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???????? Fluorescent DNA-based enzyme
sensors Molecular Engineering of DNA Molecular
Beacons Biological Applications of Molecular
Beacons
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Molecular Engineering of DNA Molecular Beacons
???? (Molecular Beacons) ??????????
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Biological Applications of Molecular Beacons
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????PCR?? (Real-time PCR assays)
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????DNA (Detection of triplex DNA)
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??????????? (SNP and Genetic Screening)
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?????(Monitoring proteins)
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Application of MBs in enzymatic studies. a)
Real-time monitoring of SSBDNA 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 LDHDNA interactions LDH binds the MB and
disturbs its structure, whereby fluorescence is
enhanced.
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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 11, 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
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
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
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
Figure 3. Thermal proles for MB hybrids. (Left)
Curve A is the thermal prole 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
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
Fig. 1 Principle scheme of monitoring activity of
E. coli DNA ligase catalyzing DNA ligation based
on molecular beacon.
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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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