Label-free Chemiluminescent Biosensor For Sensitive Detection Of 5-hydroxymethylcytosine In Genomic DNA

Epigenetic inheritance is a heritable change in gene expression without the involvement of DNA sequence change.In the mammalian genomes,5-methylcytosine(5mC)and5-hydroxymethylcytosine(5hmC)are the two predominant epigenetic markers.5hmC is an oxidative intermediate in ten-11 translocation-mediated oxidation of 5mC.It is a modified base presented at low levels in diverse cell types in mammals,and plays key roles in DNA demethylation,genomic reprogramming,and gene expression.Aberrant DNA hydroxymethylation has been associated with various diseases and is a predictive biomarker for cancers,neurological abnormalities,and perilous diseases.Therefore,the accurate detection of5 hmC in genomic DNA is very important for the study of epigenetic regulation of disease development and the early diagnosis of cancer.Since 5hmC exists in lower abundance and has a similar structure to 5mC and cytosine C,the methods for the 5hmC assay should possess high sensitivity and good specificity with the capability of eliminating the interference from 5mC and cytosine C.However,reported 5hmC assays often involve expensive and complex instruments,radiologically labeled substrates,and specific antibodies.Recently,some amplification strategies have been introduced to improve the sensitivity,including boronic acidmediated polymerase chain reaction and ligation-mediated rolling circle amplification,but they inevitably suffer from nonspecific amplification and even false positivity and require the careful design of primers,labeled nucleic acid probes,and specific templates.Moreover,these methods enable only locus-specific and fragment-specific detection of 5hmC.Therefore,new strategies for 5hmC detection are highly desirable.Chemiluminescence is the optical radiation produced by chemical reactions,and its signal rintensity is less disturbed by autofluorescence,scattered light,and photobleaching,enabling the achievement of high sensitivity and high signal-to-noise ratio.Herein,we develop a label-free and template-free chemiluminescent biosensor for sensitive detection of 5hmC in genomic DNA samples based on glucosylation,periodate oxidation,biotinylation,and terminal deoxynucleotidyl transferase-assisted isothermal amplification strategy.This strategy exhibits distinct advantages of bisulfite-free,improved sensitivity,and genome-wide analysis of 5hmC at constant reaction temperature without the involvement of either specially labeled nucleic acid probes or specific templates for signal amplification.The method can sensitively detect 5hmC with a detection limit of 0.21 p M,and it can detect 5hmC in the whole genome DNA with a detection limit of 39 fg/μL.Moreover,this method can distinguish 5hmC from 5mC and cytosine C,and even discriminate 0.1% 5hmC in the mixture of5 hmC-DNA and 5mC-DNA.Importantly,this proposed biosensor enables genome-wide analysis without the involvement of either isotope-labeled substrates or specific antibodies,providing a powerful platform for the detection of 5hmC in real genomic DNA with high reproducibility and accuracy.