Volume 4 Number 5 1977 Volume4
Number
5
1977
Nucleic Acids Research
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+ and Bernard P.RoquesH
+Lab.Pharmacol.Mol. Associe au CN RS, Institut Gustave Roussy, Villejuif 94800, and ++Dep.Chim., Fac.Pharm., Universite Rene Descartes, 4 Avenue de l'Observatoire, Paris 75005, France Received 10 January 1977
ABSTRACT
The rate of energy transfer between DNA interc lated ethidium cations calculated by Paoletti and Le Pecq 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 analoguesand 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 According to the Forster theory 2,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 methodology3.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 C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
1361
Nucleic Acids Research the unwinding angle of the DNA helix caused by the intercalation1. The knowledge of the value of this angle is of importance for studies related to the physical chemistry of circular DNA4-5. Such a determination was first attempted using ethidium bromide (EthBr)1. In that case the rate of energy transfer between identical intercalated molecules was deduced from steady state fluorescence polarization measurements analysed according to the Forster theory. With a refractive index of 1.4 for DNA, the rate of energy transfer deduced in this experiment was much smaller than the expected one. This led Paoletti and Le Pecql to question the value of the unwinding angle assumed to be correct at that time. It was later well demonstrated by several authors 6-8 that the modification of torsion of the DNA helix caused by the ethidium intercalation was between -26° to - 280. With this value, the rate of energy transfer computed by Paoletti and Le Pecq differs by a factor of 22 from the apparent rate of transfer. Later, other authors9 tried to solve this problem using the same experimental system but measured the fluorescence anisotropy decay instead of steady state anisotropy. A larger value of the refractive index of DNA which had been obtained at that time 10 was used. This reduced the discrepancy, but the apparent rate of energy transfer observed was still smaller than expected. Deducing the rate of energy transfer between two identical chromophores from measurements made on a array of numerous DNA intercalated molecules is in fact difficult because the interpretation of the experimental data depends on several assumptions and uncertain parameters: a) the distribution of the chromophores along the DNA. In the two preceding studies 19, the excluded site model11 was assumed to be correct. All the allowed sites were supposed to have the same probability of occupancy independently of sequence effect because EthBr DNA affinity does not depend on DNA base composition12. Nevertheless recent studies suggest some type of sequence specificity 13 b) the orientation of the ethidium molecule in its site. The crystallographic data show that the phenyl substituent 1362
Nucleic Acids Research lies in the small groove8. However this still leaves two possible orientations. Furthermore, 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 molecule1. 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 (review14,15). 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 levels. The incoherent coupling largely predominates and the relaxation described by Forster occurs. Forster16and Bennett and Kellog17 argue that the perturbations are large enough so that the same phenomenon occurs even in the case of identical molecules. On the contrary, several other argue that for identical molecules the coherent coupling may not be negligible. Coherent oscillations occur and the Forster treatment is not valid. Moreover, as the excited state is then delocalized over the identical chromopho res, the fluorescence anisotropy of the coherently coupled chromophores is expected to be equal to that of the isolated ones. Contrarily to what has been done in the case of different molecules3'21-22, no experiments have demonstrated the validity and/or the limits of applicability of the Forster theory in the case of similar chromophores, in the biological conditions of solvent and temperature. To progress in the understanding of these problems, we ha-
authorsl8-20
1363
Nucleic Acids Research 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 bisintercalate in DNA23 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 (N02-EthBr) were obtained from the Boots Pure Drug Company. We have verified that N02-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 104 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
C
r"
I
3
C
siHpsiuiuu
LU
1 =1182
etbidhlm
(EL
2.3
I
ieUptiIiUiv U(ELU )
diusIb
(ItbU
uit,u_d -iu (112=Uthr1
KNt~3
KU3
Wel meli I l= *2 3-" 2)4 6li I I 4t12)3 -(t2
ICE3
a
AcNIt"
=
-
-
-
-1)3
-
Figure 1 ties of ellipticine (El I) and of 2-6 dimethyl ellipticinium iodide (El II) have already been described 24. Acridine mono23 mer (AcMo) and dimers (AcDi I) and (AcDi II) are described Fluorescence quantum yields were measured as previously described1 using rhodamine B (q=0.69) and/or 9 amino acridine (q=.99)in ethanol as standards (table 1). In the Forster theory the rate of energy transfer kD-_oA from a donor D to an acceptor A molecule is k D-A= C(K2/R6)/tD (1) where R is the distance between D and A, TD the fluorescence lifetime of D and K depends on the normalized transition moments mA and mD of A and D and on u the unit vector of the liTable 1: Spectral characteristics of chromophores used here.
Dye interca- Absorption Fluorescence Fluorescence Quantum yield lifetime lated in (visible) emission ns poly [d(A-T)] A maxnm Xmaxnm EthBr N02-EthBr El I El II MoAc
520 540 435 450 440
600 600 530 540 500
24 =' 17 33.4 24
.14 =0 .31 .14 .22 1365
Nucleic Acids Research ne joining the chromophores according to
K2=(mA.mD-3(u.mA)(u.mD))2
(2)
In the expression (1), C is defined as CJ/6= 9790(qn-4 SI()Ai (3) where q is the donor quantum yield of fluorescence, n the refractive index of the medium, ID(i) the normalized intensity of the fluorescence of the donor at wave number ;ande A(v) the molar decadic extinction coefficient of the acceptor at F. The characteristic length Ro is
-4dF)1/6
RO= K2
C1/6
(4)
for random orientation
(5). Ro=(2/3 C)1/6 The values of Ro for the different died here are reported in table 2.
pairs of donor acceptor stu-
Table 2: Value of Ro (Angstroms) for random orientation and n=1. 75.
donor cceptor EthBr
N02-EthBr
EthBr
El I
El II
14.7 18.8
27.7 29.7
23.7 35.8
Measurement of the relative quantum yield of a fluorescent dye in presence of a quencher. Taking the equation of Le Pecq and Paoletti12 cb= (I -I)/(V-1)k (6) (cb concentration of dye bound to DNA, I', I fluorescence intensities of the solution of dye in presence and in absence of DNA, V the ratio of the fluorescence intensity emitted by the bound and free dye when excitation is made in identical conditions and k an instrumental factor), we obtain:
ql
=
((I1-I)/I)of0+1
q0 since
1366
V1/Vo
((I'-I)/I)1f1+1
=
ql/qo
(7) (8).
Nucleic Acids Research Subscripts 1 and o refer to measurements made with and without quencher. 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 described23. 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 of dyes along a 200 base pair long DNA segment was generated as in ref.1. The orientatation factor has been calculated assuming an unwinding angle of 260 for EthBr. The two possible orientations of the intercalated dye were assumed equiprobable. The computed CNDO/S orientation of the transition moment of EthBr has been used25. The direction of the transition moments of ellipticine and acridine have been computed in the same way (Le Bret unpublished results). The excitation migration is simulated as before1 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. Multipole corrections were introduced according to the results of figure 2 (see results section). Steady state fluorescence measurements 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 ref.26) with an instrument built in this laboratory. Anisotropy of fluorescence as a function of time A(t) is A (t) =-
=
)
S(t) Iv (t)+2IH(t) the fluorescence
(9)
intensity polarized verIV(t) and IH(t) being tically and horizontally respectively when the excitation light is polarized vertically. The function D(t) is recorded by stocking alternatively IV(t) and IH(t) respectively in positive and negative in the multichannel analyser. S(t) is recorded in the same way by summing up the corresponding functions and substracting the corresponding noises. For an aggregate of N identical dyes of fluorescence lifetime T 1367
Nucleic Acids Research the concentration of the mth dye Cm(t) still excited at time t when the dye CO has first been excited is the solution of the set of equations: dCm(t)/dt = -(1/t + m' m kim) Cm + m' m kmm ICm (10)
km'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 (11) CD(t) = 0.5 exp (-t/t) (1+e-2kt) 0.5 exp (-t/l) (1e-2kt) (12) k being the rate of transfer between the two molecules, CD (t) and CA(t) the concentration of excited donor and acceptor at time t. If the angle between the transition moments of the two molecules is e, the anisotropy decay is :
CA(t)
=
A(t)/A(o) = (1 + 3 cos26 + 3 sin2 6 e-2kt)/4
(13)
The averaging of this function yields the steady state anisotropy As/A(o) = (1/4) (1 + 3 cos20 + 3 sin2O/(1+2kT)) (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 The dipole-dipole term (K2 /R6 ) in the formula approximation of: "K /R "=I 12 where the transition moments mA and mD
mA=
(1)
is the
(15) (16)
are normalized and TA, TA and T'D, T 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 phenanthridiniurn cation which were computed by the PPPSCF method. Figure 2 sho-ws the variations of (17) log((K2/R6)/(lK2/R6'1)-l) for two superposed EthBr separated by a distance h and rotated 1368
Nucleic Acids Research by different angles o. The dipole-dipole approximation almost always overestimates the rate0 of transfer and becomes really correct for h larger than 15A. K
o091R -11 1
0'=85
* S-80o
*
* O-60o
|~~~~~ **o
0
-1
U
34
68
102
238
34.0
h
(A)
Figure 2. b) Energy transfer between different chromophores intercalated in DNA The Forster formula is valid in the case of energy transfer between the ellipticine derivatives and the non fluorescent analogue of ethidium because of the 100 nm difference between the wavelenth of their lowest energy band. Because of the large value of Ro in this case, measurements can be done at high phosphate to dye ratio where the distance between two neighbors are large and vary very much. This makes the influence of the relative orientation of the dyes uncritical. Several pairs have been studied. Figure 3 shows the results obtained for El II and N02-EthBr used respectively as donor and acceptor compared to the theoritical values obtained with different values of R With this pair, energy transfer appears extremely efficient at low values of r (number of dye bound per nucleotide) and becomes apparently less efficient at higher values of r. This very probably indicates that the distribution of the dye is not random and that the dyes tend to cluster at the beginning. The fitting Ro obtained at the higher values of r is smaller 1369
Nucleic Acids Research
25A 30A 40A
10r
Figure 3 Variation of the relative quantum yield q /q of El II in presence of increasing concentration of NO -ithgr. Experiment is started at r=0.001 (bound dye per gucleotide) and r is further increased by addition of N0 2-EthBr. DNA is at 0.1 mg/ml in 3 M CsCl Tris HCl 0.1pH 7.5 than the R0 computed with a DNA refractive index n=1.75. To eliminate this discrepancy, one can either introduce a higher value of the refractive index of DNA, that is n=2.1, in the calculation of the rate of transfer or think that the quantum yield of the donor molecule was underestimated by a factor of 2. The latter possibility seems improbable because similar results were obtained with other pairs studied (results not shown). This apparent refractive index obtained here is relative to the propagation of light along the axis of the DNA double helix and is larger than the value proposed by Harrington10. Nevertheless if the larger values of the polarizability of the base pairs determined by Takashima28 are substituted in the Lorentz-Lorenz formula a larger value (n=2.0) is obtained.
c) Fluorescence lifetime of ethidium The observed lifetime decreases with the phosphate/bound dye ratio (P/D). The effect is also observed in high salt concentration and is therefore not related to quenching by ethidium bound on the outside of the DNA helix. The effect is paral 1370
Nucleic Acids Research lel to the decrease of the fluorescence anisotropy of EthBrl. 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 chromophores29 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) 24
+AcMI
23
22 21 20
19 0
01
02
r
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 nucleotide). 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 HCl 0.1 M pH 7.5 with calf thymus DNA at 0.1 mg/ml. Fluorescence excitation is at 520 nm and fluorescence emission is measured through a high pass filter ( X:600 nm). We have thus been led to study systems where the Forster theory can be more directly tested in the case of identical molecules.
d) Fluorescence quenching of ethidium by its non fluorescent analogue. 1371
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 compared to the values computed by simulation for R = 16.6 A and 0 0 R0=19 A corresponding to DNA refractive index of 1.75 and 2.1. In the second type of experiment a large excess of ethi-
dium is used and a small quantity of N02-EthBr is added. In q1/q .9
.6 .7
* a R=16. 6
.4~~~~\ Ro =9
\a
-19
1 \N'
.1 .05
.1
.15
.2
r
Figure 5. Variation of the relative quantum yield of EthBr in presence of increasing concentration of NO -EthBr. Experiment is started with EthBr alone bound at r=O.Oi and r is further increased by addition of N0 -EthBr. DNA is at 0.02 mg/ml in 3 M CsCl, Tris HCl 0.1M pH i.5.
these conditions the quencher is isolated in the middle of an array of fluorescent EthBr. The decrease of fluorescence permits 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. 1372
Nucleic Acids Research
1.
0
5
10
15
20
25
30
%
35
40
NO2EthBr
Figure 6. Variation of the relative quantum yield of EthBr (q1/q ) in presence of small amounts of NO -EthBr. Experiment is st°rted at the shown ratio phosphate/bognd dye (P/D) relative to EthBr and N02-EthBr is added at the indicated percentage. DNA is at 0.02 mg/ml in CsCl 3 M Tris HCl O.1M pH 7.5. For 10% N02-EthBr the fluorescence decreases only by a factor of 1.2 indicating that apparently only 2 ethidiums are able to transfer their energy to the quencher. This would mean that there is a negligible ethidium ethidium transfer. These two observations cannot clearly be accounted for by the Forster theory. This theory would always predict a decrease of fluorescence and a migration of the energy over seve ral ethidium molecules. At P/D=4.5 intercalated molecules are indeed very close to each other (most of the time 10A apart). The probability of transfer according to the Forster formula between two EthBr molecules and between one EthBr and one N02-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 of 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 shown). This implies, if we interpret this result along the Forster theory, that the ahgle between the chromopho 1373
Nucleic Acids Research res is either 0° or close to 90° with a very slow rate of apparent rate of transfer (equation 13). Additional informations are given by the measurement of the anisotropy decay. The anisotropy decay of AcDi II in glycerol and bound to poly [d(A-T)] at high value of P/D in presence of saturating concentration of sucrose are shown in figure 7. Similar results (not shown) are obtained with AcDi I.
A()s n 10.;
D(t)
Dn
10;
tiin
time
AC(t)
1D( DO Iml d 0.1s5.
!"
b
,
d
0.15
time axi
time
Figure 7. Anisotropy decay of AcDi II in glycerol (d) and in poly Cd(A-T)h (b). The corresponding experimental functions S(t) and D(t) defined by equation 9 are shown int(a) and (c). Solutions in glycerol contain 10 % acetic acid 1.0 M and 0.01 mg/ml of dye. Poly Cd(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 at 0.1 mg/ml. Along the time axis the tic interval is 4.4 ns.
In glycerol, if we try to interpret the results along the Forster theory, the apparentRd can be calculated from the observed rate of transfer assuming a random orientation (equations 1 and 4). A random orientation is compatible with the limit reached by the fluorescence anisotropy at infinite time (Equation 13 gives A(oo)IA(o) = 1/2 for random orientation). Because for the acridine dimers dissolved in glycerol we do not know the average distance which separates the two chromophores, we can only deduce from this anisotropy decay 1374
Nucleic Acids Research rate a maximum Ro value assuming a maximum extension of the chain which separates the two chromophores 23. This value can be compared to the R0 computed from the spectral properties (equations 3 and 4). Results are shown in table III.
(A) phoperties
Computed R
spectral AcDi AcDi
I II
from
20 23
Maximum R (A) deduced from anis8tropy decay 10 14
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.
X
nm
1375
Nucleic Acids Research DISCUSSION The experimental results presented in this paper show clearly that several of the assumptions made by Paoletti and Le Pecq1 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 -re not accurate enough to propose a definite value, but they would favor a value (2.I) which is larger than the value (I.75) proposed by Harrington 10 - 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 20% 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 1376
Nucleic Acids Research 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 of the AT percentage of the DNA23.This shows that the excitation of a fluorescent 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.It is therefore interesting to observe that when the two chromophores are just slightly different like in the case of EthBr and NO2-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)I.The so-called red edge effect30 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. 1377
Nucleic Acids Research The interpretation of the phenomenon of apparent energy transfer between identical or even relatively similar chromophores appears therefore very difficult at the present time. The experimental system constituted of dimeric molecules bisintercalated in DNA appears as a potentially very valuable tool for the future study of apparent energy transfer between similar chromophores.Many types of chromophore can be used. Heterodimers can be constructed with chromophores having increasing difference in their absorption spectra so that the domain of applicability of the Forster theory could be better defined. It might be interesting to point out that these studies were initiated to get informations concerning the physical chemistry of circular DNA via the physical chemistry of intercalation. It might be thanks to works on these biological systems to which Dr J. Vinograd devoted with so much talent part of his life that understanding of energy transfer could progress.
ACKNOWLEDGEMENTS The authors are very grateful to Dr Guschlbauer for permission to use his dichographe. This work has been supported by the CNRS (ATP 1980), l'UniversitePierre et Marie Curie Paris VI, Fondation pour la Recherche Medicale Frangaise.
REFERENCES * dedicated to Jerome Vinograd 1. Paoletti J. and Le Pecq J.B. (1971) J. Mol. Biol. 59,43-62 2. Forster Th. (1959) Disc. Faraday Soc. 27, 7-17 3. Stryer L. and Haugland R.P. (1967) Proc. Nat. Acad. Sci. USA 58, 719-726 4. Bauer W. and Vinograd J. (1968) J. Mol. Biol. 33, 141-171 5. Bauer W. and Vinograd J. (1970) J. Mol. Biol. 47, 419-435 6. Wang J. (1974) J. Mol. Biol. 89, 783-801 7. Pulleyblank D.E. and Morgan A.R. (1974) J. Mol. Biol. 91, 1-13 8. TsaiC.C., Jain S.C. and Sobell H.M. (1975) Proc. Nat. Acad. Sci. USA 72, 628-632 9. Genest D., Wkhl Ph. and Auchet J.C. (1974) Biophysical Chemistry 1, 266-278 10. Harrington R.E. (1970) J. Am. Chem. Soc. 92, 6957-6964 1378
Nucleic Acids Research 11. Crothers D.M. (1968) Biopolymers 6, 575-584 12. Le Pecq J.B. and Paoletti C. (1967) J. Mol. Biol. 27, 87106 13. Krugh T.R. and Reinhardt C.G. (1975) J. Mol. Biol. 97,133162 14. Kasha M. (1963) Radiations Res. 20, 55-71 15. Steinberg J.Z. (1975) in Biochemical fluorescence concepts Chen R. and Edelhoch H. ed. Vol. 1 pp 79-113 16. Forster Th. (1965) in Modern Quantum Chemistry Sinanoglu 0. Ed. Part 3 pp 93-137 Academic Press New York 17. Bennet R.G. and Kellogg R.E. (1966) Prog Reaction Kinetics 4, 215-238 18. Simpson W.T. and Peterson D.L. (1957) J. Chem. Phys. 26, 588-593 19. Robinson G.W. and Frosch R.P. (1963) J. Chem. Phys. 38, 1187-1203 20. Aslangul C., Constanciel R., Daudel R. and Kottis P.(1972) in Advances in Quantum Chemistry 6, 93-141 21. Latt S.A., Cheung H.T. and Blout E.R. (1965) J. Am. Chem. Soc. 87, 995-1003 22. Haugland R.P., Yguerabide J. and Stryer L. (1969) Proc. Nat Acad. Sci. USA 63, 23-30 23. Le Pecq J.B., Le Bret M., Barbet J. and Roques B.P. (1975) Proc. Nat Acad. Sci. USA 72, 2915-2919. 24. Le Pecq J.B., Nguyen-Dat-Xuong, Gosse Ch. and Paoletti C. (1974) Proc. Nat. Acad. Sci. USA 71, 5078-5082 25. Le Bret M. and Chalvet 0. (1977) J. Mol. Struct. in press 26. Yguerabide J. (1972) in Methods in Enzymology, Hirs C.H.V. and Timasheff S.N. Eds, Vol 26 part C pp 498-578 Academic Press New York 27. Giacomoni P.U. and Le Bret M. (1973) FEBS Letters 29, 227-230 28. Takashima S. (1969) Biopolymers 8,199-216 29. Mc Rae E.G. and Kasha M. (1958) J. Chem. Phys. 28, 721-722 30. Weber G. and Shinitzky M. (1970) Proc. Nat. Acad. Sci. USA 65, 823-830
1379
Number
5
1977
Nucleic Acids Research
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+ and Bernard P.RoquesH
+Lab.Pharmacol.Mol. Associe au CN RS, Institut Gustave Roussy, Villejuif 94800, and ++Dep.Chim., Fac.Pharm., Universite Rene Descartes, 4 Avenue de l'Observatoire, Paris 75005, France Received 10 January 1977
ABSTRACT
The rate of energy transfer between DNA interc lated ethidium cations calculated by Paoletti and Le Pecq 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 analoguesand 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 According to the Forster theory 2,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 methodology3.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 C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
1361
Nucleic Acids Research the unwinding angle of the DNA helix caused by the intercalation1. The knowledge of the value of this angle is of importance for studies related to the physical chemistry of circular DNA4-5. Such a determination was first attempted using ethidium bromide (EthBr)1. In that case the rate of energy transfer between identical intercalated molecules was deduced from steady state fluorescence polarization measurements analysed according to the Forster theory. With a refractive index of 1.4 for DNA, the rate of energy transfer deduced in this experiment was much smaller than the expected one. This led Paoletti and Le Pecql to question the value of the unwinding angle assumed to be correct at that time. It was later well demonstrated by several authors 6-8 that the modification of torsion of the DNA helix caused by the ethidium intercalation was between -26° to - 280. With this value, the rate of energy transfer computed by Paoletti and Le Pecq differs by a factor of 22 from the apparent rate of transfer. Later, other authors9 tried to solve this problem using the same experimental system but measured the fluorescence anisotropy decay instead of steady state anisotropy. A larger value of the refractive index of DNA which had been obtained at that time 10 was used. This reduced the discrepancy, but the apparent rate of energy transfer observed was still smaller than expected. Deducing the rate of energy transfer between two identical chromophores from measurements made on a array of numerous DNA intercalated molecules is in fact difficult because the interpretation of the experimental data depends on several assumptions and uncertain parameters: a) the distribution of the chromophores along the DNA. In the two preceding studies 19, the excluded site model11 was assumed to be correct. All the allowed sites were supposed to have the same probability of occupancy independently of sequence effect because EthBr DNA affinity does not depend on DNA base composition12. Nevertheless recent studies suggest some type of sequence specificity 13 b) the orientation of the ethidium molecule in its site. The crystallographic data show that the phenyl substituent 1362
Nucleic Acids Research lies in the small groove8. However this still leaves two possible orientations. Furthermore, 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 molecule1. 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 (review14,15). 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 levels. The incoherent coupling largely predominates and the relaxation described by Forster occurs. Forster16and Bennett and Kellog17 argue that the perturbations are large enough so that the same phenomenon occurs even in the case of identical molecules. On the contrary, several other argue that for identical molecules the coherent coupling may not be negligible. Coherent oscillations occur and the Forster treatment is not valid. Moreover, as the excited state is then delocalized over the identical chromopho res, the fluorescence anisotropy of the coherently coupled chromophores is expected to be equal to that of the isolated ones. Contrarily to what has been done in the case of different molecules3'21-22, no experiments have demonstrated the validity and/or the limits of applicability of the Forster theory in the case of similar chromophores, in the biological conditions of solvent and temperature. To progress in the understanding of these problems, we ha-
authorsl8-20
1363
Nucleic Acids Research 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 bisintercalate in DNA23 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 (N02-EthBr) were obtained from the Boots Pure Drug Company. We have verified that N02-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 104 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
C
r"
I
3
C
siHpsiuiuu
LU
1 =1182
etbidhlm
(EL
2.3
I
ieUptiIiUiv U(ELU )
diusIb
(ItbU
uit,u_d -iu (112=Uthr1
KNt~3
KU3
Wel meli I l= *2 3-" 2)4 6li I I 4t12)3 -(t2
ICE3
a
AcNIt"
=
-
-
-
-1)3
-
Figure 1 ties of ellipticine (El I) and of 2-6 dimethyl ellipticinium iodide (El II) have already been described 24. Acridine mono23 mer (AcMo) and dimers (AcDi I) and (AcDi II) are described Fluorescence quantum yields were measured as previously described1 using rhodamine B (q=0.69) and/or 9 amino acridine (q=.99)in ethanol as standards (table 1). In the Forster theory the rate of energy transfer kD-_oA from a donor D to an acceptor A molecule is k D-A= C(K2/R6)/tD (1) where R is the distance between D and A, TD the fluorescence lifetime of D and K depends on the normalized transition moments mA and mD of A and D and on u the unit vector of the liTable 1: Spectral characteristics of chromophores used here.
Dye interca- Absorption Fluorescence Fluorescence Quantum yield lifetime lated in (visible) emission ns poly [d(A-T)] A maxnm Xmaxnm EthBr N02-EthBr El I El II MoAc
520 540 435 450 440
600 600 530 540 500
24 =' 17 33.4 24
.14 =0 .31 .14 .22 1365
Nucleic Acids Research ne joining the chromophores according to
K2=(mA.mD-3(u.mA)(u.mD))2
(2)
In the expression (1), C is defined as CJ/6= 9790(qn-4 SI()Ai (3) where q is the donor quantum yield of fluorescence, n the refractive index of the medium, ID(i) the normalized intensity of the fluorescence of the donor at wave number ;ande A(v) the molar decadic extinction coefficient of the acceptor at F. The characteristic length Ro is
-4dF)1/6
RO= K2
C1/6
(4)
for random orientation
(5). Ro=(2/3 C)1/6 The values of Ro for the different died here are reported in table 2.
pairs of donor acceptor stu-
Table 2: Value of Ro (Angstroms) for random orientation and n=1. 75.
donor cceptor EthBr
N02-EthBr
EthBr
El I
El II
14.7 18.8
27.7 29.7
23.7 35.8
Measurement of the relative quantum yield of a fluorescent dye in presence of a quencher. Taking the equation of Le Pecq and Paoletti12 cb= (I -I)/(V-1)k (6) (cb concentration of dye bound to DNA, I', I fluorescence intensities of the solution of dye in presence and in absence of DNA, V the ratio of the fluorescence intensity emitted by the bound and free dye when excitation is made in identical conditions and k an instrumental factor), we obtain:
ql
=
((I1-I)/I)of0+1
q0 since
1366
V1/Vo
((I'-I)/I)1f1+1
=
ql/qo
(7) (8).
Nucleic Acids Research Subscripts 1 and o refer to measurements made with and without quencher. 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 described23. 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 of dyes along a 200 base pair long DNA segment was generated as in ref.1. The orientatation factor has been calculated assuming an unwinding angle of 260 for EthBr. The two possible orientations of the intercalated dye were assumed equiprobable. The computed CNDO/S orientation of the transition moment of EthBr has been used25. The direction of the transition moments of ellipticine and acridine have been computed in the same way (Le Bret unpublished results). The excitation migration is simulated as before1 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. Multipole corrections were introduced according to the results of figure 2 (see results section). Steady state fluorescence measurements 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 ref.26) with an instrument built in this laboratory. Anisotropy of fluorescence as a function of time A(t) is A (t) =-
=
)
S(t) Iv (t)+2IH(t) the fluorescence
(9)
intensity polarized verIV(t) and IH(t) being tically and horizontally respectively when the excitation light is polarized vertically. The function D(t) is recorded by stocking alternatively IV(t) and IH(t) respectively in positive and negative in the multichannel analyser. S(t) is recorded in the same way by summing up the corresponding functions and substracting the corresponding noises. For an aggregate of N identical dyes of fluorescence lifetime T 1367
Nucleic Acids Research the concentration of the mth dye Cm(t) still excited at time t when the dye CO has first been excited is the solution of the set of equations: dCm(t)/dt = -(1/t + m' m kim) Cm + m' m kmm ICm (10)
km'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 (11) CD(t) = 0.5 exp (-t/t) (1+e-2kt) 0.5 exp (-t/l) (1e-2kt) (12) k being the rate of transfer between the two molecules, CD (t) and CA(t) the concentration of excited donor and acceptor at time t. If the angle between the transition moments of the two molecules is e, the anisotropy decay is :
CA(t)
=
A(t)/A(o) = (1 + 3 cos26 + 3 sin2 6 e-2kt)/4
(13)
The averaging of this function yields the steady state anisotropy As/A(o) = (1/4) (1 + 3 cos20 + 3 sin2O/(1+2kT)) (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 The dipole-dipole term (K2 /R6 ) in the formula approximation of: "K /R "=I 12 where the transition moments mA and mD
mA=
(1)
is the
(15) (16)
are normalized and TA, TA and T'D, T 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 phenanthridiniurn cation which were computed by the PPPSCF method. Figure 2 sho-ws the variations of (17) log((K2/R6)/(lK2/R6'1)-l) for two superposed EthBr separated by a distance h and rotated 1368
Nucleic Acids Research by different angles o. The dipole-dipole approximation almost always overestimates the rate0 of transfer and becomes really correct for h larger than 15A. K
o091R -11 1
0'=85
* S-80o
*
* O-60o
|~~~~~ **o
0
-1
U
34
68
102
238
34.0
h
(A)
Figure 2. b) Energy transfer between different chromophores intercalated in DNA The Forster formula is valid in the case of energy transfer between the ellipticine derivatives and the non fluorescent analogue of ethidium because of the 100 nm difference between the wavelenth of their lowest energy band. Because of the large value of Ro in this case, measurements can be done at high phosphate to dye ratio where the distance between two neighbors are large and vary very much. This makes the influence of the relative orientation of the dyes uncritical. Several pairs have been studied. Figure 3 shows the results obtained for El II and N02-EthBr used respectively as donor and acceptor compared to the theoritical values obtained with different values of R With this pair, energy transfer appears extremely efficient at low values of r (number of dye bound per nucleotide) and becomes apparently less efficient at higher values of r. This very probably indicates that the distribution of the dye is not random and that the dyes tend to cluster at the beginning. The fitting Ro obtained at the higher values of r is smaller 1369
Nucleic Acids Research
25A 30A 40A
10r
Figure 3 Variation of the relative quantum yield q /q of El II in presence of increasing concentration of NO -ithgr. Experiment is started at r=0.001 (bound dye per gucleotide) and r is further increased by addition of N0 2-EthBr. DNA is at 0.1 mg/ml in 3 M CsCl Tris HCl 0.1pH 7.5 than the R0 computed with a DNA refractive index n=1.75. To eliminate this discrepancy, one can either introduce a higher value of the refractive index of DNA, that is n=2.1, in the calculation of the rate of transfer or think that the quantum yield of the donor molecule was underestimated by a factor of 2. The latter possibility seems improbable because similar results were obtained with other pairs studied (results not shown). This apparent refractive index obtained here is relative to the propagation of light along the axis of the DNA double helix and is larger than the value proposed by Harrington10. Nevertheless if the larger values of the polarizability of the base pairs determined by Takashima28 are substituted in the Lorentz-Lorenz formula a larger value (n=2.0) is obtained.
c) Fluorescence lifetime of ethidium The observed lifetime decreases with the phosphate/bound dye ratio (P/D). The effect is also observed in high salt concentration and is therefore not related to quenching by ethidium bound on the outside of the DNA helix. The effect is paral 1370
Nucleic Acids Research lel to the decrease of the fluorescence anisotropy of EthBrl. 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 chromophores29 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) 24
+AcMI
23
22 21 20
19 0
01
02
r
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 nucleotide). 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 HCl 0.1 M pH 7.5 with calf thymus DNA at 0.1 mg/ml. Fluorescence excitation is at 520 nm and fluorescence emission is measured through a high pass filter ( X:600 nm). We have thus been led to study systems where the Forster theory can be more directly tested in the case of identical molecules.
d) Fluorescence quenching of ethidium by its non fluorescent analogue. 1371
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 compared to the values computed by simulation for R = 16.6 A and 0 0 R0=19 A corresponding to DNA refractive index of 1.75 and 2.1. In the second type of experiment a large excess of ethi-
dium is used and a small quantity of N02-EthBr is added. In q1/q .9
.6 .7
* a R=16. 6
.4~~~~\ Ro =9
\a
-19
1 \N'
.1 .05
.1
.15
.2
r
Figure 5. Variation of the relative quantum yield of EthBr in presence of increasing concentration of NO -EthBr. Experiment is started with EthBr alone bound at r=O.Oi and r is further increased by addition of N0 -EthBr. DNA is at 0.02 mg/ml in 3 M CsCl, Tris HCl 0.1M pH i.5.
these conditions the quencher is isolated in the middle of an array of fluorescent EthBr. The decrease of fluorescence permits 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. 1372
Nucleic Acids Research
1.
0
5
10
15
20
25
30
%
35
40
NO2EthBr
Figure 6. Variation of the relative quantum yield of EthBr (q1/q ) in presence of small amounts of NO -EthBr. Experiment is st°rted at the shown ratio phosphate/bognd dye (P/D) relative to EthBr and N02-EthBr is added at the indicated percentage. DNA is at 0.02 mg/ml in CsCl 3 M Tris HCl O.1M pH 7.5. For 10% N02-EthBr the fluorescence decreases only by a factor of 1.2 indicating that apparently only 2 ethidiums are able to transfer their energy to the quencher. This would mean that there is a negligible ethidium ethidium transfer. These two observations cannot clearly be accounted for by the Forster theory. This theory would always predict a decrease of fluorescence and a migration of the energy over seve ral ethidium molecules. At P/D=4.5 intercalated molecules are indeed very close to each other (most of the time 10A apart). The probability of transfer according to the Forster formula between two EthBr molecules and between one EthBr and one N02-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 of 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 shown). This implies, if we interpret this result along the Forster theory, that the ahgle between the chromopho 1373
Nucleic Acids Research res is either 0° or close to 90° with a very slow rate of apparent rate of transfer (equation 13). Additional informations are given by the measurement of the anisotropy decay. The anisotropy decay of AcDi II in glycerol and bound to poly [d(A-T)] at high value of P/D in presence of saturating concentration of sucrose are shown in figure 7. Similar results (not shown) are obtained with AcDi I.
A()s n 10.;
D(t)
Dn
10;
tiin
time
AC(t)
1D( DO Iml d 0.1s5.
!"
b
,
d
0.15
time axi
time
Figure 7. Anisotropy decay of AcDi II in glycerol (d) and in poly Cd(A-T)h (b). The corresponding experimental functions S(t) and D(t) defined by equation 9 are shown int(a) and (c). Solutions in glycerol contain 10 % acetic acid 1.0 M and 0.01 mg/ml of dye. Poly Cd(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 at 0.1 mg/ml. Along the time axis the tic interval is 4.4 ns.
In glycerol, if we try to interpret the results along the Forster theory, the apparentRd can be calculated from the observed rate of transfer assuming a random orientation (equations 1 and 4). A random orientation is compatible with the limit reached by the fluorescence anisotropy at infinite time (Equation 13 gives A(oo)IA(o) = 1/2 for random orientation). Because for the acridine dimers dissolved in glycerol we do not know the average distance which separates the two chromophores, we can only deduce from this anisotropy decay 1374
Nucleic Acids Research rate a maximum Ro value assuming a maximum extension of the chain which separates the two chromophores 23. This value can be compared to the R0 computed from the spectral properties (equations 3 and 4). Results are shown in table III.
(A) phoperties
Computed R
spectral AcDi AcDi
I II
from
20 23
Maximum R (A) deduced from anis8tropy decay 10 14
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.
X
nm
1375
Nucleic Acids Research DISCUSSION The experimental results presented in this paper show clearly that several of the assumptions made by Paoletti and Le Pecq1 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 -re not accurate enough to propose a definite value, but they would favor a value (2.I) which is larger than the value (I.75) proposed by Harrington 10 - 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 20% 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 1376
Nucleic Acids Research 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 of the AT percentage of the DNA23.This shows that the excitation of a fluorescent 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.It is therefore interesting to observe that when the two chromophores are just slightly different like in the case of EthBr and NO2-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)I.The so-called red edge effect30 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. 1377
Nucleic Acids Research The interpretation of the phenomenon of apparent energy transfer between identical or even relatively similar chromophores appears therefore very difficult at the present time. The experimental system constituted of dimeric molecules bisintercalated in DNA appears as a potentially very valuable tool for the future study of apparent energy transfer between similar chromophores.Many types of chromophore can be used. Heterodimers can be constructed with chromophores having increasing difference in their absorption spectra so that the domain of applicability of the Forster theory could be better defined. It might be interesting to point out that these studies were initiated to get informations concerning the physical chemistry of circular DNA via the physical chemistry of intercalation. It might be thanks to works on these biological systems to which Dr J. Vinograd devoted with so much talent part of his life that understanding of energy transfer could progress.
ACKNOWLEDGEMENTS The authors are very grateful to Dr Guschlbauer for permission to use his dichographe. This work has been supported by the CNRS (ATP 1980), l'UniversitePierre et Marie Curie Paris VI, Fondation pour la Recherche Medicale Frangaise.
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