Detection Of MiRNA Based On RNA Binding Protein

MicroRNAs (miRNAs) are a class of non-protein coding short RNA (19～23nucleotides) which evolved conservatively in eukaryotes and have a regulating function. Large data demonstrate that the development of cancer is often accompanied by abnormal expression of miRNA. Therefore, miRNA is of great importance to be a biomarker for cancer diagnosis, cancer prediction and feedback of treatment. RNA binding proteins (RNA binding protein, RBPs) are a class of proteins which can directly binds to the RNA molecules. Based on that, RNA binding protein can be used to detect and enrich miRNA.Based on the characteristics of double-stranded RNA binding protein p19, we have developed a method which can rapid detect miRNA by enhancing fluorescence polarization signal. P19proteins don’t bind to ssRNA, rRNA, mRNA, ssDNA or dsDNA, but can bind to double-stranded RNA of19-21bases with high affinity. Take advantage of this feature, we can raised the fluorescence polarization signal of miRNA-fluorescent probe duplex. At room temperature, the isothermal process in rapid "mixed-measurement" format can detect10pM miRNA within3min.The reason miRNA in blood is stable because of Ago2-miRNA complex mostly. Based on this characteristic we figure out that we can enrich miRNA in blood and cancer cells by immunoprecipitating Ago2-miRNA complex. By optimizing the condition of the method, we can get over0.15μg Ago2-miRNA complex in2×105cancer cells which means the enrichment efficiency is beyond85%.