【作者】 周桂萍；

【导师】 黄锡荣；

【作者基本信息】 山东大学 ， 分析化学， 2010， 博士

【摘要】 序列决定了DNA分子特性,DNA序列的变化导致人类个体间的差异。约90%的DNA序列变化归因于单核苷酸多态性(SNP),即基因组水平上单个位点碱基的改变。SNP与人类进化、遗传性疾病、个体药物敏感性等困扰人类从而引起普遍关注的问题紧密相关,对SNP的研究将会对人类进化过程、基因定位、药物开发、疾病诊疗等方面产生深远影响。其中,建立适用于遗传性疾病、肿瘤的早期诊断以及诊疗过程的简便快速的SNP检测方法在临床诊疗中具有十分重要的意义。本论文研究目标就是采用普通的分析仪器,建立简便快速识别具有不匹配碱基的核苷酸链的方法,使之能应用于普通诊疗机构。一、染料放大DNA杂交信号的研究插入式荧光染料与DNA双链结合后产生荧光,可以放大DNA的杂交信号,但需要采用荧光光度计检测信号。花青染料在可见光区具有强吸收,如果能与DNA双链特异性结合,就可以将DNA杂交信号转化为可见光区的光吸收信号,用普通分光光度计就可进行检测。本章基于花青染料3-乙基-2-[7-(3-乙基-2-苯并噻唑啉)-1,3,5-庚三烯]碘化苯并噻唑与DNA杂交双链的特异性结合作用,将DNA的杂交信号转化为染料在可见光区的光吸收信号。实验结果表明,与DNA杂交双链结合后,该染料在760 nm处产生强吸收峰,吸光度数值及其稳定性受杂交链长度和体系温度的影响。在25℃条件下,采用24-mer探针,可以检测μg·mL-1数量级的互补DNA链,比DNA自身杂交信号灵敏度提高两个数量级。在优化条件下,利用该染料还可识别短链DNA中存在的SNP。与其它DNA片段的检测以及SNP的识别方法相比,该方法更为简便迅速,在临床诊疗中具有潜在的应用价值。二、反胶束介质调控DNA杂交反应的研究DNA杂交反应前后,在260 nm处的吸光度会有变化。杂交双链匹配度不同,吸光度的变化幅度不同。这一光吸收信号可以采用紫外分光光度计检测。但是,水溶液中进行的DNA杂交反应,浓度高至100μmol/L时,DNA分子的紫外吸光度超出仪器检测范围。浓度低至1 u mol/L,杂交前后吸光度的变化幅度小,也观测不到吸光度的变化。反胶束介质使局部浓集于水核中的DNA分子杂交,同时DNA的表观浓度大大降低,反胶束介质还降低了DNA的杂交反应速度和杂交双链的稳定性,使不同匹配度DNA双链的紫外吸光度产生差异,因此用紫外分光光度计即可检测SNP。但是文献研究表明反胶束中DNA杂交反应速率太慢,单个样品检测周期约4小时。本章采用合成的24个碱基的DNA片段,在AOT反胶束中研究了影响DNA杂交反应速度的因素。采用析相的方法,使反胶束溶液两相分离,析相后DNA仍存在于有机相(反胶束)中。析相后DNA链仍能进行杂交反应,杂交反应速度加快。根据不同匹配度DNA链杂交后紫外吸光度,可以识别存在不匹配碱基的DNA链。利用库仑滴定法测定有机相中水分含量,双链不匹配度越高,有机相中水分含量越高,根据有机相中水分含量,也可以识别具有不匹配碱基的核苷酸链。三、DNA杂交信号的酶放大研究近年来,利用酶催化反应转换和放大各种检测信号的方法越来越受到关注。2002年,Alan S.等人报道了利用生物工程修饰酶放大DNA检测信号的方法,首次将DNA的杂交反应信号转换并放大为酶催化反应信号。但是,该方法的局限性在于对酶、抑制剂和DNA的修饰连接过程太过复杂。核酸适配体是1990年被发现的一类特殊的寡核苷酸链,可以通过一种称之为SELEX (systematic evolution of ligands by exponential enrichment)的离体选择过程获得。1992年通过SELEX筛选出凝血酶核酸适配体,可以抑制凝血酶对纤维蛋白原的水解活力。本章基于凝血酶与G15D核酸适配体的特异性结合原理,在G15D核酸适配体上修饰DNA分子信标结构,修饰后的探针在溶液中形成G-四聚体和分子信标两个结构域,G-四聚体结构域与凝血酶结合,抑制了凝血酶的水解活力。当溶液中存在与分子信标环部互补的DNA链时,由于DNA的杂交反应,使分子信标结构被打开,G-四聚体结构被破坏,凝血酶复活。不同匹配度DNA链与探针链杂交后,探针的G-四聚体结构被破坏的程度不同,凝血酶的活力不同,通过测定凝血酶的活力可以识别具有不匹配碱基的目标链。凝血酶的活力用凝血酶水解纤维蛋白原的初凝时间表示。由于凝血酶凝血时间是临床诊断中一种测定人体凝血功能的常规检测方法,将SNP的识别信号转化为凝血酶凝血时间,将易于在普通的诊疗机构推广应用。四、核酸适配体对凝血酶的变构作用研究凝血酶与底物复合物结晶、凝血酶定点突变、分子模拟等方法常常用来研究凝血酶变构作用。通过对凝血酶与凝血酶调制蛋白(thrombomodulin, TM)的结晶复合物检测,发现TM与凝血酶结合并没有引起凝血酶构象改变。但又有研究表明,TM结合于凝血酶使凝血酶对蛋白质C的识别能力增强。对于外结合位点Ⅰ和Ⅱ之间是否有相互作用这一重要问题,也始终没有一致的结论,有的研究认为两个位点之间有构象相关性,即外结合位点Ⅰ与配体的结合会使外结合位点Ⅱ失去与配体的结合能力,但是有些研究又认为两个位点之间没有关联。G15D核酸适配体特异性结合于凝血酶外结合位点Ⅰ,抑制凝血酶对纤维蛋白原的水解活力,抑制凝血酶与TM的结合。DNA 60-18(29)核酸适配体结合于外结合位点Ⅱ,也抑制凝血酶对纤维蛋白原的水解活力。由于G15D核酸适配体和DNA 60-18(29)核酸适配体本身并不被凝血酶水解,并且凝血酶水解发色底物的反应不受核酸适配体的抑制,因此本章用核酸适配体来研究外结合位点对凝血酶的变构作用。本章我们选用核酸适配体研究外结合位点对凝血酶的变构作用。首先,核酸适配体可以分别结合于凝血酶的两个位点,本身不被凝血酶水解。其次,相对于TM、纤维蛋白原等大分子蛋白类底物,核酸适配体对酶构象和活力检测的干扰大大减小。此外,凝血酶水解小分子发色底物的反应不被核酸适配体抑制,可以直接检测凝血酶水解活力的变化。研究结果表明,核酸适配体与凝血酶的结合会引起凝血酶构象的轻微改变,这种改变可以促进凝血酶对发色底物的水解。这一现象似乎可以说明,大分子底物结合于凝血酶外结合位点,不仅有利于底物的酶切位点与凝血酶活性中心的接触,还可以促进凝血酶的水解活力。五、基于核酸适配体与凝血酶的相互作用测定凝血酶本章基于核酸适配体和凝血酶相互作用的研究结果,利用核酸适配体制作生物传感器来检测凝血酶。用方波电势脉冲(SWPP)法制备纳米多孔金电极,将巯基修饰的G15D核酸适配体固定在纳米多孔金电极上,之后利用核酸适配体结合溶液中的凝血酶,凝血酶的另一个结合位点连接与金纳米颗粒结合的巯基-DNA60-18[29]核酸适配体,形成三明治式结构。为了减少凝血酶与金纳米颗粒上核酸适配体的交互作用,还在金纳米颗粒表面固定了不与凝血酶结合的寡核苷酸链。利用电极外层吸附的阳离子还原剂[Ru(NH3)6]3+产生电化学信号。由于纳米多孔金和金纳米颗粒的放大作用,所形成的传感器可以非常灵敏地用于凝血酶的检测。更多还原

【Abstract】 DNA sequence determines the characteristics of DNA molecule. The change of DNA sequence affects not only the coding for a specific polypeptide, but also the coding for RNA molecules such as ribosomal RNA and transfer RNA, thus resulting in individual differences in human being. About 90% of human DNA polymorphism is single nucleotide polymorphism (SNP). SNP is single base pair position at which different sequence alternatives (alleles) exist in normal individuals in some population(s) and has close relationship with genetic diseases and drug sensitivity. Therefore, it is very important to detect SNP in clinical diagnosis and therapy.Most DNA assays are based on DNA hybridization reacted in aqueous solution or solid-supported system. Compared with solid-supported DNA hybridization, homogeneous reactions are fitter for automation because no separation or purification is needed after the allele discrimination reaction. However, Most of the homogeneous SNP typing methods need the label of fluorephore or radioactive isotope, which makes them expensive, special-apparatus-depending and time-consuming, thereby often beyond the reach of clinical laboratory.The thesis aims to develop fast and easy SNP assays in homogeneous solution which can be used routinely in disease diagnosis and therapy in clinics.1. Using a Cyanine Dye to Transmit DNA Hybridization Signal to Visible AbsorbanceIntercalating fluorescent dyes have been used to transmit dsDNA signal because of their high affinity for and large fluorescence enhancement upon binding dsDNA, but special optical apparatus is needed. The cyanine dyes are also able to recognize dsDNA structures by intercalation, exterior stacking, major groove binding, and minor groove binding. With the specific binding of cyanine dyes, DNA hybridization signal can be transmitted into visible absorbance, thus realizing easy and fast assay using ordinary spectrophotometer.A kind of cyanine dye,3,3’-diethylthiatricarbocyanine iodide (DTTC) was used to transmit the DNA hybridization signal to visible absorbance of the cyanine dye based on the specific binding interaction of duplex DNA and DTTC. The result indicated that, when binding with DNA duplex, the cyanine dye produced two absorbing peaks, in which the absorbance peak at 760nm resulted from intercalation binding mode, while the other absorbance peak at 675 nm due to groove binding mode. The absorbance at 760nm and its stability were influenced by the length of the hybrids and the experimental temperature. When the temperature was set at 25℃and 24-mer oligonucleotide was used as the probe, target DNA with concentration being as low as 1μg·mL-1 could be detected, thus realizing a more sensitive assay with the sensitivity being two orders lower than that of DNA at 260nm. Under appropriate conditions, the cyanine dye could be used not only to detect target single stranded DNA, but also to recognize SNP in target DNA fragments. Compared with other target DNA or SNP-typing assays, the present assay had advantages in simplicity and rapidity, and had an applicable potential in clinical diagnosis.2. Monitoring DNA Hybridization by UV spectra in AOT reverse micellesDNA hybridization could be monitored by measuring UV absorbance at 260 nm. But when DNA hybridized in aqueous solution at a high concentration, the absorbance would be too high to be detected. While DNA hybridized at a low concentration, the change of the absorbance at 260 nm could not be detected either.Reverse micelles, which are formulated by surfactant molecules in organic solvents, provide nanostructural water pools which can dissolve DNA at rather high concentration but the whole concentration is still low. Reverse micelles decrease DNA hybridization rate and the stability of dsDNA, thus amplify the differences between fully matched and partially matched DNA targets. Goto et al reported a 20-mer DNA hybridization in reverse micelles, but results showed the assay was time consuming.Using a 24-mer oligonucleotide as a model, we studied the influence of AOT reverse micelles on DNA hybridization rate and the methods to amplify the signal differences among different complementary DNA fragments. In order to increase DNA hybridization rate in reverse micelles, we separated the two phases by freezing the reverse micelles.After the freeze of AOT reverse micelles, DNA remained in organic solvent, but the bulk of water decreased deeply. Because fully complementary dsDNA folded more closely than those partially complementary dsDNAs, after the freezing, the water content in fully complementary dsDNA system was much lower than those partially complementary dsDNAs. Thus by measuring water content, a mutation in target DNA fragment could also be recognized.3. Transmit DNA Hybridization Signal to Catalytic Activity of an EnzymeTo develop a feasibly low cost SNP assay in a homogeneous solution, we designed a DNA probe, which had a thrombin-inhibiting aptamer domain and a stem-and-loop structure (like a molecular beacon). In this way, the hybridization signal produced at the loop could be switched directly to the signal of thrombin activity. The stem-and-loop structure was carefully designed so that under appropriate hybridization conditions, different target DNAs with sequences either fully or partially complementary to the loop sequence resulted in different degrees of the damage of the aptamer domain on the probe. The conformational changes of the probe after its hybridization with target DNAs were confirmed by CD spectra and FRET studies. Under selected conditions, different target DNAs resulted in obvious differences in thrombin activity toward fibrinogen, thereby, realizing an easy discrimination of SNPs using routinely clinical fibrinogen clotting assay. Compared with other SNP-typing assays, the present assay has advantages in simplicity and rapidity, and has potential application in clinical diagnosis.4 Binding effects of aptamers on thrombinOn the surface of thrombin, there exist two positively charged binding sites-exosite I and exosite II. The two exosites facilitate thrombin to bind various negatively charged substrates or ligands so as to regulate the different role of thrombin. Many reports have been released on the recognition mechanism of thrombin for its natural substrates or ligands through the two exosites, but there is a controversy on the allostery of thrombin during the exositeⅠand/or exositeⅡbinding(s).Crystallographic study on thrombin-thrombomodulin complex indicated no change of the conformation of thrombin’s active site. Study on thrombin-fibrinogen crystal did not provide any information about the allosteric effect of fibrinogen on thrombin. However, some studies showed that the binding of TM fragment, hirudin fragment or other ligands to exositeⅠand exositeⅡproduced allosteric changes. ExositesⅠandⅡwere reported to be closely linked allosterically, i.e., binding of a ligand to one exosite resulted in nearly total loss of affinity of the other exosite for ligands, but other studies supported the independence of the interactions. The contradictory results may be due to the following facts:Firstly, thrombin and its macromolecular substrates or ligands lost flexibility in crystal, so the slight allosteric change of thrombin may be undetectable. Secondly, the binding ligands used were usually from thrombin’s natural substrates, inhibitors, or the fragments of the substrates or their derivatives. Their binding to one exosite may affect the binding of other ligands to another exosite.For thrombin, two kinds of aptamers are obtained. One binds to exositeⅠ, the other binds to exositeⅡ. Because the two aptamers are non-protein, soluble, and specific for binding exositeⅠorⅡ, the conformation of thrombin with exosites empty or occupied by one or two aptamer(s) can be observed in aqueous solution using CD spectra and intrinsic fluorescence without any modifications or marks of thrombin and/or the aptamers. In addition, the aptamers have no direct linkage with the active site of thrombin. The use of small chromogenic substrate, (3-Ala-Gly-Arg-p-nitroanilide diacetate, which is cleaved by thrombin at a slow rate, for the determination of the hydrolysis activity of thrombin also facilitated the present study, making the phenomenon straightforward and obvious.CD and intrinsic fluorescence spectra indicated that after binding with aptamers the secondary structure of thrombin seemed unchanged, but the whole conformation of thrombin changed. The binding of aptamers on thrombin also made the catalytic activity of thrombin toward the chromogenic substrate (β-Ala-Gly-Arg-p-nitroanilide diacetate) increased. The present study indicated that the allostery of the two exosites seemed to be independent.5 Sensitive thrombin detection based on the interaction between aptamers and thrombinBased on the interaction between aptamers and thrombin, an attempt was made to develop a nanoporous gold (NPG)-based electrochemical aptasensor for thrombin detection. The substrate electrode NPG was in situ fabricated by a facile one-step square wave potential pulse (SWPP) treatment. The treatment involved repeated gold oxidation-reduction and intensive H2 bubbles evolution. After 100 min treatment, the active surface area of Au increased greatly (34 times). The electrochemical aptasensor was fabricated using a layer-by-layer assembling strategy. A "sandwich" structure was formed via thrombin connecting the aptamer-modified NPG and the aptamer-modified gold nanoparticles (GNPs). The AuNPs was modified with two kinds of single strand DNA (ssDNA). One was aptamer of thrombin, but the other was not, reducing the crossreaction between thrombin and its aptamer on the same GNP. The electrochemical signal produced by the [Ru(NH3)6]3+ bound to ssDNA via electrostatic interaction was measured by chronocoulometry. Due to the amplification effects of both NPG and GNPs, this novel NPG-based aptasensor could detect thrombin quantitatively in the range of 0.01-22nmol/L with a detection limit as low as 30 fmol/L. The present aptasensor also exhibited excellent selectivity, stability and reusability.更多还原