Application Of Isothermal Exponential Amplification Reaction (IEXPAR) On Detection Of Plant MicroRNA And Research On Its Sequence Dependence

MicroRNAs(miRNAs)are a large family of non-protein coding small RNA molecules that can serve as a novel class of biomarkers for diseases.Accurate quantification of miRNA expression level has become more and more important for meeting the ever-growing demand for both fundamental biological studies and early diagnosis of diseases.As well as we know,isothermal exponential amplification reaction(IEXPAR)techniques have been successively developed for the simple and rapid detection of nucleic acids with remarkably high sensitivity and selectivity,which not only is used to detect targets ranging from mRNA to cells,proteins,small molecules,and even ions but also well-suited to the efficient amplification of miRNA.However,the bottlenecks of IEXPAR,that are target sequence-dependent amplification bias and non-sepcific amplification inherent in IEXPAR,which obviously restrict IEXPAR in terms of analytical sensitivity,specificity,and speed.Hence,the introduction of the modified traditional IEXPAR template alleviates the target sequence-dependent amplification bias,which was tagged with a Poly(dT)10 tail using 3’-thiol-cappe.And then,the additives of single-strand binding protein(SSB)also suppress non-specific amplification to some extent.The simple strategy that have played an important role in highly sensitive detection of all kinds of miRNAs by combining modified traditional IEXPAR template with SSB.The results of this study further provides us with a powerful tool to study and thoroughly solve bottleneck problem of IEXPAR.On the other hand,most isothermal amplification strategies including efficient IEXPAR where target animal miRNAs serve as the primers during the nucleic acid replication reactions,have been employed to fabricate highly sensitive miRNA assays at present.However,the results of study have indicated that plant miRNAs differ from animal miRNAs in that they are 2’-O-methylated at the 3’ end nucleotide which limits the ability of plant miRNAs to serve as primers for polymerization reactions.Therefore,the traditional efficient amplification reactions such as IEXPAR cannot be used for directly detection of plant miRNAs.In order to sovle intrinsic limitations of IEXPAR,herein,we wish to report a new and simple strategy that enables the highly sensitive and specific detection of plant miRNAs by combining the 3 WJ structure with highly efficient IEXPAR(3WJ-IEXPAR).According to the sequence of target plant miRNA,two 3WJ DNA probes including 3WJ primer and 3WJ template are rationally designed,without target miRNA,3WJ probes do not interact with each other and 3WJ primer extension and signal primer generation do not occur.In the presence of target miRNA,the sequence of 3WJ primer and 3WJ template will respectively hybridize with target miRNA and in such a case,the short sequence of 3WJ primer and 3 WJ template can also form a stable miRNA-tethered complex 3WJ structure.Then,the sequence of the 3WJ primer will extend along the sequence of the 3WJ template and genreate well-performed IEXPAR trigger to initiate the subsequent IEXPAR.This new design shares several unique advantages that was a well-performed IEXPAR initiating sequence rather than the plant miRNA itself is amplified by IEXPAR through the formation of target miRNA-tethered 3WJ structure,which not only overcome the intrinsic limitation of plant miRNAs cannot be directly served as primers for polymerization reactions,but also can effectively avoid the target sequence-dependent amplification bias to some extent.Due to the high sensitivity and specificity of the 3WJ-IEXPAR assay for ath-miR156a,the amount of ath-miR156a in total RNA extracted from Arabidopsis thaliana can be quantitatively evaluated by this 3 WJ-IEXPAR method.