Study On The New Biosensor For The Detection Of N~6-Methyladenosine In RNA

Eukaryotic RNAs contain a variety of epigenetic modifications.Among them,N⁶-methyladenosine(m6A)is the most common and abundant methylation modification,especially in messenger RNA(mRNA)and non-coding RNA(lncRNA).Recent studies have shown that m6A plays an important role in regulating DNA replication,protein translation,gene expression,and other life processes.However,the specific regulatory mechanisms of methylation on various diseases(e.g.tumorigenesis,myeloid differentiation,stem cell differentiation,bacterial infection and drug resistance regulation)are still unclear.Therefore,the construction of accurate and sensitive m6A modification assays is important to promote further research on RNA epigenetic modifications in precision medicine.Since the m6A modification in RNA(m6A-RNA)has a small effect on the nucleic acid base properties and hardly interferes with Watson-Crick base pairing.Therefore,it cannot be directly and effectively detected by sequencing and nucleic acid hybridization alone.Given the irreproducible nature of methylated bases,the use of conventional amplification methods(e.g.polymerase chain reaction(PCR)or nucleic acid isothermal amplification strategies)results in the loss of methylation information.Moreover,the relative low content of m6A modification in actual samples makes it difficult to achieve sensitive detection of hypomethylation samples.m6A samples are unstable and easily degraded in pretreatment sessions,which affects the accuracy of detection results.Based on the current problems faced by m6A detection,this thesis developed an electrochemical biosensing detection method for m6A-RNA with analogue-modified nanoprobes by combining biomolecular technology,electrochemical sensing technology,nanotechnology,and fluorescence technology,the methylation-specific PCR template amplification technique for the detection of site-specific m6A modifications in RNA,and the integration of CRISPR/Cas12a technology for ultrasensitive detection of hypermethylated samples.The specific studies are as follows.1.electrochemical biosensing strategy based on analogue-modified nanoprobes for detection of m6A modifications in RNAThe studies in this section use methylation-specific antibodies as the recognition element of m6A.Based on the antibody's ability to recognize m6A modifications in both RNA and DNA.Here,we designed a analogue-modified DNA probes(m6A-DNA)that can compete with m6A-RNA to bind antibodies on the electrode surface.Porous platinum-cobalt nanospheres(PtCo-MPNs)with excellent catalytic properties were synthesized by a one-step method,and the m6A-DNA was assembled into a analogue-modified nano-signal probe by binding to PtCo-MPNs.The nanoprobes catalyze H₂O₂ to generate DPV signal output for quantitative detection of m6A-RNA through competitive reaction with the target on the electrode surface.The constructed electrochemical sensing method has good linearity in the range of 0.005?100 n M with a detection limit as low as 2.1 p M.Otherewise,this method achieved the validation of the detection of RNA samples extracted from multiple cells with high agreement with the results of the m6A immunoassay kit.2.Xeno nucleic acids probe mediated methylation-specific RT-qPCR for the detection of site-specific m6A in RNA at single-base resolutionThe previously constructed electrochemical immunosensing is an effective method for the quantitative detection of total m6A-RNA.With the in-depth research of RNA modification in life science,the quantitative detection of total RNA modification has been difficult to meet the needs of fine dissection of disease regulation mechanism and clinical detection and diagnosis,and the site-specific m6A single-base resolution quantitative detection has become a research trend in the recent years.Due to the irreproducibility of m6A during amplification,it is difficult to effectively detect m6A by conventional amplification methods without complex pretreatment,and sensitive of the site-specific m6A detection remains difficult.Here,we propose a simple xeno nucleic acids(XNA)probe mediated methylation-specific quantitative reverse transcription polymerase chain reaction(MsRT-qPCR).In this experiment,an XNA probe was designed to close the m6A corresponding adenine(A)site of the unmodified RNA template.Based on the difference in unstranding temperature between m6A-RNA and unmodified RNA(A-RNA)and lock nucleic acid(LNA),the reverse transcription primer selectively bound to the XNA probe on m6A-RNA in a strand displacement reaction on the template,achieving selective PCR amplification of the m6A-RNA template.To further ensure the accuracy and sensitivity of the method,a novel nucleic acid-modified magnetic nanoprobe was used instead of the antibody-dependent immunoprecipitation(IP)method to isolate and purify the cell-derived RNA samples.The constructed method allows linear detection of m6A-RNA methylation fraction in the range of 0.1?1.0.Using this method,we also successfully quantified the m6A fraction of 2577 loci in Hela cell lncRNA.The assay was also validated for multiple known methylation sites in mRNA and lncRNA of Hela and HEK393T cells,and was in high agreement with the results reported in the literature.The constructed method is inexpensive and requires only a conventional real-time PCR instrument for detection,which is expected to enable translational applications in clinical and routine laboratories.3.MsRT-PCR integration of CRISPR/Cas12a signal amplification for ultrasensitive detection of site-specific m6A modifications in RNASince most m6A sites in human cellular lncRNAs and mRNAs have methylation fractions between 6%and 80%,the pre-constructed MsRT-qPCR method has small differences in amplification cycling curves?Ct for low methylation fraction sample detection,which makes it difficult to effectively distinguish these low methylation site differences.Clustered regularly interspaced short palindromic repeats(CRISPR)based gene editing systems are gene-specific editing tools that have been studied more in recent years.In this study,the pre-constructed methylation-specific PCR reaction and CRISPR/Cas12a system were integrated,and the PCR-amplified double-stranded DNA(ds DNA)products were used to activate the slicing of the reporter probe by the CRISPR/Cas12a system to achieve the amplified output of the fluorescent signal,which further improved the sensitivity of the detection method.The detection of m6A-RNA samples with methylation fraction of 0.05?0.2 had good detection linearity by signal amplification.And multiple hypomethylation sites in cellular mRNA and lncRNA samples were detected,and the sensitivity was significantly improved compared with the previous qPCR results.