Locked Nucleic Acid Modified Molecular Beacons For Nucleic Acid And Protein Detection

Benefited from their sophisticate switchable hairpin structure, high efficient signaling transduction mechanism and flexibility of chemical modification, molecular Beacons (MBs) have been increasingly applied into a broad range of fields far more than the PCR monitoring or allele analysis. By teaming with other platforms and biotechnologies, MBs have been used for protein assay, enzyme monitoring, mRNA tracing in living cells, biosensors. It's very initial to design and synthesis new kinds of MBs which can overcome disadvantages of common MBs to meet the requirements coming from biology, biomedicine, chemistry and biotechnology. A novel locked nucleic acid modified molecular beacon (LMB) which has good hybridization kinetics and superior resistance to enzymatic cleavage was designed. This thesis utilized the advantageous of LMB and DNase I to develop a series of methods for simple/fast/sensitive nucleic acid and protein assay. The main points of this thesis are summarized as follows:1. DNA assay based on locked nucleic acid modified molecular beaconA novel method for DNA detection was developed based on LMB and DNase I. In the presence of DNase I, the hybridization products of DNA and LMB was enzymolysised and restored the fluorescence of LMB. While in the absence of DNA, the fluorescence of LMB was constant. The LMB and DNase I-based approach for DNA detection demonstrated in this work promises a simple and agile design of probes as well as considerable sensitivity with a detection limit of 160 pM, which achieved a 25-fold enhancement compared with the common molecular beacon-based methods for DNA detection.2. RNA detection based on locked nucleic acid modified molecular beaconA novel method for cycling signal amplification RNA detection was developed based on LMB and DNase I. In the presence of DNase I, the hybridization products of RNA and LMB were enzymolysised. With the cycling of released target RNA, more and more molecular beacons were opened and enzymolysised. This assay could determine RNA sensitively and simplely with a detection limit of 0.1 pM.3. Protein detection based on locked nucleic acid modified molecular beaconA novel method for protein detection was developed based on LMB and steric-hindrance effect. Aptamer probes were used to specifically bind with thrombin that would protect the probes from the hydrolyzed by the DNase I and Exo I. However, the free aptamer probes were easily hydrolyzed without any protection. The protected aptamer probe was quantified without denatured thrombin by the LMB which can offered a fluorescent signal change with and without binding with the single-strand probes. This work presented the simple, selective and sensitive assay for thrombin detection based on LMB and steric-hindrance effect with a detection limit of 0.1 nM.