Sensitive Detection Of Epigenetic Biomarkers DNA Methylation AndmicroRNA Point Mutation

Epigenetics refers to the heritable changes in gene expression without a change in the DNA sequence itself.The abnormal expression of epigenetic modifiers is related to various diseases.Thus,the quantitative analysis of epigenetic modifiers is crucial to early diagnosis of disease,drug screening,prognosis of disease treatment,discovery of new biological function.Epigenetics mainly includes DNA methylation,histone modification and non-coding RNA.The traditional detection methods for epigenetic biomarkers mainly include enzyme-linked immunosorbent assay,mass spectrometry,Western blot and isotope assay.However,the above methods have unavoidable disadvantages,such as radioactive pollution,low sensitivity,cumbersome operation and expensive instruments,which limit their practical application.In this thesis,a series of epigenetic biomarker detection methods with high sensitivity and specificity were developed based on DNA methylation and RNA point mutation,combined with chemiluminescence and real-time fluorescence detection technology.The main contents of this thesis are as follows:1.Taking advantage of methylation-dependent cleavage and encoding flexibility in nucleic acid functions and structures,we demonstrate for the first time the cooperative in-situ assembly of G-quadruplex DNAzyme nanowires for one-step sensing of CpG methylation in human genomes.The presence of CpG methylation catalyzes the site-specific cleavage of 5-mCs by a new methyldirected endonuclease GlaI to create two incisions with 3′-OH termini.Then template-free TdT repairs the cleaved incisions,initiating single-base extension to generate the poly-T chains.The resultant poly-T products can hybridize with the poly-A loops of hairpins 1 to activate autonomous cross-opening of hairpins 2 and 3 through the toehold-mediated strand displacement(TMSD)cascades.The subsequent binding of DNAzymes with cofactor hemin forms the hemin/Gquadruplex nanostructures which can produce chemiluminescence readout for the amplified sensing.The proposed nanodevice exhibits good specificity and high sensitivity with a detection limit of 0.565 a M in vitro and 1 cell in vivo.It can distinguish 0.001% CpG methylation level,quantify different CpG methylation targets from diverse human cancer cells,and even discriminate CpG methylation expressions between lung tumor and precancerous tissues.Importantly,this nanodevice can be performed isothermally in one step within 2 h in a label-free manner without the involvement of any bisulfite conversion,fluorescence tagging and PCR amplification process,promising a simple,robust and universal platform for genomic methylation-related biomedical research and clinical diagnosis.2.MicroRNA(miRNA)-related single-nucleotide polymorphisms(miR-SNPs)are genetic variants that may alter miRNAs expression and/or maturation with multiple functional consequences,and they are promising biomarkers for cancer prognosis.Herein,we develop a ligation-initiated loop-mediated isothermal amplification(LAMP)strategy for label-free and realtime detection of miR-SNPs with zero background.In the presence of mutant targets(miR-196a2T),the dumbbell-shaped probe(DSP)is formed by the specific ligation of two substrates(HP1 and HP2),which acts as the template to initiate the subsequent LAMP.Once the DSP is formed,the forward primer and the backward primer-primed polymerization extension and strand displacement DNA synthesis can be repeated automatically and continuously,generating the double-strand DNA(dsDNA)in an exponential manner under isothermal conditions,which can be simply and label-free monitored with SYBR Green I as fluorescent indicator.Due to the specificity of ligation reaction,the exponential amplification efficiency of LAMP,and the nearzero background resulting from the efficient suppression of nonspecific amplification by employing multi-primer recognition region-directed amplification,this strategy displays ultrahigh sensitivity with a detection limit of 2.46 a M.Moreover,it can accurately distinguish as low as0.001% mutation level in the large pool of wild-type miRNAs.Furthermore,this strategy can be successfully applied for quantitative analysis of miR-196a2 T expression level in non-small cell lung cancer(NSCLC)patient tissues.