Construction And Analytical Applications Of Abasic-Site Containing DNA Molecular Beacon And Functional Gold Nanoclusters

Our work mainly study on construction and analytical applications of abasic-site containing DNA molecular beacon and functional gold nanoclusters. First of all, abasic site was artificially embedded into DNA at specific site. By changing molecular beacon with different sequence, the methods could successfully detect melamine and amyloid beta with high selectivity and sensitivity. It was found that gold naonclusters could be effective carriers for drug delivery because of low toxicity and stable fluorescence, therefore, gold nanoclusters with surface modifications were successfully applied to siRNA delivery and target release in our work, which have extremely good clinical applications.Chapter 1. IntroductionIn this paper, we firstly introduced three kinds of molecular beacons and their applications in biological analysis. Synthesis of abasic site and its combination with small molecules was also simply presented. In addition, strand displacement reaction (SDA) was emphatically introduced. We presented the synthesis and biological applications of gold nanoclusters. At the end of the paper, we pointed out the purpose and significance of the thesis.Chapter 2. Sensitive Detection of Melamine Based on Abasic Site-containing Triplex Molecular BeaconOn the basis of hydrogen bonding interaction between thymine and melamine and nucleic acid molecular recognition function, in our work, we construct sensitive melamine detection system with the help of abasic site. Melamine is a high nitrogen content of industrial material, which are harmful to animals and human beings when excessively exist in food and water. It’s well known that uptaking of excessive melamine for long time can lead to calculus of bladder and kidney, damage of reproductive and urinary system, and can further cause bladder cancer. In this way, it is of great importance to detect melamine quantitatively and sensitively. Strong fluorescence could be detected in our system for the reason that the double labeled DNA do not interact with abasic site-containing DNA. However, addition of melamine into the system can induce the formation of triplex molecular beacon, leading to fluorescence quenching. Furthermore, the detection limitation of melamine could reach 0.3μM by optimizing the base number of molecular beacon and reaction temperature.Chapter 3. Selective and Enzyme-free Amplified Detection of DNA and Amyloid β Oligomers Based on Abasic Site-containing Molecular BeaconA label-free molecular beacon (MB) system integrated with enzyme-free amplification strategy was developed for simple and highly selective assay of DNA and Aβ oligomers. Amyloid-beta (Aβ) oligomers are highly toxic species in the process of Aβ aggregation and are regarded as potent therapeutic targets and diagnostic markers for Alzheimer’s disease (AD). The detection system was const ucted with two abasic site (AP site)-containing label-free molecular beacon MB1 and MB2. The fluorescent of ligand 2-amino-5,6,7-trimethyl-1,8-naphyridine (ATMND) was quenched upon binding to the AP site in DNA stem through hydrogen bonding. As expected, target DNA could trigger continuous opening of two delicately designed MBs (MB1 and MB2), leading to release of ATMND molecules. Combining with Aβ oligomers aptamers, the strategy was applied to analyze Aβ oligomers and the results showed that it could quantify Aβ oligomers with high selectivity and monitor the Aβ aggregation process.Chapter 4. Cationic Fluorescent Gold Nanoclusters for siRNA DeliveryIn this work, we develop a novel assay which uses cationized gold nanoclusters as hypotoxicity vectors for siRNA delivery to implement fluorescence imaging and diagnostic therapy. Small interfering RNA (siRNA) is one of the most commonly used tools for disease treatment on account of the special ability of silencing gene expression. Traditional drug delivery systems for siRNA exhibit several key limitations including undesirable toxicity, low efficiency and lacking of tracking capability, which is still a challenge for biomedical application. Herein, our work focuses on the development of employing BSA-protected GNCs (DMA/EDA-BSA-Au) as a dual functional nano-platform for siRNA delivery and tracing. The siRNA delivery procedure of DMA/EDA-BSA-Au complex was mediated by potential of hydrogen based on the acid degradation characteristics of BSA. The systemic delivery using siRNA as a model drug resulted in a significantly reduced GFP expression in the lung cancer cells. In this way, cationized gold nanoclusters were thought to be developed as efficient vectors for siRNA therapeutics of various cancer diseases.