A label-free analysis strategy of miRNA is designed using the rolling circle amplification (RCA) and fluorescence resonance energy transfer (FRET) with cationic conjugated polymer (CCP) as donor. In this strategy, the cationic poly [(9, 9-bis(6'-N, N, N-triethylammonium)hexyl) fluorenylenephenylene dibromide] (PFP) serves as the donor of FRET and SYBR Green I (SG) serves as the acceptor. PFP is a water-soluble pi-conjugated polymer with cationic charged side chain functionalities. Its structure allows for efficient intrachain and interchain energy transfer mechanisms. It can be combined with DNA by electrostatic interaction. SG is an asymmetrical cyanine dye which preferentially binds to double-stranded DNA (ds-DNA) and stains single-stranded DNA with lower performance. The fluorescence of SG is weak in the free state, but greatly enhanced once the DNA-SG-complex is formed. Let 7a is used as the target molecule. A padlock probe matched with let 7a and the DNA probes matched with the RCA product are designed. In the presence of loret 7a, the hybridization of the padlock probe and target sequences brings two ends of padlock probe close together and can be covalently ligated into a loop in the catalyzing of T4 DNA ligase. When phi29 DNA polymerase and dNTPs are added, the rolling circle amplification of the circularized padlock probe is initiated from the target molecules and then a long single strand DNA with a lot of repetitive sequences is generated. When DNA probes and SG are added, a long ds-DNA is produced and stained by SG. The DNA-SG-complex and PFP are absorbed together through electrostatic interaction and the strong FRET from PFP to SG occurs due to the overlapping between the fluorescent emitting spectrum of the PFP and the absorption spectrum of SG. In the absence of let 7a, the padlock probe is not circularized, which induces the inhibition of rolling circle amplification and hybridization process. Therefore, the FRET also cannot occur. As a result, the let 7a can be quantitatively determined by monitoring the change of FRET signal. The results show that the let 7a concentrations in the range of 0. 05 similar to 5 nmol . L-1 are linearly proportional to the detection signals. The specificity of this method is studied and the most of tested interfering substances have no influence on the test result of let 7a except let 7b and let 7c. Additionally, by the detection of let 7a concentration in the extract solution of cells, it is indicated that the strategy can be applied to the practical samples analysis. Because fluorescent labeling is not required, this strategy reduces the detection cost and simplifies the operation steps. Therefore, this protocol shows certain potential in the study of miRNA-related biological processes as well as disease diagnosis.
