Applications Of Nucleic Acids Probes In MRNA Detection

The essential information on RNA synthesis, processing, transport, and localization obtained in real time will offer unprecedented opportunities for advancement in molecular biology, disease pathophysiology, drug discovery, and medical diagnostics. As a powerful tool, nucleic acid probes have been utilized in RNA study and explored wealth of essential information of RNA. However,how to further extend the applications of nucleic acid probes in biology and medicine research, how to develop novel nucleic acid probes to resolve new problems, how to get the dynamic data of these life processes sensitively and accurately in real time are still great challenges to analysts.In this dissertation, a series of mRNA detection methods based on molecular beacon and adjacent probe have been developed. The main researches of this dissertation are summarized as follows.1. RNA-templated single-base mutation detection based on T4 DNA ligase and reverse molecular beacon. A novel RNA-templated single-base mutation detection method was developed and applied to identification of single-base mutation in codon 273 of the p53 gene taking advantage of the high specificity of T4 DNA ligase and the capability of RNase H to digest RNA in RNA/DNA complex. One-base mismatch can be discriminated by analyzing the change of fluorescence intensity before and after RNase H digestion. This method has several advantages for practical applications, such as ease of design of detection probes; direct discrimination of single-base mismatch of the RNA extracted from cells; no requirement of PCR amplification; and performance of homogeneous detection.2. Design and synthesis of the superquencher molecular beacon. Improving the quenching efficiency is one of the useful strategies to improve the signal-to-background ratio and to optimize the sensitivity of the molecular beacon. In this work, normal molecular beacon and superquencher molecular beacon were synthesized and purified using the DNA autosynthesizer and liquid chromatogram.The products were identified by MALDI-TOF-MS. Our results showed that, the signal-to-background ratio of superquencher molecular beacon was 112, which is 5 times higher than that of normal molecular beacon with the same sequence. The significantly increased signal-to-background ratio of the superquencher molecular beacon was due to the decreasing of the background signal of the probe. Choosing cytidine as the final nucleotide base next to the fluorophore, or let one arm of molecular beacon be complementary to the target can further increase the signal-to-background ratio of the superquencher molecular beacon.3. Detection of nucleic acid in cell lysate using phosphorothioate-modified adjacent probe. In order to eliminate the false-negative signals resulting from nuclease degradation of the convenient adjacent probe in biological sample analysis, phosphorothioate modified adjacent probe was designed and synthesized for mRNA detection in cell lysate. Results showed that phosphorothioate modified adjacent probe can efficiently reduce the false-positive and false-negative signals resulting from nuclease degradation, thus, improving the accuracy in mRNA detection in biological samples. Phosphorothioate modified adjacent probes are easy to design, very sensitive and selective in monitoring nucleic acid targets, easy to hybrid to the target sequence, and allow rapid detection in complex sample matrix. As the light-up signaling approach is used to detect the presence of target sequence, removing of unbound probes are not necessary and tedious washing and separating procedures are avoided.4. Monitoring p21 mRNA expression in living cell based on molecular beacon fluorescence increasing rate. The normal molecular beacons tend to generate false positive signals due to nuclease degradation, when used for intracellular mRNA expression level analysis. A specific molecular beacon (p21 MB) for the important tumor suppressor gene p21 has been designed and synthesized. The fluorescence signal was detected in real-time after injecting the p21 MB into nasopharyngeal carcinoma cell and p33-transfected nasopharyngeal carcinoma cell. In both cases, the fluorescence intensities achieved maximum in about 15 minute. However, the fluorescence increasing rate within 4 minutes right after microinjection was significantly different between these two cell lines, indicating different p21 mRNA expression levels. The results obtained in the real-time detection were also validated by RT-PCR. Analysis of the initial fluorescence increasing rate can efficiently reduce the nuclease degradation and improve the accuracy in living cell mRNA detection.5. mRNA detection in living cell using phosphorothioate-modified molecular beacon. In order to resolve the intracellular biostability problem of molecular beacons, phosphorothioate modified DNA, which can efficiently improve the nuclease resistance of the backbone of the DNA, were chosen to design and synthesize molecular beacons. Phosphorothioate modified molecular beacon can efficiently improve the accuracy in living cell mRNA detection while keeping the advantages of molecular beacons, such as excellent selectivity, high sensitivity, and without-separation detection, and has been applied in real time detection of GFP mRNA in COS-7 cell and GFP tansfected COS-7 cell.