High Sensitive And Homogeneous Detection Of Microrna Based On Ligase Chain Reaction And Cationic Conjugated Polymers

MicroRNA (miRNA) exists in cells of animals and plants widely and plays a regulationrole to messenger RNA (mRNA) which is formed through the process of gene transcription.Some researches show that cancer has close relationship with some miRNAs’ abnormalexpression. Therefore, a high sensitive method to detect miRNA is practically meaningful tothe further research of the regulation mechanism of miRNA and the endogenous diseaseinitiated by miRNA.As the low expression quantity of miRNA in human cells, the technology of nucleic acidamplication is usually applied to detect miRNA qualitatively or quantitatively. However,traditional nucleic acid amplication technology such as reverse transcription polymerase chainreaction (RT-PCR) has shortcomings such as complex primer design, weak sensitivity.Another nucleic acid amplication technology, ligase chain reaction (LCR), needs long timeand thus reduce the experimental efficiency as it must applied polyacrylamide gelelectrophoresis (PAGE) to realize the assay of miRNA quantitatively or semi-quantitatively.In this paper, a simple and homogeneous microRNA assay is developed by integration ofligase chain reaction (LCR) and lambda exonuclease-assisted cationic conjugated polymer(CCP) biosensing. Two pairs of probs for LCR were designed. One single-strand DNA ofeach probs was modified by fluorophore at3’-end and phosphate group at5’-end. Thetemplate for LCR is formed by the unique bind function of T4RNA ligase2based on RNAtemplate. LCR is utilized for exponential amplification of microRNA, and lambdaexonuclease is introduced to degrade excess fluorescein-labeled probes in LCR foreliminating background signal. By addition of CCP, positively charged CCP is close to thenegatively charged fluorescein labeled LCR products through strong electrostatic interaction,leading to efficient FRET from CCP to fluorescein. In contrast, the degraded mononucleotidesby lambda exonuclease are far away from CCP because of the much weaker electrostaticinteractions between mononucleotides and CCP. Accordingly, effective FRET from CCP tothe fluorescein-labeled mononucleotides does not occur. As a result, it is possible to perform homogeneous miRNA detection via integration of LCR and lambda exonuclease-assisted CCPdetection by monitoring FRET signal from CCP to fluorescein. The method is sensitiveenough to detect0.1fM target microRNA and specific to discriminate one-base difference ofmiRNAs, which paves a new way for homogeneous LCR-based nucleic acids detection andmolecular diagnosis.