The Synthesis And Investigation Of Novel Molecular Beacons

The first DNA autosynthesizer based on the solid-phase synthesis technology was set up in 1980s, which broke the bottleneck of DNA chemical synthesis, and people could be free to design and synthesize the optional nucleic acid molecular probes, which were applied in the fields of biology and medicine. However, the high sensitivity, stability and signaling mechanism of nucleic acid molecular probes are required constantly to advance the novel research methods of molecular biology. So the design and synthesis of these molecular probes is indispensable. In this thesis, the DNA synthesis platform was set up, and some novel probes were synthesized and investigated. Three parts of work were finished in this thesis.1.The foundation of DNA synthesis platformBased on the DNA autosynthesizer and liquid chromatogram technology, three kinds of nucleic acid molecular probes were synthesized and purified, which included cDNA, normal molecular beacon (MB), super-quencher molecular beacon (SQ-MB). The products were identified by MALDI-TOF-MS. The hybridization result of SQ-MB with cDNA showed that the signal/background ratio increased 40 times, and it was 4 times higher than normal molecular beacon. The distinct change of hybridization signal proved that we can synthesize and purify the DNA and dye-modified DNA probes.2.The synthesis and investigation of locked nucleic acid molecular beaconIn order to detect the mRNA in vivo, a novel locked nucleic acid molecular beacon (LNA-MB) was synthesized which contained three base-pairings in stem structure. The hybridization kinetics with cDNA was fast. Comparing with DNA molecular beacon (DNA-MB), LNA-MB showed a higher capability to resist nuclease digestion and high thermodynamic stability.3.The investigation of hybridization property of LNA-MB with shared-stem cDNA.The reported work showed that the shared-stem cDNA could improve the sensitivity of molecular beacon. So we designed the shared-stem cDNA, and investigated the hybridization of LNA-MB with shared-stem cDNA. The results showed that the sensitivity of shared-stem cDNA was superior to cDNA, the S/B was up to 200 times, the linear range was 20 pmol·L-1– 20 nmol·L-1, the detection limit was 9.8 pmol·L-1, and its initial fluorescence increasing velocity of hybridization with shared-stem cDNA was 5 times quicher than the mismatched shared-stem DNA. The LNA-MB with these prominent characteristics can provide a potential in high sensitive detection of mRNA in vivo.