| RNA plays an indispensable role in gene expression as a bridge of genetic information from genes to functional proteins.In particular,messenger RNA(mRNA)is the.translation template that can directly determine the amino acid sequence of the corresponding protein.Therefore,mRNA variation will directly alter the structure or function of the corresponding protein,which is associated with a variety of diseases,including cancers.In addition,RNA modifications are also involved in cell growth,metabolism and apoptosis.N~6-methyladenosine(m~6A)is one of the most common and abundant post-transcriptional modifications in RNA,which is the methylation of the sixth nitrogen atom of adenosine(A).With the continuous understanding of m~6A it is found that m~6A is not only related to the dynamic gene regulation,including the mRNA translation,shearing and degradation,but also closely related to cancers,leukemia and other diseases.Recently,tumor-related specific-site mRNA mutations have been biomarkers for early diagnosis and prognosis of cancers,and the identified-site m~6A have also received increasing attention in related aspects.So,highly sensitive detection of cancer-related mRNA mutations and m~6A of RNAs are of great significance to biology and biomedicine.In this paper,we have developed a method based on the Ribonuclease H(RNase H)-cleavage assisted reverse transcription polymerase chain reaction(RT-PCR)strategies and the rational designs for the detection of the specific-sites mRNA mutations and m~6A of RNAs.The main research contents of this paper are as follows:(1)Accurately quantifying specific-site mRNA mutations in single cells or peripheral blood is of great significance for the research of genetic diseases and cancer,but how to avoid false positive interference from abundant normal mRNA is still a great challenge.Herein,we have proposed a RNase H cleavage-assisted high-specificity RT-PCR strategy for detection of specific-site mRNA mutations.In this method,a properly designed DNA probe contains a Locked Nucleic Acid(LNA)exactly opposite to the mutant site,is completely complementary to wild-type mRNA(wtRNA),which can selectively guide the RNase H to cleave only the wtRNA while the mutant mRNA(mutRNA)will remain intact.The intact mutRNA can be amplified and detected by real-time RT-PCR but the disconnected wtRNA will be not replicated at all.Based on the highly selective depletion of wtRNA,this strategy can identify 0.1%of mutated mRNAs in the background of a large number of wild-type mRNAs with high specificity.Besides for the excellent specificity,ultrahigh sensitivity is also achieved for this proposed assay,which allows the quantification of mutRNA at single molecule and single cell level.Because of its high sensitivity and specificity,this method has a good application prospect in the clinical diagnosis of diseases and biomedical research targeted by mutant mRNA.(2)The distribution and expression of m~6A are organized difference,so both sensitive and accurate detection of m~6A in organisms is very important for biological research and the diagnosis of m~6A-related cancers.However,since the methyl of m~6A is inert and has a small steric hindrance,the base pairing principle of A/T or A/U can still be applied.Therefore,it is impossible to sensitively distinguish between m~6A and A in the same-site by conventional methods.Inspired by the previous study,a series of nucleic acid-modified probes that are fully complementary to the target RNA were designed to distinguish between the specific-site m~6A and A.Fortunately,a four 2'-0-methyl ribonucleotides and a ribonucleotide inserted DNA probe can guide RNase H to eliminate most of the interference from unmethylated RNA,but the RNA-containing m~6A is less affected.Combined with RT-PCR,we preliminarily achieve the distinction between m~6A and A with only one methyl group difference in structure,providing a new strategy to efficiently distinguish the m~6A of RNA. |
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