Integration Of Rolling Circle Amplification And Cationic Conjugated Polymer For The Homogeneous Detection Of MicroRNA

MicroRNAs (miRNAs) are a class of non-coding small RNA genes, and their sequences are22-24nt that play important roles in the regulation of translation and expression. As its role in gene expression and control in the ontogeny, it is important for us to detect miRNAs to study its biological function and clinical diagnosis. In the test, we develop a novel and homogeneous assay for the detection of miRNAs by integration of rolling circle amplification (RCA) and cationic conjugated polymer (CCP). MiRNA-221serves as the template for specifically circularizing the padlock probe (PLP) with a sequence that is complementary to the miRNA-221, and T4RNA ligase2connect the both ends of the padlock probe to form a ring. Afterwards, miRNA-221directly acts as the primer to initiate the RCA reaction in the presence of phi29DNA polymerase that generates a long, tandem single-strand DNA product. When products fully hybridied with labeled-DNA strands, we introduced the CCP, efficient FRET from CCP to fluorescein occurs as a result of the strong electrostatic interactions between the CCP and the hybridied products. By measuring the change of the emission intensities of CCP and fluorescein, it was possible to detect miRNAs in a homogeneous manner. There was a good linear correlation between the FRET efficiency and the amount of miRNA211in the concentration range of0.5-20pM, The detection limit was estimated to be200fM.