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《Journal of molecular recognition : JMR》2017,30(2)
To monitor the specificity of Staphylococcus aureus aptamer (SA‐31) against its target cell, we used enzyme‐linked aptamer assay. In the presence of target cell, horseradish peroxidase–conjugated streptavidin bound to biotin‐labeled SA‐31 showed specific binding to S aureus among 3 different bacteria with limit of detection of 103 colony‐forming unit per milliliter. The apparent K a was 1.39 μM−1 ± 0.3 μM−1. The binding of SA‐31 to membrane proteins extracted from cell surface was characterized using isothermal titration calorimetry, and the effect of changes in binding temperature and salt concentrations of binding buffer was evaluated based on thermodynamic parameters (K a, ΔH , and ΔG ). Since binding of aptamer to its targets solely depends on its 3‐dimensional structure under experimental conditions used in selection process, the change in temperature and ion concentration changed the affinity of SA‐31 to its target on surface of bacteria. At 4°C, SA‐31 did not show an affinity to its target with poor heat change upon injection of membrane fraction to aptamer solution. However, the apparent association constants of SA‐31 slightly varied from K a = 1.56 μM−1 ± 0.69 μM−1 at 25°C to K a = 1.03 μM−1 ± 0.9 μM−1 at 37°C. At spontaneously occurring exothermic binding reactions, affinities of S aureus aptamer to its target were also 9.44 μM−1 ± 0.38 μM−1 at 50mM, 1.60 μM−1 ± 0.11 μM−1 at 137mM, and 3.28 μM−1 ± 0.46 μM−1 at 200 mM of salt concentration. In this study, it was demonstrated that enzyme‐linked aptamer assay and isothermal titration calorimetry were useful tools for studying the fundamental binding mechanism between a DNA aptamer and its target on the outer surface of S aureus . 相似文献
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Chin-Yu Wang Chung-Yi WuTing-Chun Hung Chi-Huey WongChung-Hsuan Chen 《Biochemical and biophysical research communications》2014
We proposed to use a novel stepwise sequence-constructive SELEX method to develop DNA aptamers that can recognize Globo H which is a tumor-associated carbohydrate antigen. A combinatorial synthetic library that consisted of DNA molecules with randomized regions of 15-bases was used as the starting library for the first SELEX procedure. The input DNA library for the second round of SELEX consisted of the extension of the 5′ and 3′-ends with 7-bases that were randomized from four selected aptamers. The third round of SELEX was performed following the same procedures as described for the second round of SELEX. The experimental results indicate that the binding affinity of DNA aptamers to Globo H was enhanced when using the sequence-constructive SELEX approach. The selectivity of the DNA aptamers for related disaccharides, mannose derivatives, and Globo H analogs demonstrated the ability of the DNA aptamers to discriminate the presence of various glycans with different structures. 相似文献
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核糖核酸酶HII (RNaseHII)能有效降解RNA和DNA杂交链中的RNA链。为进一步研究其功能 ,利用大肠杆菌XL1blue为模板 ,相应的寡聚脱氧核苷酸为引物 ,PCR扩增大肠杆菌RNaseHII(rnh 2 )基因 ,并将目的基因连接到克隆载体 pUC18上 ,经测序确认无误 ,分别亚克隆到能够进行IPTG诱导的表达载体pTrcHisC和进行温度诱导的表达载体pBV2 2 0上。重组质粒转化到大肠杆菌DH5α细胞中获得高效表达。在载体pTrcHisC和 pBV2 2 0中目的蛋白RNaseHII的表达量均超过菌体总蛋白的 2 0 % ,且表达产物以稳定的包涵体形式存在。此项工作为以后目的蛋白的纯化提供了有利条件 ,并为研究其结构和功能奠定了基础。 相似文献
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核酶对人巨细胞病毒mRNA片段的体外切割 总被引:6,自引:1,他引:6
引导序列GSs(GuideSequences)是能与mRNA互补,引导核酶RNaseP催化核心M1RNA对互补区域特异切割的小片段游离RNA。针对人巨细胞病毒HCMV(humancytomegalovirus)DNA聚合酶mRNA序列设计GS,共价结合到大肠杆菌来源M1RNA中,构建成M1GST7核酶。通过对巨细胞病毒DNA聚合酶亚克隆片段转录产物体外切割实验,表明该核酶具备对DNA聚合酶mRNA片段的特异切割能力 。 相似文献
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Eric Ouellet Eric T. Lagally Karen C. Cheung Charles A. Haynes 《Biotechnology and bioengineering》2014,111(11):2265-2279
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Patel D Tock MR Frary E Feng M Pickering TJ Grasby JA Sayers JR 《Journal of molecular biology》2002,320(5):1025-1035
The flap endonucleases, or 5' nucleases, are involved in DNA replication and repair. They possess both 5'-3' exonucleolytic activity and the ability to cleave bifurcated, or branched DNA, in an endonucleolytic, structure-specific manner. These enzymes share a great degree of structural and sequence similarity. Conserved acidic amino acids, whose primary role appears to be chelation of essential divalent cation cofactors, lie at the base of the active site. A loop, or helical archway, is located above the active site. A conserved tyrosine residue lies at the base of the archway in phage T5 flap endonuclease. This residue is conserved in the structures of all flap endonucleases analysed to date. We mutated the tyrosine 82 codon in the cloned T5 5' nuclease to one encoding phenylalanine. Detailed analysis of the purified Y82F protein revealed only a modest (3.5-fold) decrease in binding affinity for DNA compared with wild-type in the absence of cofactor. The modified nuclease retains both structure-specific endonuclease and exonuclease activities. Kinetic analysis was performed using a newly developed single-cleavage assay based on hydrolysis of a fluorescently labelled oligonucleotide substrate. Substrate and products were resolved by denaturing HPLC. Steady-state kinetic analysis revealed that loss of the tyrosine hydroxyl function did not significantly impair k(cat). Pre-steady state analysis under single-turnover conditions also demonstrated little change in the rate of reaction compared to the wild-type protein. The pH dependence of the kinetic parameters for the Y82F enzyme-catalysed reaction was bell-shaped as for the wild-type protein. Thus, Y82 does not play a role in catalysis. However, steady-state analysis did detect a large (approximately 300-fold) defect in K(M). These results imply that this conserved tyrosine plays a key role in ternary complex formation (protein-DNA-metal ion), a prerequisite for catalysis. 相似文献
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Cheng C Dong J Yao L Chen A Jia R Huan L Guo J Shu Y Zhang Z 《Biochemical and biophysical research communications》2008,366(3):670-674
New therapeutics are urgently needed for the treatment of pandemic influenza caused by H5N1 influenza virus mutants. Aptamer was a promising candidate for treatment and prophylaxis of influenza virus infections. In this study, systemic evolution of ligands through exponential enrichment (SELEX) was used to screen DNA aptamers targeted to recombinant HA1 proteins of the H5N1 influenza virus. After 11 rounds of selection, DNA aptamers that bind to the HA1 protein were isolated and shown to have different binding capacities. Among them, aptamer 10 had the strongest binding to the HA1 protein, and had an inhibitory effect on H5N1 influenza virus, as shown by the hemagglutinin and MTT assays. These results should aid the development of new drugs for the prevention and control of influenza virus infections. 相似文献
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核酸适配体生物传感器是利用固定在电极表面的适配子与被测溶液中心肌肌钙蛋白Ⅰ(cTnⅠ)发生特异性结合,从而达到检测的目的.我们对玻碳电极进行阳极氧化、氨基化修饰,通过碳二亚胺盐酸盐(carbodiimide hydrochloride,EDC)、N-羟基琥珀酰亚胺(N-hydroxysuccinimide,NHS)活化作用将适配子结合在电极表面.cTnⅠ最佳检测范围是0.05~5 nmol/L,最低检测限为0.05 nmol/L,检测时间为5 min. 相似文献
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We have developed an aptameric enzyme subunit (AES) which can detect the DNA in a homogeneous solution. The AES is an artificial
enzyme subunit composed of an enzyme-inhibiting aptamer bearing a target-molecule binding site. We connected a probe DNA to
a thrombin-inhibiting aptamer at its 5′ or 3′ end. The inhibitory activity of the thrombin-inhibiting aptamer bearing the
probe DNA decreased compared to that of the original aptamer; however, it recovered upon hybridization with the target DNA.
Using this AES, we were able to detect target DNAs by measuring the thrombin activity in a homogeneous solution.
K. Ikebukuro and W. Yoshida have contributed equally to this work. 相似文献
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《DNA Repair》2017
Y-family DNA polymerase iota (Pol ι) possesses both DNA polymerase and dRP lyase activities and was suggested to be involved in DNA translesion synthesis and base excision repair in mammals. The 129 strain of mice and its derivatives have a natural nonsense codon mutation in the second exon of the Pol ι gene resulting in truncation of the Pol ι protein. These mice were widely used as a Pol ι-null model for in vivo studies of the Pol ι function. However whether 129-derived strains of mice are fully deficient in the Pol ι functions was a subject of discussion since Pol ι mRNA undergoes alternative splicing at exon 2. Here we report purification of mouse Pol ι lacking the region encoded by exon 2, which includes several conserved residues involved in catalysis. We show that the deletion abrogates both the DNA polymerase and dRP lyase activities of Pol ι in the presence of either Mg2+ or Mn2+ ions. Thus, 129-derived strains of mice express catalytically inactive alternatively spliced Pol ι variant, whose cellular functions, if any exist, remain to be established. 相似文献
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A. S. Prakasha Gowda George-Lucian Moldovan Thomas E. Spratt 《The Journal of biological chemistry》2015,290(26):16292-16303
DNA polymerase ν (pol ν) is a low fidelity A-family polymerase with a putative role in interstrand cross-link repair and homologous recombination. We carried out pre-steady-state kinetic analysis to elucidate the kinetic mechanism of this enzyme. We found that the mechanism consists of seven steps, similar that of other A-family polymerases. pol ν binds to DNA with a Kd for DNA of 9.2 nm, with an off-rate constant of 0.013 s−1and an on-rate constant of 14 μm−1 s−1. dNTP binding is rapid with Kd values of 20 and 476 μm for the correct and incorrect dNTP, respectively. Pyrophosphorylation occurs with a Kd value for PPi of 3.7 mm and a maximal rate constant of 11 s−1. Pre-steady-state kinetics, examination of the elemental effect using dNTPαS, and pulse-chase experiments indicate that a rapid phosphodiester bond formation step is flanked by slow conformational changes for both correct and incorrect base pair formation. These experiments in combination with computer simulations indicate that the first conformational change occurs with rate constants of 75 and 20 s−1; rapid phosphodiester bond formation occurs with a Keq of 2.2 and 1.7, and the second conformational change occurs with rate constants of 2.1 and 0.5 s−1, for correct and incorrect base pair formation, respectively. The presence of a mispair does not induce the polymerase to adopt a low catalytic conformation. pol ν catalyzes both correct and mispair formation with high catalytic efficiency. 相似文献
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对一种耐热性古茵--詹氏甲烷球茵(Methanocaldococcus jannaschii)的DNA连接酶进行了克隆、表达、纯化,并对其生物化学特性和酶学活性进行了初步研究.詹氏甲烷球菌DNA连接酶重组蛋白在ATP及Mg<'2+>二价阳离子存在的条件下具有连接酶活性,能够封闭DNA链上的切割.通过不同温度下的测试,50~80℃为较适合连接温度,其耐热性强,甚至在90℃下加热5 min后仍有连接酶活性;其发挥活性的pH值范围比较宽泛.最适pH值为6.0~9.0.这是国际上对詹氏甲烷球菌DNA连接酶的首次报导. 相似文献
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Mélanie Lemor Ziqing Kong Etienne Henry Raphaël Brizard Sébastien Laurent Audrey Bossé Ghislaine Henneke 《Journal of molecular biology》2018,430(24):4908-4924
Consistent with the fact that ribonucleotides (rNTPs) are in excess over deoxyribonucleotides (dNTPs) in vivo, recent findings indicate that replicative DNA polymerases (DNA Pols) are able to insert ribonucleotides (rNMPs) during DNA synthesis, raising crucial questions about the fidelity of DNA replication in both Bacteria and Eukarya. Here, we report that the level of rNTPs is 20-fold higher than that of dNTPs in Pyrococcus abyssi cells. Using dNTP and rNTP concentrations present in vivo, we recorded rNMP incorporation in a template-specific manner during in vitro synthesis, with the family-D DNA Pol (PolD) having the highest propensity compared with the family-B DNA Pol and the p41/p46 complex. We also showed that ribonucleotides accumulate at a relatively high frequency in the genome of wild-type Thermococcales cells, and this frequency significantly increases upon deletion of RNase HII, the major enzyme responsible for the removal of RNA from DNA. Because ribonucleotides remain in genomic DNA, we then analyzed the effects on polymerization activities by the three DNA Pols. Depending on the identity of the base and the sequence context, all three DNA Pols bypass rNMP-containing DNA templates with variable efficiency and nucleotide (mis)incorporation ability. Unexpectedly, we found that PolD correctly base-paired a single ribonucleotide opposite rNMP-containing DNA templates. An evolutionary scenario is discussed concerning rNMP incorporation into DNA and genome stability. 相似文献
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Spiridonova V. A. Rog E. V. Dugina T. N. Strukova S. M. Kopylov A. M. 《Russian Journal of Bioorganic Chemistry》2003,29(5):450-453
The formation of complexes between various thrombin preparations and 30-mer aptamer DNA was comparatively studied, and a correlation between the complex formation and the fibrinogen-hydrolyzing activity of thrombin was found. The aptamer DNA was shown to inhibit the formation of fibrin from fibrinogen. 相似文献
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