research proposal libo cao ph.d., analytical (dr. peter de b. harrington) ohio university department...
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Research Proposal
Libo CaoPh.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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Ohio University Center for Intelligent Chemical Instrumentation
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
O
N
NH2
ON
O
HO
HH
HH
OP
O-
O
Cytosine
O
NH
O
ON
O
HO
HH
HH
OP
O-
O
Thymine
O
N
NN
N
NH2
O
HO
HH
HH
PO
O
O-
CH3
Adenine
O
NH
N
N
O
NH2N
O
H
HH
HHO
PO
O
O-
CH3
Guanine
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Ohio University Center for Intelligent Chemical Instrumentation
Label Dyes with the Biomolecular
Dye COOH N
O
O
OHDye COO N
O
O
DCC, DMF
Dye SO3H N
O
O
OHDCC, DMF
Dye S N
O
OO
O S
O
NHNH2 Bio
BioDye
O O
Davidson, R. S.; Hilchenbach, M. M. Photochem. Photobiol. 1990, 52, 431.
Carboxylic group
Sulfonyl group
N-Hydroxysuccinimide1,3-dicycolhexylcarbodiimideDimethyl formamide
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How Dye Ester Linked with Sequencing Primer
N OO
H2N(CH2)6OH
P O(i-Pr)2N
Cl
CN
P O(i-Pr)2N
(i-Pr)2N
CN
PO
N(i-Pr)2
O
O Dye
O
O
HO
PO
O
-OOH
O
NC
OOxidation
Linker
i).
ii).
Linker
Chloro Reagent
Bis Reagent
Dye
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The Structure of a Molecular Beacon
A GAA
ACC
TA
TG
C
T
T
G
A
AG
A T
G C
C G
G C
OO 3'5'P OO
O
Dye1-5’-GCGAGAAGTTAAGAACCTATGCTCGC-3’-Dye2
Can be written as:
Dye1Dye2
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Ohio University Center for Intelligent Chemical Instrumentation
Efficiency Evaluation of the MBs
One fluorophore Two fluorophores
Mechanism
Efficiency
Parameter,
,
fl open
fl close
IE
I
1, 2,
1, 2,
1, 2,
1, 2,
/
/f open f open
f close f close
f open f close
f close f open
I IE
I I
or
I IE
I I
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Fluorescence Resonance Energy Transfer (FRET)
60
6 60
RE
R R
Hillisch, A.; Lorenz, M.; Diekmann, S. Curr. Opin. Struc. Biol. 2001, 11, 201.
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
0R
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Effect of Auto fluorescence
http://www.diatronscience.com/AutoFluor.html.
Auto fluorescence of Gray Snapper (L.griseus) Oocyte
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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
Ishii, Y.; Yoshida, T.; Funatsu, T.; Wazawa, T.; Yanagida, T. Chem. Phys. 1999, 247, 163.
α-tropomyosin (αTm)
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 α-helix and thin lines random coil polypeptide.
Donor (D) and acceptor (A) fluorephores are labeled to a single cysteine residue at 190th position
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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
Ishii, Y.; Yoshida, T.; Funatsu, T.; Wazawa, T.; Yanagida, T. Chem. Phys. 1999, 247, 163.
(a) The donor (Cy3) images taken with a band-pass filter of 545-595 nm on excitation at the donor
(b) The acceptor (Cy5) images due to FRE T taken with a band-pass filter of 650-710 nm on excitation at the donor
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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 αTm 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 αTm 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-GCTCGTCCATGCCCAGGAAGGAGGCAACGACACGAGC-Dye2-3’
Target: 5’-GTCGTTGCCTCCTTCCTGGGCATGG-3’________________________________________________________________________
Dye1=Cy3
Dye2=Cy5
Dye2
Dye2
Dye1
Dye1
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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.
..
u..
u..
u
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Schematic Diagram of the Optical Setup
Dietrich, A.; Buschmann, V.; Müller, C.; Sauer, M. Rev. Mol. Biotechnol. 2002, 82, 211
1) Frequency-doubled Nd:YAG (Neodymium) laser emitting at 532 nm2) The collimated laser beam was directed into an inverted microscope and coupled into the microscope objective with high numerical apertures via a dichroic beam splitter3) Within the microscope objective, the beam was focused into the sample to detect freely diffusing FRET constructs4) The fluorescence light was collected through the same objective and imaged onto the active areas of two avalanche photodiodes5) Need additional band pass filters in front of the APDs
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Spectroscopic Characteristics of the Different FRET Constructs In Aqueous Buffer
,Df rel DE
,Df rel
Dietrich, A.; Buschmann, V.; Müller, C.; Sauer, M. Rev. Mol. Biotechnol. 2002, 82, 211.
DEAE
-- Relative fluorescence quantum yield of donor
-- FRET energy of donor decreased
-- FRET energy of acceptor increased
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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:1) The DNA sensor based on a MB does not need
labeled analyte or intercalation reagents.
2) Can be used to directly detect, in real time target DNA/RNA molecules without using competitive assays.
3) 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
(a) Noncomplementary oligonucleotide(b) One-base-mismatched oligonucleotide(c) Complementary oligonucleotideAll 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: CGAGCGGTACGGGTCCTTCCTCCGTTGCTGGCTCG
One-base-mismatch DNA: CGAGCGGTACGGGTCCTACCTCCGTTGCTGGCTCG
Two-base-mismatch DNA: CGAGCGGTACGGGTCCTAGCTCCGTTGCTGGCTCG
Non-complementary DNA: CGAAACCTGCGAATGGTAGCTCCAATGTGGAATCG
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Estimation of the FRET parameters
60
6 60
RE
R R
5 2 4
0 8.79 10 ( )DR k n J
D -- quantum yield of the donor
-- orientation factork²-- overlap integralJ -- refraction index of the medium n
R0 of the four investigated D/A pairs are: 63.5ο
Afor (R6G/Cy5)ο
Afor (TMR/Cy5)ο
Afor (Cy3/Cy5)ο
Afor (TMR/JA133)
64.5
55.8
59.0
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Schematic of Experimental Setup
Laser source
Sample
Microscope objective
Beam splitter
Beam splitter
APD
APD
Band-passfilters
Counting board
Personal computer
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Evaluation of efficiency for MBs
1, 2,
1, 2,
1, 2,
1, 2,
/
/f open f open
f close f close
f open f close
f close f open
I IE
I I
or
I IE
I I
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Cost Analysis
DNA sequences
• <$300 Molecular beacons
• <$5000 Optical Setup
• <$5000 Computer with software
• <$6000 Other Chemicals
• <$2000
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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