Research Proposal Libo Cao Ph.D., Analytical (Dr. Peter de B. Harrington) Ohio University Department...

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


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

http://www.ohiou.edu/


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



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



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



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



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



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



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



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



Effect of Auto fluorescence

http://www.diatronscience.com/AutoFluor.html.

Auto fluorescence of Gray Snapper (L.griseus) Oocyte

http://www.diatronscience.com/AutoFluor.html

http://www.diatronscience.com/AutoFluor.html



My Goals of Designed Molecular Beacons

Longer wavelength to avoid background noise

Two Fluorophores

The two dyes chosen should be able to FRET



Choose Donor and Acceptor Dyes



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



Fluorescence Spectrum for Tm Labeled with Cy3 and Cy5 in Bulk Measurements




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



Fluorescence Intensity of the Donor Fluorescence




Fluorescence Intensity of the Increase in the Acceptor Fluorescence Due to FRET




Fluorescence Spectrum from a single Cy3-Cy5-Labeled αTm molecule




Time Records of the Donor and Acceptor Fluorescence from a Single Cy3-Cy5 Labeled αTm molecule




Designed MB and target DNA sequences

_______________________________________________________________________MB: 5’-Dye1-GCTCGTCCATGCCCAGGAAGGAGGCAACGACACGAGC-Dye2-3’

Target: 5’-GTCGTTGCCTCCTTCCTGGGCATGG-3’________________________________________________________________________

Dye1=Cy3

Dye2=Cy5

Dye2

Dye2

Dye1

Dye1



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



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



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



Fluorescence Emission Spectra of the Different FRET Constructs in Aqueous Buffer




FRET Histograms Extracted from Single Molecule Data of the Differently

Labeled D/A Constructs and Corresponding Gaussian Fits




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.



Scheme of immobilization of biotinlyed MB DNA on optical fiber surface

Liu, X; Tan, W. Anal. Chem. 1999, 71, 5054.



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



Experiment--Donors and Acceptors for the MBs



Four MBs Designed

5’-Cy3-GCTCGCCATGCCCAGGAAGGAGGCAACGACCGAGC-Cy5-3’

5’-TMR-GCTCGCCATGCCCAGGAAGGAGGCAACGACCGAGC-Cy5-3’

5’-R6G-GCTCGCCATGCCCAGGAAGGAGGCAACGACCGAGC-Cy5-3’

5’-TMR-GCTCGCCATGCCCAGGAAGGAGGCAACGACCGAGC-JA133-3’



DNA Sequences Needed

Complementary DNA: CGAGCGGTACGGGTCCTTCCTCCGTTGCTGGCTCG

One-base-mismatch DNA: CGAGCGGTACGGGTCCTACCTCCGTTGCTGGCTCG

Two-base-mismatch DNA: CGAGCGGTACGGGTCCTAGCTCCGTTGCTGGCTCG

Non-complementary DNA: CGAAACCTGCGAATGGTAGCTCCAATGTGGAATCG



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



Schematic of Experimental Setup

Laser source

Sample

Microscope objective

Beam splitter

Beam splitter

APD

APD

Band-passfilters

Counting board

Personal computer



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



Cost Analysis

DNA sequences

• <$300 Molecular beacons

• <$5000 Optical Setup

• <$5000 Computer with software

• <$6000 Other Chemicals

• <$2000



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.



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



Future Work

Studying protein-DNA/RNA interactions

Fluorescent immunoassay

DNA sequencing`

Other bio-molecular analyses



Acknowledgements

• Dr. Pete B Harrington

• Mariela Ochoa

• Preshious Rearden

• Bryon Moore


Research Proposal Libo Cao Ph.D., Analytical (Dr. Peter de B. Harrington) Ohio University Department...

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