Development Of Ultrasensitive Methods For The Detection Of Epigenetic Biomarkers

Epigenetic inheritance is the mitotically or meiotically heritable change in gene expression or cellular phenotype that occurs without changes in base-pairing of DNA.It can regulate many cellular processes（e.g.cellular embryogenesis,and genomic imprinting）of the organisms through controlling gene expression and transcription.Enormous researches have shown that the abnormal level of epigenetic inheritance in human cells can lead to the occurrences of developmental disorder,senile dementia,and carcinogenesis progression.Thus,epigenetic inheritance can provide new opportunities for disease diagnosis,treatment and preventionThe molecular basis of heritable epigenetics has been studied in a variety of organisms,and a series of epigenetic biomarkers have been discovered and studied,including DNA methylation,DNA hydroxymethylation,noncoding RNA（ncRNA）,RNA methylation and histone modification.The accurate detection of epigenetic biomarkers is not only of importance to epigenetics-related biochemical research,but also of significance to diagnosis,prognosis,and therapy of cancers.Conventional biosensors for epigenetic biomarkers detection mainly include northern blotting,radiometric assay,mass spectrometry,and immunosorbent assay.But they suffer from poor sensitivity,time-consuming steps,large sample consumption,and the involvement of radioactive materials,expensive instruments,complicated sample preparations,labourconsuming operations.In this thesis,we use DNA methylation,DNA hydroxymethylation,ncRNA and RNA methylation as the models to develop a series of simple,selective and sensitive methods for detection of a variety of epigenetic biomarkers in combination with novel material（quantum dot（QD）,magnetic bead（MB））and nucleic acid amplification technology,fluorescence spectrum analysis,single-molecule detection technology.The detailed contents are as follows:1.We develop a single QD-based nanosensor for sensitive detection of DNA methylation at both cytosine/guanine dinucleotides（CpG）and non CpG sites using tricyclic ligase chain reaction（LCR）-mediated QD-based fluorescence resonance energy transfer（FRET）.We design two sets of DNA probes（X and Y,X’and Y’）for methylated DNA assay.In the presence of thermostable DNA ligase,probes X and Y may adjacently hybridize with the methylated DNA to obtain the ligated XY products which may function as the templates for probes X’and Y’to generate the X’Y’products.The resultant X’Y’products may in turn act as the templates to ligate probes X and Y for the generation of XY products,consequently inducing tricyclic LCR amplification under thermal denaturation conditions to generate a large number of XY products.The subsequent hybridization of XY products with the capture and reporter probes results in the formation of sandwich hybrids which may assemble on the 605QD surface to obtain 605QD–oligonucleotide–Cy5 nanostructures,inducing efficient FRET from the 605QD to Cy5 and the emission of Cy5.This nanosensor can detect DNA methylation at single 5-methylcytosine（5mC）resolution with a detection limit（LOD）of as low as 1.00×10^-17M and a large dynamic range of7 orders of magnitude.Moreover,this nanosensor can distinguish as low as a 0.01%methylation level,and it can detect DNA methylation in human lung cancer cells as well,holding great potential for accurate epigenetic evaluation and early cancer diagnosis2.We develop a label-free fluorescence method for sensitive discrimination of5-hydroxymethylcytosine（5hmC）and 5mC based on the specific oxidation of 5hmC to5-formylcytosine（5fC）and the subsequent conversion to uracil（U）in combination with ligation mediated rolling circle amplification（RCA）.The 5hmC-DNA are oxidized with KRu O₄ and then subjected to the bisulfite treatment/Na OH conversion protocol.The 5hmC will convert to U.Then this probe may function as the template for the ligation of padlock probe and can subsequently primer the isothermal RCA reaction,producing large numbers of single-stranded DNA fragments of variable lengths that can be simply quantified using SYBR Green II fluorescent dye.This method exhibits high sensitivity with a LOD of as low as 3.48×10^-14M.It can discriminate as low as the 0.01%5hmC level from the mixture,and exhibits good performance in serum sample analysis as well.This research opens a new avenue for ultrasensitive 5hmC assay and is useful for the study of methylation/demethylation regulation and epigenetic evaluation3.We develop a single-QD-based FRET nanosensor to accurately detect micro RNAs（miRNA）based on copper-free and enzyme-free cycling click chemistry-mediated tricyclic LCR amplification.When target miRNA is present,two DNA probes（i.e.,DNA probes1 and 2）modified with azide（N₃）and dibenzocyclooctyne（DBCO）,respectively,can proceed copper-free and enzyme-free click chemistry to generate probes 1-2 ligation product.Subsequently,two DNA probes（i.e.,DNA probes 3 and 4）modified with N₃ and DBCO,respectively,can hybridize with probes 1-2 ligation product to generate probes 3-4 ligation product.Both probes 1-2 ligation product and probes 3-4 ligation product can act as the templates to initiate cycling click chemistry-mediated tricyclic LCR amplification whose products can be easily measured by the single-QD-based FRET nanosensor.This assay does not involve any enzymatic reverse transcription,copper catalyst,and ligase enzyme,and it exhibits excellent selectivity,high sensitivity(LOD is 3.87×10^-17 M),and the capability of differentiating even single-base mismatch.This method can accurately quantify miRNA-155even at the single-cell level,and it can distinguish the miRNA-155 expression in tissues of healthy persons and nonsmall cell lung cancer（NSCLC）patients as well.4.We developed a single-molecule detection method to mesure methylation of adenosines at the N⁶ position（m⁶A）in messenger RNA（m RNA）based on m⁶A-sensitive endoribonuclease and terminal deoxynucleotidyl transferase（Td T）-induced amplification.In this assay,m⁶A site can block the toxin protein（Maz F）endonuclease-mediated cleavage of target peobe,resulting in the target peobe can hybridize with the capture probe and primer probe to form a sandwich hybridization structure.Then the MBs will combine with the sandwich hybridization structur and Td T enzyme will catalyze the efficient polymerization extension by incorporating a large number of Cy5 fluorescent nucleotides into biosensor to generate a poly Cy5 fluorescent nucleotides sequence and produce the signal amplification reaction.After magnetic separation and exonuclease I（Exo I）cleavage,the Cy5 fluorescent molecules in the Td T enzyme-mediated amplification products will be released into the solution and subsequently be quantified by single-molecule detection.This assay exhibits excellent selectivity and high sensitivity with a LOD of as low as 2.24×10^-17 M.And it has a large dynamic range of 7 orders of magnitude from 1.00×10^-16 M to 1.00×10^-9 M.It can discriminate as low as the 0.01%m⁶A-m RNA level from the mixture.This assay exhibits good performance in serum sample analysis and can accurately quantify m⁶A-m RNA in cells.This research opens a new avenue for ultrasensitive m⁶A assay and is useful for the study of methylation/demethylation regulation and epigenetic evaluation...