| Important biomolecules, such as enzymes and ATP, are the structural and functional base of organism and life's phenomena. They play an important role in metabolism, recovery, blood coagulation, cell information transport, postnatal development, tissue synthesis and so on. It is very important for researchers to expound the secrets of life, to develop new functionalized medicine to surmount many difficult diseases and promote the development informatic science. What we should do is to further penetrate the interaction of the biomolecules and to develop rapid, convince, accurate and sensitive assay for them. This project is the forward position and hot point in bio-analytical chemical research.Aiming at the research goals mentioned above, this thesis utilized the advantageous of molecular beacon and nucleic acid enzyme (ligase or polymerase) to develop a series of methods for real-time monitoring the activity of kinase, phosphatase and restriction endonuclease. Moreover, novel detection methods for adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) have been developed with high sensitivity and selectivity. It is the first example of using molecular beacon as a biosensor for the detection of biologically important small molecules. The main points of this thesis are summarized as follows:Firstly, the applications of NTFS have been extended to detect important biomolecules, such as ATP, NAD, NADP, creatine kianse and protein kinase:1. Detection of ATP using molecular beacon (MB). A highly sensitive and simple fluorimetric method for the determination of ATP based on ATP-dependent T4 DNA ligase has been developed. This approach utilized a MB, T4 DNA ligase and two short oligonucleotides. In the presence of ATP, the T4 DNA ligase catalyzed the ligation reaction and the ligation product restored the fluorescence of MB. The increase of fluorescent intensity of MB was related to the concentration of ATP. This assay could determine ATP in the range of 1~300 nM with a detection limit of 0.14 nM. Cellular ATP concentration in several cell lines has also been determined.2. Detection of creatine kinase activity using MB. A novel method for the determination of creatine kinase activity based on ATP-dependent T4 DNA ligase has been developed. The ATP was produced from adenosine 5'-diphosphate (ADP) and creatine phosphate catalyzed by creatine kinase. Then T4 DNA ligase catalyzed the ligation reaction with ATP resulting the formation of match DNA, which caused conformation changes of molecular beacon. The increase of fluorescent intensity of molecular beacon was related to the concentration of creatine kinase. This assay could determine creatine kinase in the range of 1~50 U/L with a detection limit of 0.64 U/L. Moreover, the method was shown to be suitable for the sensitive detection of creatine kinase inhibitors.3. Detection of protein kinase A using MB. Protein kinase A was detected by quantifying the ATP after the protein kinase reaction. ATP assay was achieved by using T4 DNA ligase and molecular beacon. This assay could determine protein kinase A in the range of 12.5~150 nM with a detection limit of 1.25 nM. Moreover, the assay was available to investigate the effect of genistein on protein kinase A. It was a universal, non-radioisotope and homogeneous method for protein kinase A assay.4. An enzymatic cycling and signal amplification assay for ATP using MB. A sensitive assay for ATP was developed by enzymatic cycling and signal amplification assay. T4 DNA ligase consumed ATP and produced AMP, which was phosphorylated to ATP with 2'-deoxycytidine 5'-triphosphate (dCTP) as the phosphate donor catalyzed by adenylate kinase and nucleoside-diphosphate kinase. The ATP was detected by molecular beacon and T4 DNA ligase mentioned above. With the cycling of ATP→AMP→ATP, more and more molecular beacons were opened. This assay could determine ATP in the range of 0.01~10 nM with a detection limit of 5 pM. The sensitivity of the method developed here is comparable with bioluminescence.5. Detection of NAD based on DNA ligation using MB. A highly sensitive and simple fluorimetric method for the determination of NAD based on NAD-dependent E.Coli DNA ligase has been developed. This approach utilized a MB, E.Coli DNA ligase and two short oligonucleotides. In the presence of NAD, the E.Coli DNA ligase catalyzed the ligation reaction and the ligation product restored the fluorescence of MB. The increase of fluorescent intensity of MB was related to the concentration of NAD. This assay could determine NAD in the range of 0.3~40 nM and 40~300 nM with a detection limit of 0.3 nM. The calorie restriction effect on intracellular NAD level of the MCF7 cell has also been investigated.6. A high-sensitive method for the detection of NADP using MB. NADP assay was completed based on high-dependence of E. coli DNA ligase on coenzyme, nicotinamide adenine dinucleotide (NAD). First, NADP was transformed to NAD by alkaline phosphatase, and then NAD product was introduced into molecular beacon system and quantified. NADP concentration was represented by the initial enhancement rate of fluorescence intensity. This assay could determine NADP in the range of 5~200 nM with a detection limit of 2 nM. Compared with current assay methods, this approach was convenient, quick and highly sensitive.At last, the NTFS has been improved in investigating the interactions between nucleic acids and proteins (enzymes) by using MB based on polymerase extension reaction:7. Real-time monitoring of DNA polymerase activity using MB. Traditionally, polymerase is assayed by denaturing gel electrophoresis and autoradiography, which are complex and discontinuous, and incapable of providing the dynamic data. Here, a novel DNA polymerase assay based on MB has been proposed and developed. The polymerization process has been indicated by fluorescence singal in real-time. Under optimized conditions, this assay could determine Klenow Fragment exo- in the range of 0.003~6.25 U/mL with a detection limit of 0.003 U/mL. To our knowledge, there were no other methods having comparable sensitivity. The effect of drugs on the activity of polymerase has been investigated. This method was shown to be suitable for the sensitive detection of polymerase inhibitors.8. Real-time monitoring of nucleic acids dephosphorylation process using MB. A novel approach has been developed to monitor the dephosphorylation catalyzed by T4 polynucleotide kinase (PNK). The dephosphorylation process has been indicated by fluorescence singal in rea-time. This assay could determine T4 PNK in the range of 0.1~15 U/mL with a detection limit of 0.1 U/mL. The effect of inhibitor on the phosphatase activity of T4 PNK has been investigated. This method was shown to be suitable for screening potential inhibitors of T4 PNK.9. Real-time monitoring of restriction endonuclease activity using MB. Traditional methods to assay the activity of restriction endonuclease are discontinuous and time consuming. Here, a novel, continuous fluorescence assay for restriction endonuclease activity based on MB has been developed. This assay could determine Rsa I endonuclease in the range of 1~250 U/mL with a detection limit of 1 U/mL. The effect of inhibitor on the activity of Rsa I endonuclease has been investigated. This method was shown to be suitable for the sensitive detection of Rsa I endonuclease inhibitors.10. Detection of alkaline phosphatase activity using MB. A novel, continuous fluorescence assay for alkaline phosphatase activity based on MB has been developed. This assay could determine alkaline phosphatase in the range of 4×10-16~4×10-14 M with a detection limit of 4×10-16 M. It is comparable with or better than some commonly used techniques. And it is the first example of using DNA as a substrate for alkaline phosphatase assay. Furthermore, this method could be further improved by combing DNA amplification technique, such as PCR and LCR. The effect of inhibitor on the activity of alkaline phosphatase has been investigated. This method was shown to be suitable for screening potential inhibitors of alkaline phosphatase. |
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