Fluorescent DNA-based enzyme sensors

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1 Fluorescent DNA-based enzyme sensors
高分子传感材料—六 Fluorescent DNA-based enzyme sensors Molecular Engineering of DNA: Molecular Beacons Biological Applications of Molecular Beacons

2  Molecular Engineering of DNA: Molecular Beacons
1 2 分子信标的工作原理是什么？ 3 分子信标设计的基本原则有哪些？

3 Biological Applications of Molecular Beacons
 Biological Applications of Molecular Beacons 实时检测PCR过程 (Real-time PCR assays) 1 2 探测三股DNA (Detection of triplex DNA) 3 单核酸多态性与基因筛选 (SNP and Genetic Screening) 4 监测蛋白质(Monitoring proteins) 5 生物传感器与生物芯片 (Biosensors and biochips)......

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3.4 监测蛋白质(Monitoring proteins) Application of MBs in enzymatic studies. a) Real-time monitoring of SSB–DNA binding: The MB binds to the SSB protein whereby its structure is disrupted and its fluorescence is restored. b) Detection of the enzymatic digestion of DNA: The enzyme cleaves the MB and destroys the hairpin structure to restore fluorescence. c) Detection of LDH–DNA interactions: LDH binds the MB and disturbs its structure, whereby fluorescence is enhanced.

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3.4 监测蛋白质(Monitoring proteins) The interactions between two key macromolecular species, nucleic acids and proteins, control many important biological processes. There have been limited effective methodologies to study these interactions in real time. In this work, we have applied a newly developed molecular beacon (MB) DNA probe for the analysis of an enzyme, lactate dehydrogenase (LDH), and for the investigation of its properties of binding with single-stranded DNA. Molecular beacons are single-stranded oligonucleotide probes designed to report the presence of specific complementary nucleic acids by fluorescence detection. The interaction between LDH and MB has resulted in a significant fluorescence signal enhancement, which is used for the elucidation of MB/LDH binding properties. The processes of binding between MB and different isoenzymes of LDH have been studied. The results show that the stoichiometry of LDH-5/MB binding is 1:1, and the binding constant is 1.910-7 M-1. We have also studied salt effects, binding sites, temperature effects, pH effects, and the binding specificities for different isoenzymes. Our results demonstrate that MB can be effectively used for sensitive protein quantitation and for efficient protein-DNA interaction studies. MB has a signal transduction mechanism built within the molecule and can thus be used for the development of rapid protein assays and for real-time measurements.

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3.4 监测蛋白质(Monitoring proteins) Application of MBs in phosphorylation and ligation studies. a) Real-time monitoring of nucleic acid ligation: Two oligonucleotides that are complementary to opposite halves of the MB loop hybridize with the MB, whereby a nick is formed, and the stem may be opened slightly. The DNA ligase binds to the nick and catalyzes the ligation of the two short oligonucleotides to form a longer oligonucleotide. The ligation product hybridizes with the MB to restore fluorescence. b) Monitoring of nucleic acid phosphorylation: Oligonucleotide A is first phosphorylated at the 5’-hydroxy group by the polynucleotide kinase. The nick formed upon the hybridization of oligonucleotide B and phosphorylated oligonucleotide A with MB can be sealed by the DNA ligase, whereupon the stem helix of the MB is opened, and fluorescence is restored.

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3.4 监测蛋白质(Monitoring proteins) Figure 1. `Ligate and light'. Schematics diagram of real-time monitoring of the nucleic acid ligation process by a MB.

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3.4 监测蛋白质(Monitoring proteins) Figure 2. Real-time fluorescence scans and corresponding gel electrophoresis. (Left) Curve A is a time scan of fluorescence intensity of MB with N1; B is of MB with N2 and N4; C is of MB with N2 and N3; curve D is of MB itself. t0 is the time when T4 DNA ligase is added into the MB/oligo solution. (Right) Gel electrophoresis images. Lanes 1 and 2 are for sample D; 3 and 4 for sample C; 5 and 6 for sample B; and 7 and 8 for sample A. Lanes 1, 3, 5 and 7 represent samples D, C, B and A before the addition of T4 DNA ligase, while lanes 2, 4, 6 and 8 represent corresponding samples obtained at 360 s after the addition of ligase. There is a major difference between lanes 5 and 6, while there is basically no difference for all the other pairs.

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3.4 监测蛋白质(Monitoring proteins) Figure 3. Thermal pro®les for MB hybrids. (Left) Curve A is the thermal pro®le of MB with N1, and curve B is that of MB with N2 and N4. (Right) Fluorescence intensity ratio is plotted as a function of temperature. The ratio is calculated using the fluorescence intensity of MB with N1 over that of MB with N2 and N4 without ligase. This profle can be used for determining the optimal temperature for ligase studies using MBs.

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3.4 监测蛋白质(Monitoring proteins) Figure 4. Effects of molecular species on ligation process. (A) Ligation velocity in the presence of various concentrations of ATP. (B) Effects of Mg2+ on ligation. (C) Effects of dATP on ligation. (D) Effects of K+ and Na+ on ligation. Initial ligation velocity in each chart was normalized in each experiment. Therefore, each plot can only be compared within itself, and cannot be used to draw comparisons with plots.

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3.4 监测蛋白质(Monitoring proteins) Fig. 1 Principle scheme of monitoring activity of E. coli DNA ligase catalyzing DNA ligation based on molecular beacon.

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3.4 监测蛋白质(Monitoring proteins) Fig. 2 Correlation of gel eletrophoresis assay and fluorescence assay for DNA ligation. (a) Polyacrylamide gel assay for ligation reaction. (b) Real time monitoring curve of DNA ligation. The ligase added into sample at time t.

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