【作者】 马昌杯；

【导师】 王柯敏；

【作者基本信息】 湖南大学 ， 分析化学， 2007， 博士

【摘要】 生物酶和ATP等重要生物分子是生物体和生命现象的结构基础和功能基础,在物质代谢、机体防御、血液凝固、肌肉收缩、细胞信息传递、个体生长发育、组织修复等方面发挥着不可替代的重要作用,它们的含量和活性直接关系到生物体的健康。因此,深入研究这些生物分子之间的相互作用,特别是建立这些生物分子快速、简便、准确、灵敏的分析方法,对分子水平上阐明生命的奥秘、新型药物的开发,攻克许多疑难病症以及促进信息科学的发展都有重要的意义,是当前生物分析化学研究的前沿和热点。本论文以上述科学问题为目标,结合当前发展的核酸探针技术和分子生物学手段,巧妙地利用了核酸酶(连接酶或聚合酶)、核酸以及分子信标核酸探针的特殊性质,发展了激酶、磷酸酶和限制性内切酶等生物酶活性实时检测的新方法。更重要的是,首次将分子信标探针用于三磷酸腺苷(ATP)、烟酰胺腺嘌呤二核苷酸(NAD)、烟酰胺腺嘌呤二核苷酸磷酸(NADP)等重要生物小分子的检测。主要内容归纳如下:首先,拓展了NTFS平台技术的应用,将其用于ATP、NAD、NADP、肌酸激酶、蛋白激酶等重要生物分子的检测,具体包括以下六个部分:1、分子信标技术结合T4 DNA连接酶用于ATP的检测。利用T4 DNA连接酶对ATP的高度依赖性,我们发展了一种ATP检测新方法。在ATP存在的条件下,T4 DNA连接酶以分子信标为模板,将两段短核酸片段连接,连接产物将分子信标打开,荧光信号增强,从而达到检测ATP的目的。该方法灵敏,简单,其线性响应范围为1~300 nM,检测下限为0.14 nM。并利用该方法对细胞内的ATP水平进行了初步分析。2、分子信标技术用于肌酸激酶活性的检测。二磷酸腺苷(ADP)与磷酸肌酸在肌酸激酶催化作用下生成肌酸和ATP,由于T4 DNA连接酶对ATP的高度依赖性,T4 DNA连接酶就会识别并利用生成的ATP进行DNA连接反应,产生的长链DNA将分子信标打开,使荧光信号增强,达到检测肌酸激酶活性的目的。其线性响应范围为1~50 U/L,检测下限为0.64 U/L。并用此方法考察了药物对CK活性的影响。该方法快速、简便、灵敏度高。3、分子信标技术用于蛋白激酶A活性的检测。以蛋白激酶A为模型,通过测定蛋白激酶催化后所剩余的ATP的量来检测蛋白激酶的活性。T4 DNA连接酶和分子信标底物被用于ATP的检测,其线性响应范围为12.5~150 nM,检测下限为1.25 nM。并考察了药物染料木素对蛋白激酶A的抑制作用。该方法不用进行同位素标记、操作简便、具有通用性。4、分子信标结合酶信号放大检测ATP。结合连接酶、腺苷酸激酶和核苷二磷酸激酶对ATP进行了检测。连接反应消耗ATP生成单磷酸腺苷(AMP),AMP与三磷酸脱氧胞苷酸(dCTP)在腺苷酸激酶的催化下生成ADP和2-脱氧胞苷5-二磷酸(dCDP),ADP和dCTP在核苷二磷酸激酶催化下生成ATP,使得ATP得到循环。利用T4 DNA连接酶对ATP的高度依赖性,ATP的不断产生使得被打开的分子信标不断增加,检测的线性范围为0.01~10 nM,检测下限为5 pM,与最灵敏的ATP检测方法生物发光法的灵敏度相当。5、基于分子信标的NAD高灵敏检测方法。本章利用大肠杆菌DNA连接酶对NAD的高度依赖性,发展了一种NAD检测新方法。在NAD存在的条件下,大肠杆菌DNA连接酶被激活,并以分子信标为模板,将两段短核酸片段连接,连接产物将分子信标打开,荧光信号增强,从而达到检测NAD的目的。该方法灵敏,简单,其线性范围为0.3~40 nM及40~300 nM,检测下限为0.3 nM。并利用该方法考察了卡路里限制对MCF7细胞内NAD水平的影响。6、基于分子信标的NADP高灵敏检测方法。首先利用碱性磷酸酶将NADP的磷酸根切除转化生成NAD,然后将NAD加入到分子信标及大肠杆菌DNA连接酶连接体系中进行检测,通过记录并计算荧光增强的初速度来定量,检测的线性范围为5~200 nM,检测下限为2 nM。这是一种简单、快速、高灵敏度的NADP分析新方法,且具有良好的特异性。其次,对NTFS平台技术进行了改进,基于聚合酶延伸反应发展了一种更简便快速的用于实时研究核酸与蛋白质(酶)相互作用的新技术,主要包括以下四个部分:7、分子信标用于DNA聚合酶活性的实时监测。研究聚合酶活性的传统方法主要是放射性同位素标记结合凝胶电泳,操作复杂费时,并且不能提供实时信息。本章利用分子信标技术,建立了一种简单、快速、灵敏地实时监测聚合酶活性的方法。其线性响应范围为0.003~6.25 U/mL,检测下限为0.003 U/mL。并探讨了聚合酶抑制剂对酶活性的影响,为以聚合酶为作用靶标的新药开发及筛选提供了一种新的思路。8、分子信标用于核酸3’端去磷酸化过程的实时监测。利用分子信标技术,结合DNA聚合酶发展了一种核酸3’端去磷酸化实时监测方法,建立了T4多聚核苷酸激酶(T4 PNK)的酯酶活性分析新方法。其线性响应范围为0.1~15 U/mL,检测下限为0.1 U/mL。并探讨了酶抑制剂对T4 PNK酯酶活性的影响,为T4 PNK抑制剂筛选提供了新的思路和方法。9、分子信标用于限制性内切酶活性的实时监测。传统的检测限制性内切酶活性的方法是不连续的,费时的。本章利用分子信标技术,建立了一种简便快捷、灵敏地连续监测限制性内切酶活性的方法。其线性响应范围为1~250 U/mL,检测下限为1 U/mL。并探讨了限制性内切酶抑制剂对酶活性的影响,为以限制性内切酶为作用靶标的新药开发及筛选提供了新的手段。10、分子信标用于碱性磷酸酶活性的分析。利用分子信标技术,建立了一种简单、快速、灵敏地监测磷酸酶活性的新方法,首次利用DNA作为酶作用底物对其活性进行检测。其线性响应范围为4×10-16~4×10-14 M,检测下限为4×10-16 M。并探讨了碱性磷酸酶抑制剂对酶活性的影响,为以碱性磷酸酶为作用靶标的新药开发及筛选提供了新的思路和手段。更多还原

【Abstract】 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.更多还原