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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
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
Ohio University
Center for Intelligent Chemical Instrumentation
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
Ohio University
Center for Intelligent Chemical Instrumentation
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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Center for Intelligent Chemical Instrumentation
Structure of A, T, C, G
NH2
O
N
Cytosine
N
O
O
P
O
O
O
H
H
O
H
H
P
O
H
O
H
N
O
H
NH 2
Adenine
NH
N
NH 2
P
H
O
H
H
O
H
H
O-
O
H
P
O-
O
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N
N
CH3
O
H
O
N
N
O
H
H
H
O
N
Guanine
O
H
H
CH3
O
O
OH
O
N
O
O
O-
NH
Thymine
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Label Dyes with the Biomolecular
O
O
Dye
DCC, DMF
N OH
COOH
Dye
COO
O
O
Carboxylic group
N-Hydroxysuccinimide
O
DCC, DMF
Dye SO3H
N OH
O
1,3-dicycolhexylcarbodiimide
Dimethyl formamide
O
O
Dye S O N
O
O
NH2 Bio
O
Dye S NH Bio
Sulfonyl group
Davidson, R. S.; Hilchenbach, M. M. Photochem. Photobiol. 1990, 52, 431.
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N
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O
How Dye Ester Linked with Sequencing Primer
Cl
P O
(i-Pr) 2N
CN
Chloro Reagent
HO
dye
Linker
O
OH
Linker
H2N(CH 2)6OH
dye
N
O
dye
COO
O
dye
N(i-Pr)2
O P
O
i).
O
ii).
Dye
Linker
Oxidation
NC
P
-O
O
O
O
O
Bis Reagent
(i-Pr) 2N
P O
(i-Pr) 2N
CN
Ohio University
O
Center for Intelligent Chemical Instrumentation
Dye
The Structure of a Molecular Beacon
A
A
G
T
A
A
T
C
C
T
A
G
A
A
G
C
A
T
G
C
C
G
G
C
5' O
O P O
O
G
Can be written as:
T
Dye1-5’-GCGAGAAGTTAAGAACCTATGCTCGC-3’-Dye2
O
3'
Dye2
Dye1
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Center for Intelligent Chemical Instrumentation
Efficiency Evaluation of the MBs
One fluorophore
Two fluorophores
Mechanism
Efficiency
Parameter
E
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I fl ,open
I fl ,close
E 
I f 1,open / I f 2,open
I f 1, close / I f 2,close
or
E 
I f 1, open I f 2,close
I f 1, close I f 2,open
Center for Intelligent Chemical Instrumentation
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
6
0
R
E 6
6
R0  R
R0 – 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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Center for Intelligent Chemical Instrumentation
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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Center for Intelligent Chemical Instrumentation
Choose Donor and Acceptor Dyes
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Center for Intelligent Chemical Instrumentation
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 α-helix and thin lines
random coil polypeptide.
α-tropomyosin (αTm)
Donor (D) and acceptor (A)
fluorephores are labeled to a
single cysteine residue at
190th position
Ishii, Y.; Yoshida, T.; Funatsu, T.; Wazawa, T.; Yanagida, T. Chem. Phys. 1999, 247, 163.
Ohio University
Center for Intelligent Chemical Instrumentation
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.
Ohio University
Center for Intelligent Chemical Instrumentation
Fluorescence images of FRET within a Single Protein Molecule
(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
Ishii, Y.; Yoshida, T.; Funatsu, T.; Wazawa, T.; Yanagida, T. Chem. Phys. 1999, 247, 163.
Ohio University
Center for Intelligent Chemical Instrumentation
Fluorescence Intensity of the Donor Fluorescence
Ishii, Y.; Yoshida, T.; Funatsu, T.; Wazawa, T.; Yanagida, T. Chem. Phys. 1999, 247, 163.
Ohio University
Center for Intelligent Chemical Instrumentation
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.
Ohio University
Center for Intelligent Chemical Instrumentation
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.
Ohio University
Center for Intelligent Chemical Instrumentation
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.
Ohio University
Center for Intelligent Chemical Instrumentation
Designed MB and target DNA sequences
_______________________________________________________________________
MB:
5’-Dye1-GCTCGTCCATGCCCAGGAAGGAGGCAACGACACGAGC-Dye2-3’
Target: 5’-GTCGTTGCCTCCTTCCTGGGCATGG-3’
________________________________________________________________________
Dye1
Dye1=Cy3
Dye2=Cy5
Dye2
Dye1
Dye2
Ohio University
Center for Intelligent Chemical Instrumentation
..
u
Model of the D/A DNA Constructs with Varying Distance
Dietrich, A.; Buschmann, V.; Mller, C.; Sauer, M. Rev. Mol. Biotechnol. 2002, 82, 211.
Ohio University
Center for Intelligent Chemical Instrumentation
Schematic Diagram of the Optical Setup
1) Frequency-doubled Nd:YAG (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
Ohio University
Center for Intelligent Chemical Instrumentation
Spectroscopic Characteristics of the Different FRET Constructs
In Aqueous Buffer
 Df,rel
ED
 Df,rel
-- Relative fluorescence quantum yield of donor
ED
-- FRET energy of donor decreased
EA
-- FRET energy of acceptor increased
Dietrich, A.; Buschmann, V.; Müller, C.; Sauer, M. Rev. Mol. Biotechnol. 2002, 82, 211.
Ohio University
Center for Intelligent Chemical Instrumentation
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.
Ohio University
Center for Intelligent Chemical Instrumentation
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.
Ohio University
Center for Intelligent Chemical Instrumentation
A Fiber-Optic Evanescent Wave DNA Biosensor Based on This
MB
Advantages:
1)
2)
3)
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.
Ohio University
Center for Intelligent Chemical Instrumentation
Scheme of immobilization of biotinlyed MB DNA on optical
fiber surface
Liu, X; Tan, W. Anal. Chem. 1999, 71, 5054.
Ohio University
Center for Intelligent Chemical Instrumentation
Dynamics of Hybridization of MB Evanescent Wave Sensor
(a) Noncomplementary oligonucleotide
(b) One-base-mismatched oligonucleotide
(c) Complementary oligonucleotide
All in aqueous buffer containing 1 M NaCl
Ohio University
Center for Intelligent Chemical Instrumentation
Experiment--Donors and Acceptors for the MBs
Ohio University
Center for Intelligent Chemical Instrumentation
Four MBs Designed

5’-Cy3-GCTCGCCATGCCCAGGAAGGAGGCAACGACCGAGC-Cy5-3’

5’-TMR-GCTCGCCATGCCCAGGAAGGAGGCAACGACCGAGC-Cy5-3’

5’-R6G-GCTCGCCATGCCCAGGAAGGAGGCAACGACCGAGC-Cy5-3’

5’-TMR-GCTCGCCATGCCCAGGAAGGAGGCAACGACCGAGC-JA133-3’
Ohio University
Center for Intelligent Chemical Instrumentation
DNA Sequences Needed

Complementary DNA:
CGAGCGGTACGGGTCCTTCCTCCGTTGCTGGCTCG

One-base-mismatch DNA:
CGAGCGGTACGGGTCCTACCTCCGTTGCTGGCTCG

Two-base-mismatch DNA:
CGAGCGGTACGGGTCCTAGCTCCGTTGCTGGCTCG

Non-complementary DNA:
CGAAACCTGCGAATGGTAGCTCCAATGTGGAATCG
Ohio University
Center for Intelligent Chemical Instrumentation
Estimation of the FRET parameters
6
0
R
E 6
6
R0  R
5
2 4
R0  8.79 10 ( D k n J )
 D -- quantum yield of the donor
k² -- orientation factor
J -- overlap integral
n -- refraction index of the medium
ο
R0 of the four investigated D/A pairs are:
63.5
forA(R6G/Cy5)
ο
64.5forA(TMR/Cy5)
ο
55.8forA(Cy3/Cy5)
ο
59.0forA(TMR/JA133)
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Center for Intelligent Chemical Instrumentation
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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Center for Intelligent Chemical Instrumentation
Evaluation of efficiency for MBs
E 
I f 1,open / I f 2,open
I f 1, close / I f 2,close
or
E 
I f 1, open I f 2,close
I f 1, close I f 2,open
Ohio University
Center for Intelligent Chemical Instrumentation
Cost Analysis





DNA sequences
•
<$300
Molecular beacons
•
<$5000
Optical Setup
•
<$5000
Computer with software
•
<$6000
Other Chemicals
•
<$2000
Ohio University
Center for Intelligent Chemical Instrumentation
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.
Ohio University
Center for Intelligent Chemical Instrumentation
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
Ohio University
Center for Intelligent Chemical Instrumentation
Future Work

Studying protein-DNA/RNA interactions

Fluorescent immunoassay

DNA sequencing`

Other bio-molecular analyses
Ohio University
Center for Intelligent Chemical Instrumentation
Acknowledgements
•
Dr. Pete B Harrington
•
Mariela Ochoa
•
Preshious Rearden
•
Bryon Moore
Ohio University
Center for Intelligent Chemical Instrumentation