Plant MicroRNA Analysis Based On Click Nucleic Acid Ligation Reaction And T7 RNAP Transcription Mechanism

Plant microRNAs(miRNAs)are a class of non-coding small RNA molecules,20 to 24 nucleotides in length.In plant,miRNAs usually regulate gene expression either on transcriptional level or post-transcriptional level by cleavage of target mRNAs and inhibition of mRNAs translation.With the development of latest high-throughput sequencing technology,more than 2,000 miRNAs have been discovered in different plant species,but there are still many unsolved problems about its mechanisms of action and biological functions.Many studies have shown that plant miRNAs play an important role in the life process of plant growth and plant response to abiotic stress.And some researchers have also found that exogenous plant miRNAs can enter the animal from food and may regulate the target mRNAs genes of certain mammals.Therefore,the establishment of high sensitivity and high specificity for plant miRNAs analysis is particularly essential for the in-depth study of its biological functions and impact on human health.Due to its short sequence and low content in cells.miRNAs have brought certain difficulties to the establishment of analytical methods.What's more,a large number of studies have confirmed that the structure of plant miRNAs is quite different from animal miRNAs.The 3' terminal base of animal miRNAs has a 2'-OH group,but the 3'terminal base of the plant miRNAs sequence has a 2'-OCH3 group.Due to the steric hindrance of the 2'-OCH3 group,it is difficult to occur base polymerization at the 3'-OH end of plant miRNAs.which leads to plant miRNAs cannot be used as primers for polymerization extension.This greatly limits the use of a variety of nucleic acid amplification methods,which poses new challenges for plant miRNAs detection.Now the methods for detecting plant miRNAs have been established,mainly including Northern blotting,electrochemical methods and some novel nucleic acid amplification methods.However,these methods have many limitations,so it is still very important to construct a simple and sensitive method for detecting plant miRNAs.Although plant miRNAs cannot be used as primers for the polymerization extension reaction,they can serve as an excellent template for click chemistry-mediated ligation chain reaction.In this paper,we constructed a highly efficient click chemistry-mediated ligation chain reaction using plant miRNA as a template,and established a highly sensitive analytical method based on T7 RNA polymerase(T7 RNAP)transcription-binding isothermal exponential amplification reaction(IEXPAR).And we apply it to the detection of miR156a.In this method,when the target miRNA is present,two probes respectively modified with N3 and DBCO groups will hybridize with miRNA.They will form a stable sandwich duplex structure,and click reaction occurs rapidly.After reasonable design,only the ligation product can trigger T7 RNAP transcription and produce a large number of RNA transcripts.Finally these RNA transcripts can trigger the subsequent IEXPAR reaction to achieve rapid quantitative detection of the target miRNA.This assay overcomes the shortcoming that plant miRNAs cannot directly trigger the IEXPAR reaction.And we simultaneously add thermal cycling and T7 RNAP transcription in the method to achieve signal amplification for the target miRNA.The experimental results show that the method can detect miR156a as low as 10 fM,and the fluorescence response has a good linear relationship with the negative logarithm of the miR156a concentration.The detection range spans four orders of magnitude.And the assay has good specificity,which can achieve single base discrimination.