gene regulation,genomic imprinting and various aspects of gene activity control,the selective detection of 5mC in genomic DNA has great significance.A lot of research studies have been conducted to discriminate 5-methylcytosine from non-modified cytosine.Many of the strategies are based on the selective deamination of cytosine with bisulfite,which were restricted to harsh reaction conditions and limited resolution.Approaches based on the methylated DNA immunoprecipitation method were widely used to detect 5mC,but they are time-consuming and high cost.Complicated pretreatments,stringent apparatus requirements,being labor-intensive and time-consuming together restrict the development of 5mC detection.Cytosine modifications could induce numerous conformational alterations in the local structure of duplex DNA.Many reports have revealed that 5-cytosine methylation increases helical pitch and unwinding and influences the formation of DNA cruciforms.Inspired by the influence of 5mC on the conformation of duplex DNA,we designed a SER system that can differentiate 5-methylcytosine from normal cytosine.In the SER,single-stranded DNA(ssDNA)is selectively recognized by its methylated homologous double-stranded DNA(dsDNA).Then,the complementary strand is melted,paired with the ssDNA and the subsequent base pair exchange proceeds until they formed a stable heteroduplex product,while the non-methylated dsDNA is hardly exchanged.This method is fast and enzyme-free,works efficiently under physiological conditions,and is non-external-energy-dependent.It almost does not require apparatus.All the reagents used in the reaction are very common in the laboratory.Furthermore,the SER is highly sequence selective,and requires no conversion of target sequences.This feature widely broadens its applicable scope to almost any sequences more than just some finite targets.It supports simultaneous monitoring of multiple methylated sites in a single system by using different labels.Besides,the fluorescence signal is easy to be observed and calculated,and its intensity can intuitively reflect the methylation levels of target dsDNA.This detection method is so widely adaptable and low-cost that it can be easily applied to other various DNA nanotechnologies.