Reaserch Of Multiple Signal Amplification Strategies In The Detection Of Circulating MicroRNA

Currently, the research of circulating microRNA is a very active area, which is of great significance to explore the life phenomenon, disease mechanism, diagnosis and prognosis of disease, gene therapy, drug development, and so forth. However, most traditional methods for circulating microRNA detection are always restricted by many aspects such as complex operations and sensitivity, which can not meet the needs to investigate the biological function of circulating microRNA.Compared with cyclodextrin, cyclodextrin polymers not only keep the ability of envelope and recognition, but also has the increased stability and solubility, the effects of crosslinking agent and multivalent binding, and so on. In our preliminary study, we found that ?-cyclodextrin polymer had excellent fluorescence enhancement effect for pyrene, and could be combined with other detection technology to improve detection sensitivity. Based on the excellent fluorescence enhancement effect of ?-cyclodextrin polymer for pyrene, we have developed rapid and sensitive methods for circulating microRNA detection by combining the advantages of enzyme. The details are summarized as follows:1. Based on the excellent fluorescence enhancement effect of ?-cyclodextrin polymer for pyrene, we have developed a method for sensitive detection of microRNA-21. In this method, DNA polymerase and lambda exonuclease were used as the tools of enzyme reactions, and the mono-pyrene-labeled molecular beacon acted as the signal report probe, which was phosphorylated at the 5' end and labelled with a pyrene at the 3'end. In the presence of target microRNA, two reactions were initiated in order, including the polymerase-aided strand displacement amplification and Lambda exonuclease-assisted cyclic enzymatic amplification. A great deal of pyrene located on the mononucleotide was trapped into the hydrophobic cavity of ?-cyclodextrin polymer accompanying with significant fluorescence enhancement. In the absence of target microRNA, the pyrene attached onto the molecular beacon could not enter the cavity of ?-cyclodextrin polymer because of strong steric hindrance, which led to a weak fluorescence signal. This detection method eliminated the previously demanding quencher and showed good sensitivity and specificity with a detection limit of 0.3 pM. In addition, this method displayed fine adaptability in human serum samples, and would be expected to be applied in clinical test.2. Based on the excellent fluorescence enhancement effect of ?-cyclodextrin polymer for pyrene, we have developed a strategy for the multiple amplification detection of DNA or microRNA. In this strategy, DNA polymerase, nicking enzyme and exonuclease ? were used as the tools of enzyme reactions, and the mono-pyrene-labeled DNA strand acted as the signal report probe. In the presence of target DNA or microRNA, two reactions were initiated in order, including the nicking enzyme-mediated strand displacement amplification and exonuclease ?-assisted cyclic enzymatic amplification. The pyrene located on the mononucleotide was trapped into the hydrophobic cavity of ?-cyclodextrin polymer accompanying with significant fluorescence enhancement. In the absence of target DNA or microRNA, the pyrene in the middle of the DNA strand could not enter the cavity of ?-cyclodextrin polymer because of strong steric hindrance, which led to a weak fluorescence signal. The probes were easy to design, and our method was specific and sensitive with the detection limit of 41 fM. This method has been also successfully applied for microRNA detection in human serum samples, and thus would be expected to be a universal detection platform. In addition, we have investigated the influence of water environmental pollutants on exonuclease ? activity, and the result was expected to provide theoretical basis and new methods for the inhibitor screening and pesticide toxicity evaluation.