高分子传感材料一六 Fluorescent DNA-based enzyme sensors Molecular Engineering of DNA: Molecular Beacons Biological Applications of Mole cular Beacons
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 分子信标 Molecular Beacons的基本构成是怎样的? 2分子信标的工作原理是什么? 31分子信标设计的基本原则有哪些? Complementary Target
1 分子信标的工作原理是什么？ Molecular Engineering of DNA: Molecular Beacons 分子信标 (Molecular Beacons) 的基本构成是怎样的？ 2 3 分子信标设计的基本原则有哪些？
3 Biological Applications of Mole cular Beacons 实时检测PcR过程 Real-time PCR assays 2探测三股DNA( Detection of triplex DNA 31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)
Biological Applications of Molecular Beacons 1 单核酸多态性与基因筛选 (SNP and Genetic Screening) 2 探测三股DNA(Detection of triplex DNA) 实时检测PCR过程 (Real-time PCR assays) 3 4 监测蛋白质(Monitoring proteins) 5 生物传感器与生物芯片 (Biosensors and biochips)
34监测蛋白质 Monitoring proteins MB SSB Application of MBs in enzymatic Binding、Q 号埠 studies a) Real-time monitoring of SSB-DNA binding: The MB binds to the ssb protein Cleavage whereby its structure is MB Enzyme disrupted and its fluorescence is Cutting restored b)detection of the enzymatic o digestion of DNA: The enzyme cleaves the mb and destroys the hairpin structure to restore MB LDH fluorescence Binding c)Detection of LDH-DNA x interactions: LDH binds the MB oo and disturbs its structure whereby fluorescence is enhanced
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
34监测蛋白质 Monitoring proteins Detect Protein-DNA Interactions:o Molecular Beacons: A Novel approac Jianwei Jeffery Li, Xiaohong Fang, Sheldon M. schuster, and Weihong Tan s Prof, W. Tan, Dr JJ. Li, Dr. X. Fang, Prof S.M. Schuster TGC Department of Chemistry, Biochemistry and Molecular Biology and uf Brain Institute University of Florida 0=P-O Angew. Chem. Int. Ed. 2000, 39, No. 6 Q b) Q Q TAMRA DABCYI MB
3.4 监测蛋白质(Monitoring proteins)
34监测蛋白质 Monitoring proteins 800 700 500 e400 300 100 0 0100200300400500600700800 c(SSB)/nM Figure 3. Continuous fluorescence titration of the molecular beacon with SSB. Each fluorescence value(.) at a certain SSB concentration is the average of ten readings of the fluorescence after the binding has reached uilibrium(corresponding to the plateau part of the time-scan curve shown in Figure 2). The dilution effect has been considered processing. The concentration of the MB was 250 nM. The stoichiometric ratio of MB to SsB was determined by the intersection point of two straight lines(-)extended, respectively, from the initial linear part and the plateau part of the titration curve(---). The results indicate that one SSB molecule binds to one mB molecule
3.4 监测蛋白质(Monitoring proteins)
34监测蛋白质 Monitoring proteins 1.2 0.8 e06 0.4 02 0 圈 CDNA SSB 1b-DNA HIS BS Figure 4. Relative fluorescence enhancement occurring upon binding of the molecular beacon to various Dna and protein molecules. The concentrations used were: cDNA, 1 uM; SSB, 40 ugmL- 1b-DNA, 4 UM; HIS, 200 ugmL- BSA, 400 ug mL- MB, 250 nM. The concentrations of the proteins and dNa molecules are high enough to saturate the molecular beacon in all binding studies. The perfect CDNA sequence is shown in Figure 2. The one base-pair mismatch complementary sequence is: 5 CTTCCTTCTTGGGTATGGA-3(the underlined base represents the mismatch)
3.4 监测蛋白质(Monitoring proteins)
34监测蛋白质 Monitoring proteins Using Molecular Beacons To Probe Molecular Interactions between Lactate Dehydrogenase and Single-Stranded DNA Xiaohong Fang, Jianwei Jeff Li, and Weihong Tan Department of Chemistry and UF Brain Institute, University of Florida, Gainesville, Florida 32611 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 M B/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 1.9x10-7M-1. We have also studied salt effects, binding sites, temperature effects, pH effects, and the binding specificities for different isoenzy mes Our results demonstrate that MB can be effectively used for sensitive protein quantitation and for efficient protein-DNAinteraction 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 Analytical Chemistry, Vol. 72, No. 14, July 15, 2000
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 singlestranded 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 1.910-7 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
G C C 34监测蛋白质 T A 350 CGCG 300 G C 250 T A 200 C G 8150 8100 0二P B 5 10 H+4 Time(s) Figure 1.(A) The molecular beacon DNA molecule(MB1).(B) Fluorescence intensity time scan of the MB-binding reaction with LDH- TMR DABCYT 5. Measurements were taken to ensure that diffusion was not a factor in the reaction rate(15 s was required to fully mix the MB and LDH-5 solutions before the measurements). The concentrations of MB and LDH were 0.5 and 1 uM. The buffer used was 20 mM Tris/HCl, 0.05 mM MgCl2, pH=7.8
3.4 监测蛋白质
34监测蛋白质 Monitoring proteins 400 350 300 250 200 150 100 0 05 15 2 2.5 CoH uM Figure 2. Fluorescence intensity of molecular beacon 1 at different LDH concentrations. The concentration of mb was 0. 4 uM. the buffer used was 20 mM Tris/HCl, 0.05 mM MgCl2, pH=7.8
3.4 监测蛋白质(Monitoring proteins)