volume 4 Number 5 1977
Nucleic Acids Research
A reexamination of the problem of resonance energy transfer between DNA intercalated
chromophores using bisintercalating compounds
Marc Le Bret+, Jean-Bernard Le Pecq+, Jacques Barbet"1"1" and Bernard P.Roques"*"*"
+
Lab.Pharmacol.Mol. Associe au CNRS, Institut Gustave Roussy, Villejuif 94800, and "'"'"Dep.Chim.,
Fac.Pharm., Universite Rene Descartes, 4 Avenue de I'Observatoire, Paris 75005, France
Received 10 January 1977
ABSTRACT
The rate of energy transfer between DNA intercalated
ethidium cations calculated by Paoletti and Le Pecq 1 using
the Forster theory differs from the measured one by a factor
of twenty two,if the proper geometrical factors are taken
into account.By changing some of the parameters used in the
calculation ,the discrepancy can be reduced but not eliminated.
This led us to the study of other systems where experimental
and calculated results can be more directly compared.The
apparent rate of energy transfer between ethidium and one of
its non fluorescent analogues and between various pairs of
intercalated chromophores has been studied.The fluorescence
anisotropy decay of acridine dimers in glycerol or bisintercalated in DNA has been measured.These studies show that the
Forster theory of energy transfer does not apply to the case
of identical chromophores when they are relatively close to
each other.
INTRODUCTION
2
According to the Forster theory .resonance energy
transfer between two chromophores depends on the sixth power
of the distance which separates them.Therefore its measurement
could provide for a sensitive spectroscopic method of
structure study and the name of spectroscopic ruler has been
proposed for such a methodology .On the other hand the rate
of energy transfer between two chromophores depends also
in this theory on their relative orientation.In some cases,
if the distance between the two chromophores is known,their
relative orientation can in theory be deduced from energy
transfer measurements.Such a situation exists in the case of
molecules intercalated in DNA.In that case the relative
orientation of the DNA intercalated molecules is related to
© Information Retrieval Limited 1 Falconberg Court London W1V5FG England
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the unwinding angle of the DNA helix caused by the intercalat i o n 1 . The k n o w l e d g e of the value of this angle is of importance for studies related to the physical c h e m i s t r y of circular
DNA4-5.
Such a d e t e r m i n a t i o n was first attempted using ethidium
bromide ( E t h B r ) 1 . In that case the rate of energy transfer
between identical intercalated m o l e c u l e s was deduced from steady state f l u o r e s c e n c e polarization m e a s u r e m e n t s analysed according to the F o r s t e r theory. With a r e f r a c t i v e index of 1.4 for
DNA, the rate of energy transfer deduced in this experiment
was much smaller than the expected o n e . This led Paoletti and
Le Pecql to question the value of tbe unwinding angle assumed
to be c o r r e c t at that time. It w a s later well d e m o n s t r a t e d
by several a u t h o r s 6 ~ 8 that the m o d i f i c a t i o n of torsion of the
DNA helix caused by the ethidium i n t e r c a l a t i o n was between -26°
to - 2 8 ° . With this value, the rate of energy transfer computed
by Paoletti and Le Pecq differs by a factor of 22 from the a p parent rate of transfer.
Q
Later, o t h e r authors tried to solve this problem using the
same experimental system but measured the f l u o r e s c e n c e anisotropy decay instead of steady state a n i s o t r o p y . A larger value
of the r e f r a c t i v e index of DNA which had been obtained at that
time 10 was used. This reduced the d i s c r e p a n c y , but the apparent rate of energy transfer observed was still smaller than
e x p e c t e d . Deducing the rate of e n e r g y t r a n s f e r between two
identical c h r o m o p h o r e s from m e a s u r e m e n t s made on a array of
n u m e r o u s DNA i n t e r c a l a t e d m o l e c u l e s is in fact difficult because the i n t e r p r e t a t i o n of the experimental data depends on several a s s u m p t i o n s and uncertain p a r a m e t e r s :
a) the d i s t r i b u t i o n of the c h r o m o p h o r e s along the DNA.
19
11
In the two p r e c e d i n g studies ' , the excluded site model
was assumed to be correct. All the a l l o w e d sites were supposed
to have the same probability of o c c u p a n c y i n d e p e n d e n t l y of s e DNA basee f fceocmtp o sbecause
i t i o n 1 2EthBr
. Nevertheless
suggest
quence
DNA a f f i n irecent
t y doesstudies
not depend
on s o lei
me type of s e q u e n c e specificity
.13
b) the o r i e n t a t i o n of t h e e t h i d i u m m o l e c u l e in its s i t e .
The c r y s t a l 1 o g r a p h i c data show that the phenyl substituent
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lies in the small groove . However this still leaves two possible o r i e n t a t i o n s . F u r t h e r m o r e , if the transition moment of
ethidium does not coincide with a symmetry axis of the molecule apparent rate of transfer will depend on the direction of
the transition moment in the m o l e c u l e 1 .
c) the value of the refractive index of DNA along the
long axis of the DNA helix.
d) the validity of the dipole-dipole approximation when
molecules are relatively close to each other .
e) the validity of the Forster approach itself when measurements are done on identical chromophores in similar environment.
A controversy exists whether the theory of long range non
radiative energy transfer as formulated by Forster holds in
the case of identical chromophores ( r e v i e w ^ , 1 5 ) . The energy
levels of the interacting chromophores are modified by the
coherent coulombic coupling and by the incoherent coupling
which results from the perturbations induced by the vibrations
and the disturbances of the environment. For unlike chromophores, the coherent coupling provokes only a very slight change
of the energy l e v e l s . The incoherent coupling largely predominates and the relaxation described by Forster o c c u r s . Fors t e r 1 6 a n d Bennett and Kellogl? argue that the perturbations
are large enough so that the same phenomenon occurs even in
the case of identical m o l e c u l e s . On the c o n t r a r y , several o1 ft ?O
ther authors
argue that for identical m o l e c u l e s the coherent coupling may not be n e g l i g i b l e . Coherent oscillations occur and the Forster treatment is not valid. M o r e o v e r , as the
excited state is then delocalized over the identical chromopho
r e s , the f l u o r e s c e n c e anisotropy of the c o h e r e n t l y coupled
chromophores is expected to be equal to that of the isolated'
ones.
Contrarily to what has been done in the case of different
molecules '
, no experiments have demonstrated the validity and/or the limits of applicability of the Forster theory in
the case of similar c h r o m o p h o r e s , in the biological conditions
of solvent and t e m p e r a t u r e .
To progress in the understanding of these p r o b l e m s , we ha1363
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ve used experimental systems which are simpler and more appropriate to answer some of these questions.
a) the value of the DNA refractive index along its long
axis can be in principle deduced from the determination of the
long range energy transfer between pairs of different intercalating dyes. In that case one can be confident that the Forster
theory applies.
b) the apparent rate of energy transfer between DNA intercalated ethidiums surrounded by molecules of its non fluorescent nitro analogue has been measured. In that case, if the
Forster theory applies, a single energy transfer step between
the fluorescent and the quenching dye would occur and the energy transfer rate is very simply and accurately deduced from
the determination of the fluorescence quenching of the fluorescent donor ethidium molecule.
c) the decay of fluorescence anisotropy of acridine dimers
able to bisintercal ate in D N A 2 3 has been measured. This experimental system permit, a considerable simplification of the problem
because the transfer is limited to two chromophores located at
known distance and fixed orientation. These measurements will
permit to test specifically the validity of the Forster theory
applied to identical chromophores.
Furthermore in this paper the domain of validity of the
dipole-dipole approximation will be determined by comparing
the values of the rate of energy transfer computed with the
multipole and dipole approximations.
MATERIALS AND METHODS
The chemical structures of the here studied chromophores
are reported in figure 1. Ethidium bromide (EthBr) and its
nitro derivative (N0 2 -EthBr) were obtained from the Boots Pure
Drug Company. We have verified that NO--EthBr intercalates
into double stranded DNA with the same affinity constant as
EthBr. Furthermore it provokes the same unwinding of the DNA
double helix. When intercalated into DNA, its fluorescence
quantum yield is at least 1 0 4 times less than that of EthBr.
The position of its optical band of lowest energy is 20nm
red-shifted relatively to EthBr. Preparation and some proper1364
Nucleic Acids Research
«!
•Ilipiciu III I I
I . I 4 iartkn «lirtici>i<a 111 H I
R=l flthllilB (Itblrl
litn-HIMila (Hl,.lltlil
tik>ArAs**'
aim
i. _(ci,),.««-(uj)4.«qcij)j-
Figure 1
ties of e l l i p t i c i n e (El I) and of 2-6 dimethyl el 1 i p t i c i n i u m
iodide (El II) have already been described
. Acridine monom e r (AcMo) and dimers (AcDi I) and (AcDi II) a r e d e s c r i b e d 2 3 .
F l u o r e s c e n c e quantum y i e l d s were measured as p r e v i o u s l y d e s c r i b e d 1 using rhodamine B (q=0.69) and/or 9 amino a c r i d i n e
(q=.99)in ethanol as standards (table 1 ) .
In t h e Forster theory t h e rate o f energy transfer k n . f-rom
U "••A
a donor D to an acceptor A molecule is
T
C(K2/R6)/T
(1)
where R is the distance between D and A, T Q the f l u o r e s c e n c e
lifetime of D and K depends on t h e normalized transition m o ments mft and m D of A and D and on u the unit vector of the li-
Table 1: Spectral characteristics of chromophores used here.
Dye interca- Absorption Fluorescence Fl uorescence Q u a n t u m
yield
1i fetime
lated in
(visible) emission
ns
poly [d(A-T)] X m a x n m
X
nm
max
.14
24
600
EthBr
520
=•0
NO 2 -EthBr
600
540
~o
17
.31
530
435
El I
33.4
.14
540
El CI
450
.22
24
500
440
MoAc
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ne j o i n i n g t h e chromophores a c c o r d i n g to
K2=(mA.m0-3(u.mA)(u.mD))2
In the e x p r e s s i o n ( 1 ) , C is defined as
(2)
(3)
w h e r e q is t h e donor quantum yield o f f l u o r e s c e n c e , n the r e f r a c t i v e index of t h e medium, Ig(?) the n o r m a l i z e d intensity
of the f l u o r e s c e n c e of the d o n o r at wave number v a n d t .(V) t h e
m o l a r d e c a d i c e x t i n c t i o n c o e f f i c i e n t of t h e a c c e p t o r at V .
The c h a r a c t e r i s t i c length R o is
Ro= K2 C 1 / 6
(4)
for random o r i e n t a t i o n
(5)l
Ro=(2/3 C ) 1 / 6
The values o f R o f o r the d i f f e r e n t p a i r s of donor a c c e p t o r studied here a r e r e p o r t e d in table 2 .
T a b l e 2 : V a l u e of R o (Angstroms) f o r random o r i e n t a t i o n and
n=1.75.
^^^donor
acceptor^.
EthBr
El I
El
14.7
18.8
27.7
29.7
23.7
35.8
EthBr
NO2-EthBr
II
M e a s u r e m e n t of the relative q u a n t u m y i e l d of a f l u o r e s c e n t
dye in p r e s e n c e of a q u e n c h e r .
T a k i n g t h e e q u a t i o n of L e P e c q a n d P a o l e t t i,12
(6)
cb=
(c. c o n c e n t r a t i o n o f d y e b o u n d t o D N A , I 1 , I f l u o r e s c e n c e
i n t e n s i t i e s o f t h e s o l u t i o n o f d y e i n p r e s e n c e a n d in a b s e n ce o f D N A , V the ratio o f the f l u o r e s c e n c e intensity emitted
by t h e b o u n d a n d f r e e d y e w h e n e x c i t a t i o n is m a d e in i d e n t i c a l
conditions a n d k an instrumental f a c t o r ) , w e obtain:
c <i •- i
since
1366
V 1 /V Q
>ofo+1
(7)
(8)
Nucleic Acids Research
S u b s c r i p t s 1 and o refer to m e a s u r e m e n t s made with and
without q u e n c h e r , f is the fraction of dye bound to DNA. Its
value is determined using the Scatchard equation after the
binding constant has been measured as already described .
The quenching of a dye by a non fluorescent one has been simulated on a UNIVAC 1106 computer. For a given phosphate to total dye ratio a random distribution o f dyes along a 200 base
pair long DNA segment was generated as in r e f . l . The orientatation factor has been calculated assuming an unwinding angle
of 26 for EthBr. The two possible o r i e n t a t i o n s of the intercalated dye were assumed e q u i p r o b a b l e . The computed CNDO/S
orientation of the transition moment of EthBr has been used .
The direction of the transition moments of e l l i p t i c i n e and
acridine have been computed in the same way (Le Bret u n p u b l i shed r e s u l t s ) . The excitation migration is simulated as before until either emission occurs from a fluorescent dye or
reaches the quenching dye. The ratio of the number of emissions over the total number of trials is equal to the ratio
of the quantum yields of the fluorescent dye in the presence
and in the absence of the quenching dye. M u l t i p o l e corrections
were introduced according to the results of figure 2 (see results s e c t i o n ) .
Steady state f l u o r e s c e n c e m e a s u r e m e n t s were performed using
a photon counting instrument built in this laboratory. Fluorescence decay times were measured by the time correlated
single photon counting technique (reviewed in r e f . 2 6 ) with an
instrument built in this laboratory.
A n i s o t r o p y of f l u o r e s c e n c e as a function of time A ( t ) is
A
(t
)
=
( )
S(t)
y (t)+2IH(t)
I v ( t ) and I H ( t ) being the f l u o r e s c e n c e intensity polarized vertically and h o r i z o n t a l l y respectively when the excitation
light is polarized vertically. The function D(t) is recorded
by stocking a l t e r n a t i v e l y Iw(t) and Iu(t) r e s p e c t i v e l y in positive and n e g a t i v e in the multichannel a n a l y s e r . S ( t ) is recorded in the same way by summing up the c o r r e s p o n d i n g functions
and s u b s t r a c t i n g the corresponding n o i s e s .
For an a g g r e g a t e of N identical dyes o f f l u o r e s c e n c e lifetime T
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the c o n c e n t r a t i o n of the m t h dye C (t) still excited at time t
m
when t h e dye C has
excited is the solution of the
as first been exi
set of e q u a t i o n s :
dC m (t)/dt = -(l/T + „. J „ km.ra) C m+ m. J m k M , C m , (10)
k m i m being the rate of transfer between two separate molecules
m' and m. For only two identical molecules D and A, if D has
first been excited, the equation (10) gives :
C D (t) = 0.5 exp (-t/T) ( l + e ' 2 k t )
(11)
C A (t) = 0.5 exp (-t/i) ( l - e " 2 k t )
(12)
k being the rate of transfer between the two molecules, C~ (t)
and C ft (t) the concentration of excited donor and acceptor at
time t. If the angle between the transition moments of the two
molecules is 6, the anisotropy decay is :
A(t)/A(o) = (1 + 3 cos 2 e + 3 sin 2 e e " 2 k t ) / 4
(13)
The averaging of this function yields the steady state anisotropy
A s / A ( o ) = (1/4) (1 + 3 cos 2 e + 3 sin 2 e/(l+2kx))
(14)
A(o) is in principle the fundamental anisotropy of the isolated
D molecule.
Circular dichroTsm spectra were recorded on a dichographe III
Roussel Jouan.
RESULTS
a) Validity of the dipole-dipole approximation
2 fi
The dipole-dipole term (K /R ) in the formula (1) is the
approximation o f :
"K2/R6"=i<H'A*4<Die/ri1l'A'i<* > i 2
(15)
where the transition moments m. and m D
m
A = < ¥ A ' epl ' i 'A > »
.
<16)
ape normalized and H"., V. and VQ, I" are the ground state and
first excited state wavefunctions of A and D. e/r is the coulombic operator and p is the radius vector. The wavefunctions
have been taken from those of the 3,8 diamino phenanthridinium
o7
cation
comput
cation which
which were
were computed
by the PPPSCF method
. Figure 2
shows the variations
tions of
of
llog((K
o g ( ( K 22//R
R 66))/("K
/ ( ' 2 /R 6 ")-l)
(17)
for two superposed EthBr separated by a distance h and rotated
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by d i f f e r e n t a n g l e s 8. T h e d i p o l e - d i p o l e a p p r o x i m a t i o n a l m o s t
a l w a y s o v e r e s t i m a t e s t h e rate of t r a n s f e r and b e c o m e s r e a l l y
o
correct for h larger than 15A.
85'
*
•
* 8 = 80 • 8 = 60'
• 8=
*
•
it
1
•
•
34
6.8
10.2
13.6
h(A)
Figure 2.
b) Energy t r a n s f e r b e t w e e n d i f f e r e n t c h r o m o p h o r e s i n t e r c a l a t e d
in DNA
The F o r s t e r f o r m u l a is valid in t h e case o f e n e r g y t r a n s fer b e t w e e n the e l l i p t i c i n e d e r i v a t i v e s and the non f l u o r e s cent a n a l o g u e of e t h i d i u m b e c a u s e of t h e 100 nm d i f f e r e n c e
between the w a v e l e n t h of t h e i r lowest e n e r g y b a n d . B e c a u s e of
the large v a l u e of R in this c a s e , m e a s u r e m e n t s can be done
at high p h o s p h a t e to d y e ratio w h e r e the d i s t a n c e b e t w e e n t w o
n e i g h b o r s a r e large and vary very m u c h . This m a k e s t h e i n f l u ence of the r e l a t i v e o r i e n t a t i o n of the dyes u n c r i t i c a l . S e v e ral pairs have been s t u d i e d .
F i g u r e 3 s h o w s t h e r e s u l t s o b t a i n e d f o r El II and
N O p - E t h B r used r e s p e c t i v e l y as d o n o r and a c c e p t o r c o m p a r e d to
the t h e o r i t i c a l v a l u e s o b t a i n e d with d i f f e r e n t v a l u e s of R
With this p a i r , e n e r g y t r a n s f e r a p p e a r s e x t r e m e l y e f f i c i e n t
at low v a l u e s o f r ( n u m b e r of dye bound p e r n u c l e o t i d e ) and
b e c o m e s a p p a r e n t l y less e f f i c i e n t at h i g h e r v a l u e s of r. T h i s
very p r o b a b l y i n d i c a t e s that t h e d i s t r i b u t i o n of t h e d y e is
not random and that t h e dyes tend to c l u s t e r at the b e g i n n i n g .
The f i t t i n g R Q o b t a i n e d at the h i g h e r v a l u e s o f r is s m a l l e r
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F i g u r e 3 V a r i a t i o n o f t h e r e l a t i v e q u a n t u m y i e l d a,i/q Q o f El
II in p r e s e n c e o f i n c r e a s i n g c o n c e n t r a t i o n o f N O ^ - t t h B r ;
E x p e r i m e n t is s t a r t e d at r = 0 . 0 0 1 ( b o u n d d y e p e r n u c l e o t i d e )
a n d r is f u r t h e r i n c r e a s e d by a d d i t i o n o f N O P - E t h B r . D N A is
at 0.1 m g / m l in 3 M CsCl T r i s HC1 O . l p H 7.5
t h a n t h e R c o m p u t e d w i t h a DNA r e f r a c t i v e i n d e x n = 1 . 7 5 . To
e l i m i n a t e this d i s c r e p a n c y , one can either introduce a higher
v a l u e o f t h e r e f r a c t i v e index o f D N A , t h a t is n = 2 . 1 , in t h e
c a l c u l a t i o n o f t h e r a t e of t r a n s f e r o r t h i n k t h a t t h e q u a n t u m
y i e l d o f t h e d o n o r m o l e c u l e w a s u n d e r e s t i m a t e d by a f a c t o r o f
2. T h e l a t t e r p o s s i b i l i t y s e e m s i m p r o b a b l e b e c a u s e s i m i l a r
r e s u l t s w e r e o b t a i n e d with o t h e r p a i r s s t u d i e d ( r e s u l t s n o t
s h o w n ) . T h i s a p p a r e n t r e f r a c t i v e i n d e x o b t a i n e d h e r e is r e l a t i ve to t h e p r o p a g a t i o n o f l i g h t a l o n g t h e a x i s o f t h e D N A d o u b l e h e l i x a n d is l a r g e r than t h e v a l u e p r o p o s e d by H a r r i n g ton . N e v e r t h e l e s s if t h e l a r g e r v a l u e s o f t h e p o l a r i z a b i 1 i ty o f t h e b a s e p a i r s d e t e r m i n e d by T a k a s h i m a
are substituted
in t h e L o r e n t z - L o r e n z f o r m u l a a l a r g e r v a l u e ( n = 2 . 0 ) is o b t a i ned.
c) F l u o r e s c e n c e l i f e t i m e of e t h i d i u m
The observed lifetime decreases with the phosphate/bound
d y e r a t i o ( P / D ) . T h e e f f e c t is a l s o o b s e r v e d in h i g h s a l t c o n c e n t r a t i o n a n d is t h e r e f o r e n o t r e l a t e d t o q u e n c h i n g by e t h i d i u m b o u n d o n t h e o u t s i d e o f t h e D N A h e l i x . T h e e f f e c t is p a r a !
1370
Nucleic Acids Research
lei to the decrease of the fluorescence anisotropy of E t h B r 1 .
It is not related to a change of DNA structure when intercalation proceeds since the EthBr fluorescence lifetime stays constant if another intercalating compound like an acridine, AcMo,
is added instead of more EthBr, the fluorescence excitation
is done of course selectively in the EthBr long wavelength
band where the acridine does not absorb at all. Such a phenomenon has already been described in aggregates of chromophores .
If the largest refractive index value for DNA (n=2.1) is used
in the calculations and if the corrections for dipole-dipole
approximation is taken into account as well as the decrease of
EthBr fluorescence lifetime the rate of energy transfer calculated according to Paoletti and Le Pecq is still 2 to 3 times
larger than the measured one. This reduces the discrepancy by
a factor of ten.
EthBr Fluorescence
lifetime(ns)
tcMO
Figure 4. Variation of the EthBr fluorescence lifetime when
it is bound to DNA as a function of the ratio r (number of
bound dye per n u c l e o t i d e ) . In the upper curve measurements are
started with EthBr at r=.005 and r is further increased by addition of AcMo. In the lower curve r is increased by further
addition of EthBr. Solutions are in Na Cl 0.1M tris HC1 0.1 M
pH 7.5 with calf thymus DNA at 0.1 m g / m l . Fluorescence excitation is at 520 nm and fluorescence emission is measflred
through a high pass filter ( X - 6 0 0 n m ) .
We have thus been led to study systems where the Forster
theory can be more directly tested in the case of identical
molecul e s .
d) Fluorescence quenching of ethidium by its non fluorescent
analogue.
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Nucleic Acids Research
Two types of experiments have been performed. In the
first type the relative quantum yield of EthBr is measured in
presence of a large excess of its non fluorescent analogue. In
these conditions EthBr is always surrounded by quencher and
transfer is limited to one step. The results of this type of
experiment is shown in figure 5.Experimental results are comO
pared to the values computed by simulation for R = 16.6 A and
o
0
R = 1 9 A c o r r e s p o n d i n g to DNA r e f r a c t i v e index o f 1.75 and 2 . 1 .
In the second type of e x p e r i m e n t a large excess o f e t h i dium is used and a small q u a n t i t y of N O . - E t h B r is a d d e d . In
"'Ac
. v.
RO=16.6
V.
.4
.3
.2 .
.1 -
Figure 5. Variation of the relative quantum yield of EthBr in
presence of increasing concentration of N0 2 -EthBr. Experiment
is started with EthBr alone bound at r=0.0i and r is further
increased by addition of NO,-EthBr. DNA is at 0.02 mg/ml in
3 M CsCl, Tris HC1 0.1M pH 7.5.
these conditions the quencher is isolated in the middle of an
array of fluorescent EthBr. The decrease of fluorescence perm
mits to calculate simply how far the energy migrates from ethi
dium to ethidium. The results are shown in figure 6. For
P/D= 4.5, two unexpected observations are made.
An increase of fluorescence is observed at the beginning
of the curve. This increase is relatively small. We therefore
confirmed this variation by doing parallel EthBr fluorescence
lifetime measurements which in fact increases by the same factor as the quantum yield.
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X
&
'
*
•
*
1.
.90
10
15
20
Figure 6. V a r i a t i o n o f the r e l a t i v e quantum y i e l d o f E t h B r
(q,/q ) in p r e s e n c e o f small a m o u n t s of N 0 2 - E t h B r . E x p e r i m e n t
is started at the shown r a t i o p h o s p h a t e / b o u n d dye ( P / D ) relative to EthBr and N O 9:- E t h B r is added at the indicated p e r c e n t a g e . DNA is at 0.02' mg/ml in CsCl 3 M Tris HC1 0.1M pH 7 . 5 .
For 1 0 % N O ^ - E t h B r the f l u o r e s c e n c e d e c r e a s e s only by a
f a c t o r o f 1.2 indicating that a p p a r e n t l y only 2 e t h i d i u m s are
able t o t r a n s f e r t h e i r e n e r g y t o the q u e n c h e r . This w o u l d
mean that there is a n e g l i g i b l e e t h i d i u m e t h i d i u m t r a n s f e r .
These two o b s e r v a t i o n s cannot clearly be a c c o u n t e d for
by the Forster t h e o r y . This theory would always predict a d e c r e a s e o f f l u o r e s c e n c e and a m i g r a t i o n o f the energy o v e r seve
ral e t h i d i u m m o l e c u l e s . A t P/D=4.5 i n t e r c a l a t e d m o l e c u l e s are
o
indeed very close to each other (most of the time 10A a p a r t ) .
The probability of transfer according to the Forster formula
between two EthBr molecules and between one EthBr and one
NO 2 -EthBr is in these conditions respectively 17 and 32 times
larger than the probability of direct emission.
e) Study of the apparent energy transfer in the acridine
dimers.
The steady state fluorescence anisotropy spectra o f the
acridine monomer and dimers bound to poly d(A-T) at
P/D=100 in a saturated solution of sucrose are very similar
(results non s h o w n ) . This implies, if we interpret this result
along the Forster theory, that the ahgle between the chromopho
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res is either 0° or close to 90° with a very slow rate of apparent rate o f transfer (equation 1 3 ) . Additional informations
are given by the measurement of the a n i s o t r o p y decay.
The a n i s o t r o p y decay of AcDi II in glycerol and bound to
poly [d(A-T)] at high value o f P/D in presence of saturating
c o n c e n t r a t i o n of sucrose are shown in figure 7. Similar results
(not shown) are obtained with AcDi I.
1
105
[t)
~ ^v
a
\
C
104
\
io3
102
io1
•
• ' • • ' ' : . . ,
tine
tin
A(t)
0.2
b
d
0.15
0.1
'
" ' . ' • •
. '
0.05
•
0
till
\
-
•'•'"'••
tine
Figure 7. A n i s o t r o p y decay o f AcDi II in glycerol (d) and in
poly Cd(A-T)] ( b ) . The corresponding experimental functions
S(t) and D ( t ) defined by equation 9 are shown in (a) and ( c ) .
Solutions in glycerol contain 10 % acetic acid 1.0 M and
0.01 ing/ml of d y e . Poly [ d ( A - T ) ] solutions are saturated with
sucrose at pH 5.0 acetate buffer 0.1 M. The dye concentration
is 0.005 mg/ml and poly [d(A-T)j is a t 0.1 m g / m l . Along the
time axis the tic interval is 4.4 n s .
In g l y c e r o l , if w e try to interpret the results along the
Forster theory, the apparent R can be calculated from the
observed rate o f transfer assuming a random orientation
(equations 1 and 4 ) . A random o r i e n t a t i o n is compatible with
the limit reached by the fluorescence a n i s o t r o p y at infinite
time (Equation 13 gives A(»)/A(o) = 1/2 for random orientat i o n ) . Because for the acridine dimers d i s s o l v e d in glycerol
we do not know the average d i s t a n c e w h i c h separates the two
c h r o m o p h o r e s , w e can only deduce from this anisotropy decay
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rate a maximum R value assuming a maximum extension of the
chain which separates the two chromophores
. This value can
be compared to the R computed from the spectral properties
(equations 3 and 4 ) . Results are shown in table III.
Computed R (A) from
spectral properties
AcDi
AcDi
I
II
Maximum R (A) deduced
from anisotropy decay
10
14
20
23
Table III
There results mean that the apparent rate of transfer is at
least 60 times smaller than the value computed with the Forster
formula.
In the case of AcDi I and AcDi II bound to poly d(A-T) because
the apparent energy transfer is almost not measurable, the
discrepancy is still more dramatic.
At last to detect an eventual excitonic pattern, the circular
dichroTsm spectra of the acridine monomer and dimers were
recorded. The results are shown in figure 8 and do not give any
evidence of such a pattern.
Figure 8. Circular dichroTsm of DNA-acridine complexes. Solutions are in acetate buffer 0.1 pH 5. DNA is at 0.1 mg/ml and
the ratio bound dye per phosphate is 0.1.
t '.
cr
UJ
^ 3
UJ
z
1
1 ;
I ;
1:
W\
-I
-I
ill
A nm
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Nucleic Acids Research
DISCUSSION
The experimental results presented in this paper show
clearly that several of the assumptions made by Paoletti and
Le Pecq
for the ethidium-ethidium energy transfer calculation were inadequate.
- the distribution of the intercalated dyes is probably not
always random as indicated by the measurement of the energy
transfer between the ellipticine derivative and the nitro
analogue of EthBr.
- the quantum yield of EthBr is not independent of the Dye/
Phosphate ratio.
- the refractive index of DNA along the long axis was considerably underestimated. In some conditions, as shown here, energy transfer measurements between dissimilar chromophores intercalated in DNA could permit an evaluation of this index. Our
results zre not accurate enough to propose a definite value,
but they would favor a value (2.1) which is larger than the
value (1.75) proposed by Harrington
- the dipole-dipole approximation in the Forster theory when
chromophores are close to each other (figure 2 ) .
The discrepancy between the previously calculated value of
the rate of energy transfer between ethidium molecules
and
the observed value (a factor of 22) is reduced to factor of 2
to 3 if the appropriate corrections are made. Such a discrepancy in the rate of energy transfer would lead only to a 2 0 %
inaccuracy for a distance measurement but to a difference of
25° in the determination of the angle between two intercalated
chromophores.
Finally the major problem is to know whether the Forster
theory is appropriate to account for the energy transfer between
identical chromophores.
The apparent rate of energy transfer between EthBr and its
nitro analogue is almost accounted for by the Forster theory
when measurements are done in the presence of a large excess of
the nitro derivative. In the opposite situation, that is when
EthBr is in excess the measurements cannot clearly be accounted
for by the Forster theory. The measurements of the rates of
anisotropy decay of the two acridine dimers in glycerol and in
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poly [d(A-T)] show that the apparent rate of transfer between
the two identical chromophores is considerably slower than the
value predicted by the Forster theory.In poly [d(A-T)] the
transfer can hardly be observed at all through anisotropy measurements .On the other hand the quantum yield of fluorescence
of the acridine dimers bound to DNA varies as the fourth power
23
of the AT percentage of the DNA .This shows that the excitation of afl uorescent acridine intercalated between two AT base
pairs is transferred very efficiently to the quenching acridine intercalated at the contact of a GC base pair.We are
therefore confronted with apparently conflicting experimental
results obtained on very similar experimental systems constituted of identical chromophores.Some of them cannot be interpreted using the Forster theory.Other results are not incompatible with it.
As recalled in the introduction the Forster theory is valid when the coherent coulombic coupling is ruined by the inco
herent perturbations of the medium.Therefore the Forster treat
ment is expected to apply better if molecules are far apart,in
a more disordered medium or in a different environment.lt is
therefore interesting to observe that when the two chromophores are just slightly different like in the case of EthBr and
NO_-EthBr the apparent rate of energy transfer is accounted
for by the Forster formula.Furthermore the discrepancy between
the apparent rate of energy transfer in the acridine dimers
and the rate computed from the Forster formula is smaller in
glycerol than in poly [d(A-T)] perhaps because the heterogeneity of the medium is larger in glycerol buffer solution than
in poly [d(A-T)] .The so-called red edge e f f e c t 3 0 which is an
other case where the Forster theory fails to account of transfer between identical molecules could also be explained by an
increase of the coherence at low temperature.
In this context the problem would be to get experimental
criteria which permit to define the domain of applicability of
the Forster theory.The absence of excitonic band in the spectra of circular dichroism of the acridine dimers bound to DNA
shows that the absence of excitons is not a sufficient test in
this respect.
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Nucleic Acids Research
The i n t e r p r e t a t i o n of t h e p h e n o m e n o n of a p p a r e n t energy
t r a n s f e r between identical or even r e l a t i v e l y similar c h r o m o phores a p p e a r s t h e r e f o r e very d i f f i c u l t at the present t i m e .
T h e e x p e r i m e n t a l system c o n s t i t u t e d of dimeric m o l e c u l e s bisint e r c a l a t e d in DNA a p p e a r s as a p o t e n t i a l l y very v a l u a b l e tool
for the f u t u r e study of a p p a r e n t energy t r a n s f e r between s i m i lar c h r o m o p h o r e s . M a n y types of c h r o m o p h o r e can be used. H e t e r o dimers can be c o n s t r u c t e d w i t h c h r o m o p h o r e s having increasing
d i f f e r e n c e in their absorption s p e c t r a so that the domain of
a p p l i c a b i l i t y of the Forster theory could be better d e f i n e d .
It m i g h t be interesting to point out that these studies
w e r e i n i t i a t e d to get i n f o r m a t i o n s c o n c e r n i n g the physical
c h e m i s t r y of c i r c u l a r DNA via t h e physical c h e m i s t r y of i n t e r c a l a t i o n . It m i g h t be thanks to w o r k s on these biological s y s tems to which Dr J. Vinograd d e v o t e d w i t h so much talent part
of his life that u n d e r s t a n d i n g of e n e r g y t r a n s f e r could
progress.
ACKNOWLEDGEMENTS
The a u t h o r s are very grateful to Dr G u s c h l b a u e r for p e r m i s sion to use his d i c h o g r a p h e .
This w o r k has been s u p p o r t e d by the CNRS (ATP 1 9 8 0 ) ,
1 ' U n i v e r s i t e P i e r r e et Marie C u r i e P a r i s V I , Fondation pour la
Recherche Medicale Frangaise.
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