Resonance Energy Transfer
Non-Radiative Energy Transfer driven by dipole-dipole coupling


Fluorescence Resonance Energy Transfer
Surface Energy Transfer


Multiplicity : A property of a system due to the spin, or angular
momentum, of its component particles ( e.g., electrons)
Multiplicity is the quantification of the amount of unpaired
electron spin
- Hund's rule : favors the single filling of degenerate (same energy)
Number of states with a given angular momentum :
2 S + 1, S= total spin
if all electrons are paired, S=0: multiplicity =1; singlet
if one unpaired electron, S = 1/2 ; doublet
if two unpaired electrons, S =1 ; triplet
Energy scheme used to explain the difference
between fluorescence and phosphorescence
Singlet state
Triplet state
 Phosphorescence is a process in which energy absorbed by a
substance is released relatively slowly in the form of light
Fluorescence
One of a class of luminescence phenomena
in which certain molecules may emit light
with a longer wavelength than the light
with which were excited
D + hv E
D*
ki
D
kf
D + hv F

Quantum yield Q
The ratio of the number of photons
emitted to the number of photons
absorbed

Lifetime
The average time spent in the
excited state before returning to
the ground state
Förster (or Fluorescence) Resonance Energy Transfer (FRET)
 Non-radiative energy transfer from an energy donor to an energy acceptor
 Dipole – dipole coupling
 Energy transfer efficiency :
- Degree of spectral overlap between donor fluorescence emission and
acceptor absorption
- Inversely proportional to 6th power of the distance between fluorophores
- ~ 10 nm
Energy Level Diagram
Use of FRET measurements
Calculation of the distance between fluorophores
 Detection of target analytes
 Analysis of biomolecular interactions
 Single molecule analysis
- Protein folding/unfolding
- Protein dynamics
Förster distance
The relationship between the transfer efficiency and the
distance between the two probe (R)
Ro : the Förster distance
at which the energy transfer
is (on average) 50%
Ro can be calculated using
Qd : the quantum yield of the donor,
n : the refractive index of the medium
(generally assumed to be 1.4 for proteins)
Nav : Avogadro's number (Nav= 6.02 x 10 per mole)
Kappa squared : the orientation factor
J : the overlap integral
Kappa squared
Orientation factor , kappa squared
The overlap intergral J
The degree of overlap between the donor fluorescence
spectrum and the acceptor absorption spectrum
λ : the wavelength of the light
ε(λ) : the molar extinction coefficient of
the acceptor at that wavelength
f : the fluorescence spectrum of the donor
normalized on the wavelength scale
Surface Energy Transfer

Energy transfer from a dipole to a metallic surface
Interaction of the electromagnetic field of the
donor dipole with the nearly free conduction
electrons of the accepting metal

Surface energy transfer efficiency :

KSET = (1/τD) ( do/d)4
Yun et al., JACS, 2005, 127, 3115-3119
Schematic representation of the
system, which consists of a
fluorescein moiety (FAM)
appended to ds-DNA of length R
(varying from 15 to 60bp) with a
Au nanoparticle (d = 1.4 nm)
appended to the other end.
- The flexible C6 linker produces a
cone of uncertainty ( R) for both
moieties.
Addition of EcoRI
(methyltransferase) bends the dsDNA at the GAATTC site by 128o ,
producing a new effective distance
R'.
15
20
30
60
bp
bp
bp
bp
;
;
;
;
62 A
96.4 A
130.4 A
232.4 A
10 bases per turn
3.4 A per base
Efficiency vs distance
-Energy transfer efficiency plotted
versus separation distance
between FAM and Au(NM).
-Filled circles (·) represent DNA
lengths of 15bp, 20bp, 30bp,
and 60bp. The measured
efficiencies of these strands with
the addition of M.EcoRI are
represented by the open circles.
- The dashed line is the theoretical
FRET efficiency, while the solid
line the theoretical SET efficiency

Conditions
-Overlapping of Donar emission and Acceptor Excitation spectrum.
-FRET : Donor/Acceptor; <10nm.
-SET : Donor/Metal : <20 nm
-Spectrally distinct

Applications

Pairs (http://probes.invitrogen.com/resources/;
- Biomolecular interaction study in vivo/vitro
- Tracking biomolecualr conformational change
-In vivo imaging/co-localization study
- Drug discovery
- Bio-sensing
//microscopy.biorad.com)
- Organic dye
-ALEA-488/RHOD-2; FITC/RHOD-2; FITC/TRITC; GFP/RHOD-2
- Fluorescent protein
-BFP/GFP; BFP/YFP; BFP/RFP; CFP/YFP
- Nanocrystal
-QD/QD; QD/gold
Examples of available fluorescent dye and quencher families.
 Tetramethylrhodamine (TMR), carboxytetramethylrhodamine
(TAMRA), and carboxy-X-rhodamine (ROX) are all rhodamine-based
dyes.
 The most common D/A dye combinations: coumarin/fluorescein,
fluorescein/rhodamine, and Cy3.5/Cy5.
 Popular dye/quencher combinations: rhodamine/Dabcyl and
Cy3/QSY9.
 Major suppliers:
-Molecular Probes (fluorescein, rhodamine, AlexaFluor, BODIPY
Oregon Green, Texas Red, and QSY quenchers),
-Amersham Biosciences (Cy dyes and Cy5Q/Cy7Q quenchers)
- AnaSpec (HiLyte Fluors, QXL quenchers)
- ATTO-TEC (ATTO dyes and quenchers
- Biosearch Technologies (Black Hole).
 FITC=fluorescein isothiocyanate.

Fluorephore materials used in bioanalytical FRET

Organic materials
- Available in reactive form from commercial sources : activated with N-
hydroxysuccinimide (NHS) ester, maleimide, hydrazide, amine functionality
Ex) Fluorescein dyes : very popular because of their high quantum yield,
solubility, ease of bioconjugation.
Excitation with a standard argon-ion laser (488 nm)
High rate of photo-bleaching, pH sensitive, self-quenching
- Alternatives : AlexaFluore, Cy family, BODIPY

Inorganic materials

Metal chelates, semiconductor nanocrystals
Biological origins
- Fluorescent proteins
Structures of common UV/Vis fluorescent dyes. Typical substituents at the
R position include CO2-, SO3-, OH, OCH3, CH3, and NO2; Rx marks the
typical position of the bioconjugation linker.
Target
Reactive Group
Comment
thiol
maleimide, iodoacetyl,
piridyldisulfide[a]
site-specific but requires a free
cysteine on proteins
primary amine
succinimidyl esters (NHS), sulfonyl
chlorides, iso(thio)cyanates,
carbonyl azides[a]
proteins may have many primary
amines
carboxyl
carbonyldiimidazoles,
carbodiimides[b]
allows further coupling to amines
hydroxyl
carbonyldiimidazoles, periodate,
disuccinimidyl carbonate[b]
allows further coupling to amines
carbohydrates
periodate[b]
oxidizes sugars to create reactive
aldehydes, which couple to amines
intracellular proteins
FlAsH
requires cloning
intracellular proteins
SNAP-tag/HaloTag
requires cloning and commercial
ligands
intracellular proteins
fluorescent proteins
requires cloning and formation of a
chimera
Experimental methods
Conventional filter FRET
Apply filter/emission band configurations
for donor, acceptor and FRET (donor
excitation and acceptor emission) to acquire
single images or time series.
If the donor signal decreases, acceptor and
FRET signal increases.
Acceptor photobleaching
Apply donor/ acceptor configurations to acquire
single images or time series. After some control
images, acceptor (with 514 nm) is bleached.
Donor signal increases after acceptor bleach
Analysis of FRET
Fluorescence lifetime imaging microscopy (FLIM)
Information about the interactions between, and the
structural states of, signaling molecules needs to be obtained
as a function of space and time in a living cell.
By using FLIM, the nanosecond decay kinetics of the
electronic excited-state of fluorophores can be mapped
spatially.
Fluorescence lifetime
The average amount of time that a molecule spends in the
excited state upon absorption of a photon of light.
Fluorescence lifetime is independent of fluorophore
concentration and light-path length.
Time domain FLIM
Fluorescent proteins









Green Fluorescence Protein (GFP) from jellyfish
Widespread use by their expression in other organisms
Key internal residues are modified during maturation to form
the p-hydroxybenzylideneimidazolinon chromophore, located in the
central helix and surrounded by 11 ß-strands (ß-can structure)
In-vivo labeling of cells ; Localization and tracing of target protein
GFP variants : BFP, CFP, YFP
Red fluorescent protein (DS Red) from coral reef : tetrameric, slow
maturation
Monomeric RFP by protein engineering
Quantum yield : 0.17 (BFP) ~ 0.79 (GFP)
BFP/CFP ; CFP/YFP( high change in the FRET signal ratio)
: fused to N- or C terminus of proteins by gene manipulation
GFP (Green Fluorescent Protein)




Jellyfish Aequorea victoria
A tightly packed -can (11 sheets) enclosing an -helix
containing the chromophore
238 amino acids
Chromophore


Cyclic tripeptide derived from
Ser-Tyr-Gly
The wt GFP absorbs UV and
blue light (395nm and 470nm)
and emits green light
(maximally at 509nm)
a) Normalized absorption and
b) fluorescence profiles of
representative fluorescent
proteins: cyan fluorescent
protein (cyan), GFP, Zs Green,
yellow fluorescent protein
(YFP), and three variants of
red fluorescent protein (DS
Red2, AS Red2, HC Red).
From Clontech.
Analysis of biomolecular interactions using FP
Inter-molecular FRET
FRET-based Sensors
Intra-molecular FRET
Calmodulin
-
Calcium ions : crucial for the metabolism and
physiology of eukaryotes
-
-
Regulate many cellular processes, ranging from
transcription control and cell survival to
neurotransmitter release and muscle function.
Calmodulin (CaM, 148 aa); a ubiquitous, calciumbinding protein ( typically binds 0, 2 or 4 Ca+2)
Regulate a multitude of different protein targets,
affecting many different cellular functions.
-
CaM : mediates processes such as inflammation,
metabolism, apoptosis, muscle contraction, intracellular
movement, short-term and long-term memory, nerve
growth and the immune response.
-
In the absence of Ca+2, the two main helical domains
have hydrophobic cores. On the binding of a calcium
ion, conformational changes exposes hydrophobic
regions which have the potential to act as docking
regions for target proteins ( over 100 proteins
including kinases, phosphatases etc.)
In the absence of Ca+2
In the presence of Ca+2
Modified MBP fluorescent indicator. ECFP as donor was fused to the N
terminus of MBP, and YFP as a FRET acceptor was fused to the C terminus.
- H indicates the portion of protein functioning as a hinge between the
two lobes of the MBP.
- The central binding pocket of the MBP is located between the two lobes.
- In the absence of maltose, the two FPs are at their maximum distance
from each other and FRET is minimal. Upon binding maltose, the MBP
undergoes a conformation change that brings the two FPs into close
proximity and increases FRET, which can be monitored by the change in
ratio of the YFP and CFP emission
-
a) Confocal image of a maltose-FP sensor expressed in yeast. Fluorescence is
detected in the cytosol but not in the vacuole. Scale bar=1 um.
b) Changes of the maltose concentration in the cytosol of yeast that expresses a
maltose sensor with a Kd value of 25 nM. The graph indicates emission ratio
as a function of maltose uptake for a single yeast cell.
Enzyme-generated Bioluminescence

-
BRET ( Bioluminescence RET) :
Donor : Luciferase ; Acceptor : GFP
- No excitation light source to excite the donor, which avoids
problems such as light scattering, high background noise, and
direct acceptor excitation


In-vivo monitoring of protein-protein interactions such as circadian
clock proteins, insulin receptor activity, real-time monitoring of
intracellular ubiquitination
The firefly luciferase/luciferin system : the best candidate for a
BRET-based donor ; high quantum yield ( 0.88)
Bioluminescent substrates and enzymatic reactions of
several common luciferases:
a) the aliphatic aldehyde substrate of bacterial luciferase;
b) structure and reaction of luciferin, the substrate of firefly luciferase;
c) colenterazine, the substrate for Renilla luciferase and also part of
apoaequorin.
Renilla (Sea pansy)
Enzyme-generated Chemiluminescence :
Luminophore : synthetic substrate that is excited through an enzymatically catalyzed
reactions
Chemiluminescent substrates and the enzymatic reactions of horseradish
peroxidase (HRP) and alkaline phosphatase.
a) Luminol; b) Acridan (also available as an ester); c) Adamantyl-1,2-dioxetane
(substrate for alkaline phosphatase and other enzymes).
Self-illuminating quantum dot conjugates for in-vivo imaging



Unique optical property of Qd:
- High quantum yields, large molar extinction coefficients, size-dependent
tunable emission and high photostability
 Fluorescent probes for biological imaging
Challenging issues
- Requirement for external illumination
 strong background auto-fluorescence from ubiquitous endogenous
chromophores
such as collagen, porphyrins and flavins
 little light is available for quantum dot excitation at non-superficial
locations
due to absorption and scattering of optical photons in tissues
Ideal quantum for in-vivo imaging
- Light emission with no requirement for external excitation
 Quantum dot conjugates based on the principle of BRET
So et al., Nature Biotech., 24, 339-343 (2006)
Construction of self-illuminating QDs conjugates



Use of luciferase from Renilla reniformis
Luc8 : Eight mutation variant : more stable in serum and higher
catalytic activity
- emit blue light with a peak at 480 nm upon addition of its
substrate
coelenterazine
Conjugation of Luc8 to polymer-coated CdSe/ZnS core-shell QD
655 through coupling of the amino groups on LUC8 to
carboxylated on the QD
- The hydrodynamic diameter of QD655-Luc8 : ~ 2 nm
- The conjugate contains six copies of Luc8 on average
Gold nanoparticles





Exceptional quenching ability
Plasmon resonances in the visible range
with large extinction coefficient (105 /cm/M)
Stable
Unfluctuating signal intensities
Resistant to photo-bleaching
Gold Nano Particles (AuNPs)
 Core Materials for NPs
- Au, Ag, Pt : Electron transporter, Catalysis, NPs coating for electrode
- Mg, Co, Fe : Magnetic behavior, Sample purification, MRI signal enhancing
- CdSe, ZnS, InP : Semiconductor QDs

Stabilization by surfactants in synthesis of AuNPs
- Reduction of HAuCl4 in the presence of surfactant
- Citrate, tannic acid, white phosphorus : > 3 nm
- Alkanethiol : Monolayer protected cluster (MPC), 2 ~ 3 nm
- Dendrimer : Dendrimer encapsulated nanocluster (DEN). < 2 nm
 Characteristics of AuNPs
- Surface Plasmon Resonance Band
. Absorbance band near 520 nm in 5 ~ several tens nm of AuNPs
. SPB shift responding to surface modification and environmental condition
- Photoluminescence as Gold QDs
. <2nm of AuNPs : smaller Bohr radius than semiconductor
. Size dependent excitation/emission spectrum
Synthesis of AuNPs

NP
: Nanoparticle capped with surfactant (ex) sodium citrate

MPC : Monolayer-Protected Clusters with alkanethiol (ex) 1-OT / 11-MUA

DEN / DSN : Dendrimer-Encapsulated (or Stabilized) Nanoclusters
Reduction
NaBH4
DSN
Surfactant
NP
MPC
Gold Nanoparticles
Gold Quantum Dot
before
DEN
reduction
AuCl4-
DSN
(G2-NH2/OH)
Citric
Acid
DEN
(G4)
MPC
Molecular Beacon (MB)
Single-stranded oligonucleotide molecular probe
Target strand
Quenching by FRET
 Donor : FAM (Ex 494 nm, Em 520 nm)
 Quencher : Dacyl ( Abs 380 ~600 nm)
 FRET-based probe : Sensitive and no separation step
 Real time analysis of amplicons by PCR
 Sequence specific multiplex analysis
Molecular Beacons






Single-stranded oligonucleotide hybridization probes that form a
stem-and-loop structure
The loop contains a probe sequence that is complementary arm
sequences that are located on either side of nthe probe sequence
A fluorophore is covalently linked to the end of one arm and a
quencher is covalently linked to the end of the other arm
The probe is dark in the absence of targets. When the probe
encounters a target molecule, it forms a probe-target hybrid that is
longer and more stable than the stem hybrid
The molecular beacon undergoes a spontaneous conformational
reorganization that leads the stem hybrid to dissociate and
fluorophoree and the quencher to move away form each other,
restoring fluorescence
Use : genetic screening, SNP detection, pharmacogenetic applications
Diagnosis of acquired resistance
in lung cancer using molecular beacons
Lung cancer
 One of the most common cancers : Increased rate of 8.7 %
 Leading cause of cancer death
 5-year survival rate : 60 % at stage 1
30 % at stage 2
10 % at stage 3
 Early diagnosis rate: < 25 %
Key issue in lung cancer treatment
 Acquired drug resistance
- Severe reduction in the drug efficacy during chemotherapy
- Patients who initially respond develop acquired resistance to drug
- Major cause of lung cancer death
Acquired drug resistance in lung cancer
Inhibitors against EGFR-TK domain : Gefitinib (IRESSA) and Erlotinib (Tarceva)
Unknown Mechanisms
Erlotinib
T790M secondary mutation
MET amplification
 Mutations in EGFR TK domain
- Primary mutations : L858R, Exon 19 deletion  Susceptible to TKIs
- Secondary mutation: Thr to Met at codon 790 (T790M) Resistance to TKIs
Diagnosis of acquired resistance: crucial for deciding a chemotherapy
Direct sequencing : laborious and time-consuming
Design of Molecular Beacon for T790M
Secondary mutation : Thr to Met at codon 790 (T790M) of EGFR
790
C T
CT
Sequence(5`3`)
Synthetic
target sequence(5`3`; 60bp)
WT-MB
Cy3-TCGGAGCTGCGTGATGAGTCCGA-Dabcyl
ACCTCCACCGTGCAGCTCATCACGCAGC
TCATGCCCTTCGGCTGCCTCCT
2`MT-MB
FAM-TCGGAGCTGCATGATGAGTCCGA-Dabcyl
ACCTCCACCGTGCAGCTCATCATGCAGC
TCATGCCCTTCGGCTGCCTCCT
Control MB
Cy3-CCAAGACGTTCAGATTCGCTTGG-Dabcyl
Real
(A)time PCR for detecting T790M
(B)
FAM-Mut1
Cell1line 2
350
only MB
300
W/O templ
250
PC9
(A)PC9 gDNA
200
Flu
3
4
EGFR
genotype
Exon19 del
(B)
H1975
L858R / T790M
H1975 gDNA
150
FAM-Mut1
500
100
1
350
50
W/O templ
250
-50
Flu
10
200
150
3
4
only MB
300
0
2
100
PC9 gDNA
15
20
25
H1975 gDNA
100
30
35
500
cycle
50
 Amplicon: 150 bp containing EGFR 2nd mutation
0
 40 Cycles
-50
100
10 at 95
15 °C, Annealing
20
25
- Denaturing
at 3060 °C 35
cycle
- Monitoring of fluorescence at the annealing step
- Extension at 72 °C
Oh et al., J Mol. Diag. (2011)
•Lane1 : FAM-Mut MB + buffer
•Lane2 : FAM Mut MB + PCR buffer
without template
•Lane3 : PCR with PC9 genomic DNA
•Lane4 : PCR with H1975 genomic DNA
Detection sensitivity for T790M
 Biopsy sample : a mixture of cancer and normal cells
 Relative ratio of 2nd mutated EGFR gene (T790M) to the wild type
Cell line
EGFR genotype
PC9
Exon19 del
H1975
L858R / T790M
 Detection sensitivity: ~ 2 % EGFR
gene with T790M
 Direct sequencing : ~ 25 % of 2nd
mutated EGFR gene
Oh et al., J Mol. Diag. (2011)
Test for real patient samples
Tube
No
Sample
Gefitinib
treatment
EGFR
T790M
Concentration
27P
Primary lung
cancer
Post
L858R
Y
100 ng/μl
28M1
Contralateral
lung
Post
L858R
Y
100 ng/μl

gDNA extraction from biopsy
samples from lung cancer patients
followed by real-time PCR
Positive control : H1975
Negative control : PC9
gDNA
template
100 ng
95 °C
30s
60 °C
30s
PC9
28M1
1.00
Fluorescence (A.U.)

Supplied by Mitsudomi group
at Kyoto University
27P
H1975
0.60
0.20
50 cycles
-0.20
5
Oh et al., J Mol. Diag. (2011)
15
25
Cycles
35
45
Fluorescence imaging using molecular beacons
More simple diagnosis of acquired resistance
Fixation & permeabilization
Incubation with MB
for 40 min
for 20 min
Imaging by
Confocal microscope
H1975
2nd mutation
PC9
targeted MB
(T790M-MB)
Donor : FAM (Ex. 494nm, Em 520nm)
Quencher : Dabcyl dye (Absorption 380~ 600 nm)
Drug Resistant
Fluorescence imaging
H1975
(T790M)
FAM
Cy5
Merge x60
FAM
Cy5
Merge x60
PC9
T790M-specific MB
Control MB : Cy5
: T790M MB– FAM
stem-AA…AA-stem
Analysis of biopsy samples with drug resistance by imaging
 Biopsy samples from cancer patients with drug resistance: 27
 Detection of T790M by imaging using molecular beacons : 14
- Acquired resistance via other mechanisms: MET(Receptor tyrosine
kinase) amplification
 Detection by conventional direct sequencing: 2
- Low sensitivity due to the normal cells in biopsy samples
T790M positive (Patient # 38)
T790M negative (Patient # 35)
Proteolytic activity monitored by FRET between
quantum dot and quencher
QD–peptide sensor architecture and optical
characteristics of the fluorophores used.
(a) Schematic diagram of the self-assembled QD–
peptide nanosensors; one peptide shown for clarity. Dyelabelled modular peptides containing appropriate
cleavage sequences are self-assembled onto the QD.
FRET from the QD to the proximal acceptors quenches
the QD PL. Specific protease cleaves the peptide and
alters FRET signature.
(b) Normalized absorption and emission profile of dyes
and QDs used: Cy3 dye (quantum yield=0.20,
=150,000 M-1 cm-1, ex 555 nm, em 570 nm), QXL-520
dark dye quencher ( =26,000 M-1 cm-1, ex 508 and
530 nm). The absorption of the 538 nm QDs and the
emission spectra of the 510 and 538 nm QDs are shown.
(C) Model structure for the QD–peptide conjugates. Data
were derived separately but both conformers are shown
on the same QD. The Casp1–Cy3 peptide is shown on
the right and the Thr–QXL on the left. A CdSe–ZnS
core-shell QD with a diameter of 28–29 Å is represented
by the blue inner sphere. For both peptides the His6
sequence shown in green is in contact with the QD
surface in an energy minimized conformation. Protease
recognition sequences are highlighted in yellow, and the
spacer-linker sequences are shown in grey. The Cy3
acceptor dye structure is shown in red, and the QXL-520
quencher is approximated by a magenta sphere placed
10.5 Å from the cysteine S atom. The centre-to-centre
distance determined from FRET efficiencies are 55 Å for
the QD–Casp1–Cy3 (R0=54 Å) and 56 Å for QD–Thr–
QXL (R0=43 Å). The second grey shell represents the
DHLA ligand cap whose maximum lateral extension
away from the QD surface can vary between 5 and 11 Å;
10 Å is shown here.
Advantage vs shortcomings of FRET
Advantage
- Relatively cheap
- Very efficient in measuring changes in very proximal distances
- Measure distances in molecules in solution
- Only need a few µM of labeled proteins
- Rapid detection
Shortcomings
- Uncertainty of the orientated factor
- When measuring a change in distance between two probes,
the result is a scalar and give no indications of which probe
(donor and/or acceptor) moves.
- The presence of free labels in solution could mask a change in energy transfer.

a) Structure of sulfo-NHS-BIPS (sulfo-NHS=N-hydroxysulfosuccinimide sodium salt; BIPS=1 ,3 ,3 -trimethylspiro[2H-1benzopyran-2,2 -indoline]) in the spiropyran (SP) form before (left) and merocyanine (MC) form after (right) conjugation to
a protein. b) Schematic representation of quantum dot (QD) modulation by photochromic FRET after interacting with MBPBIPS (MBP=maltose-binding protein). When BIPS is converted to the MC form by UV light, the QD emission is reduced
through FRET quenching. After photoconversion with white light to the SP form, the direct emission of the QD is
substantially increased. c) Photoluminescence spectra of the 555-nm luminescing QD 20 MBP-BIPS system with a
dye/protein ratio of 5 after photoconversion from the SP to the MC form. d) Effect of pcFRET on QD photoluminescence
(initial change from white light to UV). Figure adapted from reference [106] with permission of the American Chemical
Society.