Highly Sensitive Detection Of Messenger RNA And DNA Methylation Based On Isothermal Exponential Amplification Reaction(EXPAR)

The biological information stored in the genome is passed on to the transcriptome through the process of transcription.The transcriptome encodes proteins through the translation process,generates proteins,and releases information to cells to regulate life activities.This series of processes are called genome expression,which is the core of the central dogma of life.Both gene mutation and DNA methylation can affect the above-mentioned genome expression and are closely related to the growth,development,and disease production of the organism.The occurrence of cancer has been reported to be closely correlated with the gene mutation and the abnormal DNA methylation.Only mutations of gene are passed on to the messenger RNA through the process oftranscription will the life activity be ultimately affected at the genetic level.As a coreperformer in gene expression,mRNA constitutes the transcriptome and determines the intracellular protein composition to infect the life activities that the cells can perform.Therefore,it is more intuitive and meaningful to directly detect the mutation of cancer-related mRNA.While DNA methylation is one of the most well-studied epigenetic mechanisms in mammals,it provides a stable gene silencing mechanism and plays an important role in regulating gene expression and chromatin structure.Therefore,rapid and simple detection of mutant mRNA and methylated DNA with high sensitivity and specificity can deepen the understanding of gene expression regulation,and in particular,has great significance for early diagnosis,prediction of response to therapy,prognosis,and real-time monitoring of cancers.Pretreatment for site-specific methylated DNA and single-base mutant mRNA resulting in cumbersome and complicated procedures in conventional detection methods,and the results are susceptible to false positive signals.Isothermal exponential amplification reaction(EXPAR)shares several distinct advantages such as simple design,simple template,and no need of thermocycling processes such as PCR and LCR.With high efficiency,EXPAR can obtain up to 106-109-fold exponential amplification of target nucleic acids in minutes with only a simple DNA template strand.In this paper,we used EXPAR to establish rapid and simple methods for discriminating site-specific methylated DNA and single-base mutant mRNA with high sensitivity and specificity.The main research contents are as follows:(1)A novel methylation-dependent restriction enzyme Glal was first applied to detect site-specific methylated DNA by coupling with EXPAR.Glal can only cut site-specific methylated DNA while keep the unmethylated DNA intact.Then,only the newly exposed end fragment of methylated DNA can trigger EXPAR with the high-efficiency signal amplification but the intact unmethylated DNA will not trigger EXPAR at all.In view of this,the real-time fluorescence signal of the GlaI-EXPAR system can reliably quantify and reflect methylated DNA,and effectively eliminate the potential false positive interference from unmethylated DNA.Therefore,the GlaI-EXPAR method can directly quantify methylated DNA with ultra-high sensitivity and accuracy by combining the unique function of GlaI for highly specific differentiation of site-specific methylated DNA and EXPAR for powerful and rapid signal amplification.The Glal-EXPAR system can directly detect methylated DNA with the limit of aM level in a short time of 2 hours.More importantly,as little as 0.01%of methylated DNA can be clearly identified in the presence of a large excess of unmethylated DNA which verified the highly specificity.The GlaI-EXPAR system has also been used to determine site-specific methylated DNA in genomic DNA samples.With this unique advantages of outstanding specificity,superior sensitivity,and handy operation,the new GlaI-EXPAR strategy may provide a robust and reliable platform for discrimination of low-abundance site-specific methylated DNA.(2)RNase H can selectively cleave RNA in the RNA-DNA hybrid duplexes.With this feature,a DNA probe(LNA probe)in a suitable length with a locked nucleic acid(LNA)modification in it was rationally designed to complement to the mutant mRNA.An additional RNA blocker strand(blocker)in a proper length was introduced to be complementary with the wild mRNA.By integrating the thermodynamic difference of single-base mismatch of competitive hybridization probes and target mRNA,RNase H can cleave mutant mRNA but leave wild mRNA intact.The newly exposed mutanted mRNA fragments can trigger EXPAR with high efficiency and eliminate the false positive interference generated by wild mRNA effectively under the action of blocker.Based on the thermodynamic difference of single-base mismatch,the competitive nucleic acid probe coupled with the RNase H-EXPAR system with a low detection limit at the aM level and the whole detection can be completed within two hours.More importantly,ultra-high specificity is achieved due to the action of RNA blocker and as low as 0.01%mutant mRNA can be clearly distinguished in the presence of excess wild mRNA.With this advantages,the RNase H-EXPAR may provides great potential for the detection of cancer associated single-base mutant mRNA.