Research Proposal

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Research Proposal

• Libo Cao
• Ph.D., Analytical (Dr. Peter de B. Harrington)
• Ohio University
• Department of Chemistry and Biochemistry
• Athens, OH 45701-2979

Ohio University
Center for
Intelligent Chemical Instrumentation
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I. Introduction

• Molecular Beacon (MB)
• Single-stranded oligonucleotide probes with
  a hairpin structure that can identify the
  mutations in the human genome caused by DNA
  hybridization.
• Principle of Operation of MBs

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Advantages Over Other DNA Probes

• Extremely high selectivity with single base pair
  mismatch identification capability
• The excellent capability of studying biological
  process in real time and in vivo, and avoiding
  the inconvenience caused by using DNA
  intercalation reagents or by labeling the target
  molecules or using competitive assays

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What MBs can do?

• Detection of single-nucleotide variations
• Single-base mismatch
• DNA sequencing
• DNA biosensor based on MBs
• Sensitive monitoring of the polymerase chain
  reaction
• Real-time detection of DNA-RNA hybridization in
  living cells

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Structure of A, T, C, G
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Label Dyes with the Biomolecular
1,3-dicycolhexylcarbodiimide Dimethyl formamide
Carboxylic group
N-Hydroxysuccinimide
Sulfonyl group
Davidson, R. S. Hilchenbach, M. M. Photochem.
Photobiol. 1990, 52, 431.
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How Dye Ester Linked with Sequencing Primer
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The Structure of a Molecular Beacon
Can be written as
Dye1-5-GCGAGAAGTTAAGAACCTATGCTCGC-3-Dye2
Dye2
Dye1
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Efficiency Evaluation of the MBs
One fluorophore Two fluorophores
Mechanism
Efficiency Parameter
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Fluorescence Resonance Energy Transfer (FRET)
FRET involves non-radiative transfer of
electronic excitation from an excited donor, D
to a ground state acceptor molecule A, and occur
at distances ranging from 10 to approximately
100 Å.
E Energy transfered Förster critical
distance R Distance between the
donor and acceptor
.
Hillisch, A. Lorenz, M. Diekmann, S. Curr.
Opin. Struc. Biol. 2001, 11, 201.
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Effect of Auto fluorescence
Auto fluorescence of Gray Snapper (L.griseus)
Oocyte
http//www.diatronscience.com/AutoFluor.html.
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My Goals of Designed Molecular Beacons

• Longer wavelength to avoid background noise
• Two Fluorophores
• The two dyes chosen should be able to FRET

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Choose Donor and Acceptor Dyes

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FRET Between Cy3 and Cy5 Attached to a
Coiled-coil of Homodimer
Schematic drawing of a protein, Tyopomyosin, A
coiled-coil in the native state is denatured
into two polypeptide chains by denatureant,
temperature and lowering salt concentration at
room temperature. Thick lines represent a-helix
and thin lines random coil polypeptide. Donor
(D) and acceptor (A) fluorephores are labeled to
a single cysteine residue at 190th position
a-tropomyosin (aTm)
Ishii, Y. Yoshida, T. Funatsu, T. Wazawa, T.
Yanagida, T. Chem. Phys. 1999, 247, 163.
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Fluorescence Spectrum for Tm Labeled with Cy3 and
Cy5 in Bulk Measurements
Ishii, Y. Yoshida, T. Funatsu, T. Wazawa, T.
Yanagida, T. Chem. Phys. 1999, 247, 163.
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Fluorescence images of FRET within a Single
Protein Molecule

• The donor (Cy3) images taken
• with a band-pass filter of
• 545-595 nm on excitation at the
• donor
• The acceptor (Cy5) images due
• to FRE T taken with a band-pass
• filter of 650-710 nm on excitation
• at the donor

Ishii, Y. Yoshida, T. Funatsu, T. Wazawa, T.
Yanagida, T. Chem. Phys. 1999, 247, 163.
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Fluorescence Intensity of the Donor Fluorescence
Ishii, Y. Yoshida, T. Funatsu, T. Wazawa, T.
Yanagida, T. Chem. Phys. 1999, 247, 163.
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Fluorescence Intensity of the Increase in the
Acceptor Fluorescence Due to FRET
Ishii, Y. Yoshida, T. Funatsu, T. Wazawa, T.
Yanagida, T. Chem. Phys. 1999, 247, 163.
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Fluorescence Spectrum from a single
Cy3-Cy5-Labeled aTm molecule
Ishii, Y. Yoshida, T. Funatsu, T. Wazawa, T.
Yanagida, T. Chem. Phys. 1999, 247, 163.
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Time Records of the Donor and Acceptor
Fluorescence from a Single Cy3-Cy5 Labeled aTm
molecule
Ishii, Y. Yoshida, T. Funatsu, T. Wazawa, T.
Yanagida, T. Chem. Phys. 1999, 247, 163.
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Designed MB and target DNA sequences
__________________________________________________
_____________________ MB 5-Dye1-GCTCGTCCATG
CCCAGGAAGGAGGCAACGACACGAGC-Dye2-3 Target
5-GTCGTTGCCTCCTTCCTGGGCATGG-3 __________________
__________________________________________________
____
Dye1
Dye1Cy3
Dye2Cy5
Dye2
Dye1
Dye2
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Model of the D/A DNA Constructs with Varying
Distance
Dietrich, A. Buschmann, V. Mller, C. Sauer, M.
Rev. Mol. Biotechnol. 2002, 82, 211.
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Schematic Diagram of the Optical Setup
1) Frequency-doubled NdYAG (Neodymium) laser
emitting at 532 nm 2) The collimated laser
beam was directed into an inverted
microscope and coupled into the microscope
objective with high numerical apertures
via a dichroic beam splitter 3) Within the
microscope objective, the beam was focused
into the sample to detect freely diffusing FRET
constructs 4) The fluorescence light was
collected through the same objective and
imaged onto the active areas of two avalanche
photodiodes 5) Need additional band pass filters
in front of the APDs
Dietrich, A. Buschmann, V. Müller, C. Sauer,
M. Rev. Mol. Biotechnol. 2002, 82, 211
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Spectroscopic Characteristics of the Different
FRET Constructs In Aqueous Buffer
-- Relative fluorescence quantum yield of donor
-- FRET energy of donor decreased
-- FRET energy of acceptor increased
Dietrich, A. Buschmann, V. Müller, C. Sauer,
M. Rev. Mol. Biotechnol. 2002, 82, 211.
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Fluorescence Emission Spectra of the Different
FRET Constructs in Aqueous Buffer
Dietrich, A. Buschmann, V. Müller, C. Sauer,
M. Rev. Mol. Biotechnol. 2002, 82, 211.
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FRET Histograms Extracted from Single Molecule
Data of the Differently Labeled D/A Constructs
and Corresponding Gaussian Fits
Dietrich, A. Buschmann, V. Müller, C. Sauer,
M. Rev. Mol. Biotechnol. 2002, 82, 211.
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A Fiber-Optic Evanescent Wave DNA Biosensor Based
on This MB

• Advantages
• The DNA sensor based on a MB does not need
  labeled analyte or intercalation reagents.
• Can be used to directly detect, in real time
  target DNA/RNA molecules without using
  competitive assays.
• It is rapid, stable, highly selective, and
  reproducible.

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Scheme of immobilization of biotinlyed MB DNA on
optical fiber surface
Liu, X Tan, W. Anal. Chem. 1999, 71, 5054.
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Dynamics of Hybridization of MB Evanescent Wave
Sensor

• Noncomplementary oligonucleotide
• One-base-mismatched oligonucleotide
• Complementary oligonucleotide
• All in aqueous buffer containing 1 M NaCl

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Experiment--Donors and Acceptors for the MBs
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Four MBs Designed

• 5-Cy3-GCTCGCCATGCCCAGGAAGGAGGCAACGACCGAGC-Cy5-3
• 5-TMR-GCTCGCCATGCCCAGGAAGGAGGCAACGACCGAGC-Cy5-3
• 5-R6G-GCTCGCCATGCCCAGGAAGGAGGCAACGACCGAGC-Cy5-3
• 5-TMR-GCTCGCCATGCCCAGGAAGGAGGCAACGACCGAGC-JA133-3
  

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DNA Sequences Needed

• Complementary DNA CGAGCGGTACGGGTCCTTCCTCCGTTGCTGG
  CTCG
• One-base-mismatch DNA CGAGCGGTACGGGTCCTACCTCCGTTG
  CTGGCTCG
• Two-base-mismatch DNA CGAGCGGTACGGGTCCTAGCTCCGTTG
  CTGGCTCG
• Non-complementary DNA CGAAACCTGCGAATGGTAGCTCCAATG
  TGGAATCG

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Estimation of the FRET parameters
-- quantum yield of the donor
-- orientation factor
k²
-- overlap integral
J
-- refraction index of the medium
n
R0 of the four investigated D/A pairs are
63.5
for (R6G/Cy5)
for (TMR/Cy5)
64.5
for (Cy3/Cy5)
55.8
59.0
for (TMR/JA133)
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Schematic of Experimental Setup
Sample

Microscope objective
Beam splitter
Laser source
APD
Personal computer
Beam splitter
Band-pass filters
Counting board
APD
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Evaluation of efficiency for MBs
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Cost Analysis

• DNA sequences
• lt300
• Molecular beacons
• lt5000
• Optical Setup
• lt5000
• Computer with software
• lt6000
• Other Chemicals
• lt2000

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Novelty of my work

• FRET have been used to measure distances in
  protein structures and their assemblies in
  solution, have not been used in MB application
  yet. My proposal integrated FRET technique into
  MB.
• Idea of two fluorophore MB were created in 2001,
  no such MB are created yet. My proposal created
  several MB that will have higher efficiency than
  current MBs.

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CONCLUSIONS

• A new strategy of designing MBs which uses two
  fluorophores (Cy3 and Cy5) instead of one
  fluorophore and one quencher as the donor and
  acceptor was proposed
• MBs display high sensitivity and a large dynamic
  range
• Such MBs are able to detect target DNA with 35
  bases up to 1x10-7 M

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Future Work

• Studying protein-DNA/RNA interactions
• Fluorescent immunoassay
• DNA sequencing
• Other bio-molecular analyses

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Acknowledgements

• Dr. Pete B Harrington
• Mariela Ochoa
• Preshious Rearden
• Bryon Moore