17936 J. Phys. Chem.

1995,99, 17936-17947

Base-Content Dependence of Emission Enhancements, Quantum Yields, and Lifetimes for

Cyanine Dyes Bound to Double-Strand DNA: Photophysical Properties of Monomeric and
Bichromophoric DNA Stains

Thomas L. Netzel," Kambiz Nafisi, and Min Zhao

Department of Chemistry, Georgia State University, Atlanta, Georgia 30303

Jerome R. Lenhard
Research Laboratory, Eastman Kodak Company, Rochester, New York 14652-4708

Iain Johnson
Molecular Probes, Inc., P.O. Box 22010, Eugene, Oregon 97402-0414
Received: June 29, 1995; In Final Form: September 11, 1995@

This paper reports fluorescence quantum yield, emission enhancement, and emission lifetime measurements
for 10 cyanine dyes complexed to calf thymus DNA (CT-DNA), (dAdT)lo, and (dGdC)6 duplexes. Six of
the dyes are linked bichromophores with four cationic charges per molecule, and four are monomers with
two cationic charges per molecule. Emission enhancement is equal to the ratio of emission quantum yields
when a dye is bound to double-strand (ds) DNA to that when it is free in solution. Enhancements for these
10 dyes range from 200 to 1800. All of the dyes exhibit either bi- or triexponential emission decay kinetics
reflecting different dye/ds DNA modes of binding, and the average radiative lifetime for the bichromophores
bound to ds DNA is 5.1 f 0.8 ns. These results are consistent with expectations that binding-induced restriction
of torsion about the central methine bridge is responsible for the large emission enhancements of these dyes.
Scrutiny of the lengths of average emission lifetime for these 10 dyes on (dAdT)lo and (dGdC)6 duplexes
finds that they do not vary as expected if electron transfer (ET) emission quenching were an important process.
Predictions of the relative rates of ET quenching of excited dye emission by the four DNA nucleosides are
based on estimates of the free energy of such reactions using redox data for cyanine dye analogues of the
DNA-staining dyes. There are also differences in emission quantum yield between dyes with pyridinium
and quinolinium structural components when bound to (dAdT)lo and (dGdC)6 duplexes. These differences
are very distinct for the monomeric dyes where pyridinium dyes have 4-fold greater emission yields on (dAdT)lo
duplexes and quinolinium dyes have 2-fold greater emission yields on (dGdC)6 duplexes. A 2-fold quantum
yield increase on switching from (dAdT)lo to (dGdC)6 duplexes is also present for the quinolinium
bichromophore, TOTO-1. Very importantly, for this bichromophore and the four monomers the emission
quantum yield on CT-DNA matches very well the higher of the short duplex quantum yields. This suggests
but does not prove that the pyridinium and quinolinium cyanine dyes selectively associate with AT- and
GC-rich regions, respectively, when bound to CT-DNA. For the other five bichromophores studied, there is
little difference in emission quantum yield between the two types of short duplexes. Additionally, the emission
yields of these bichromophores when bound to CT-DNA do not clearly correspond to either of the short
duplex emission yields. Thus, for these dyes there is no clear evidence that they exhibit base-content selectivity
with respect to emission yield.

Introduction enhancement, addressing the questions of whether these pro-

Cyanine dyes have a rich history in the photographic industry'
and have recently become important in the staining of biological
samples. A particularly important application in the latter area
involves the staining of duplex or ds DNA.2 Cationic cyanine
dyes exhibit very large degrees of fluorescence enhancement
on binding to nucleic acids3x4In addition covalent linkage of
two cyanine dyes to form a bichromophore increases the nucleic
acid binding affinity by approximately 2 orders of magnitude,
as previously found in the case of phenanthridium dyes
(ethidium b r ~ m i d e ) . ~These characteristics of fluorescence
enhancement and high binding affinity are crucial for high
sensitivity nucleic acid detection application^.^-^ In this study
we investigate the mechanisms underlying the fluorescence
@Abstractpublished in Advance ACS Absrracu, November 15, 1995.
0022-365419512099-17936$09.0010 0 1995 American Chemical Society
cesses are sensitive to the base content of the nucleic acid target
and whether the photophysical properties of cyanine nucleic acid
stains are significantly modified upon coupling to form bichro-
mophores in order to obtain higher binding affinity.
In parallel with the development of biological applications
of cyanine dyes,3,4,6-'3numerous physical chemical investiga-
tions have been carried out to understand the nonradiative decay
mechanisms which control the excited-state lifetimes of these
dye^.'^-^* The generally accepted view is that the nonradiative
decay of photoexcited cyanine dyes is controlled by the rate of
rotation or torsion about the central methine bridge which links
the heterocycles at either end.15,18,22 Kemnitz et al.23 studied
the fluorescence lifetimes of monomeric 5,5'-dichloro-3,3'-
disulfopropyl-9-(ethylthia)carbocyanine in methanol and ad-
sorbed on Si02 and quartz. Presumably because of restricted
Cyanine Dyes Bound to Double-Strand DNA
motion for the adsorbed carbocyanine dye molecules, the 70-
ps fluorescence lifetime of this dye in methanol lengthened 40-
fold when adsorbed on silica and quartz. Serpone et al.24studied
the photophysics of two dithiacarbocyanine dyes in dichlo-
romethane and found that while both intersystem crossing and
internal conversion occur at comparable rates, torsional motion
of the polymethine chain is a prerequisite to both processes.
Additionally, there is evidence that the rate of isomerization of
cyanine dyes increases with decreasing length of the central
polymethine chain.25 A particularly important study in this area
showed the rate of radiationless decay rate to be inversely
proportional to the solvent's vis~osity.'~However, more
recently Sauerwein et aLI4 demonstrated a very strong cor-
relation of nonradiative lifetime with the molecular weight of
the solvent in four solvent series. In this work solvent viscosity
was not sufficient to account for the dynamics of cyanine dye
torsion among the broad number of solvents surveyed. Rather,
solvents of high molecular weight transferred less rotational
momentum per collision to the cyanine dyes than solvents of
low molecular weight and thus less readily deactivated their
excited states. Finally, there does not appear to be an intrinsic
electronic barrier to torsion about the central methine bridge in
the lowest energy, singlet excited state of these dye^.'^.^'
This paper reports fluorescence quantum yield and fluores-
cence lifetime measurements on ten different cyanine dyes each
complexed to calf thymus DNA (CT-DNA) and to (dAdT)lo
and (dGdC)6 self-complementary duplexes. Applications of
these dyes to stain ds DNA have found both no dependence of
emission enhancement upon DNA duplex base ~ontent4*~-~-"
and moderate dependence on DNA base ~ o n t e n t . ' ~To . ~ the
~ cyanine dyes complexed to ds DNA. The first is that dAdT
extent the emission enhancements are independent of DNA base
content, they can be used quantitativelyto measure total ds DNA
content in analytical protocols.
The monomeric dicationic and bichromophoric quadruply
cationic cyanine dyes bind to ds DNA more strongly than
monocationic cyanines such as thiazole orange (TO).39598J
Order-of-magnitude estimates for the dyes studied here of the
ds DNA binding constants for the monomeric and bichro-
mophoric forms, respectively, are lo6 and los M-1.27 Lee et
aL3 postulated that the fluorescence enhancement upon nucleic
acid binding of dyes such as TO (which exhibits approximately
3000-fold enhancement on RNA) is due to a change in the
relative orientation of the benzothiazole and quinolinium rings
from skewed to coplanar. TO is nearyl identical with the dye
TO-PRO-1 studied here with the difference that R' is a methyl
group for TO (see Figure 1). A recent study by Carlsson et
a1.28 agrees with these ideas and shows for YO-PRO-1 in
glycerol that the decrease in emission quantum yield with
increasing temperature has a very similar activation energy, 53
kJ/mol, to the activation energy for viscous flow, 63 kJ/mol.
Because of this result, they conclude that the increase in
emission quantum yield for YO-PRO-1 upon binding to ds DNA
is due to restricted rotation about the benzoxazole/quinoline
bridge in the bound state relative to the free state. These authors
-
also make the important point that there is only one electronic
transition in the low-energy So SI absorbance region for YO-
PRO-1 bound to ds DNA. Thus the radiative (or natural)
lifetime calculated from the absorbance spectrum, 5 ns, agrees
well with the value obtained from emission lifetime and quantum
yield measurements, 6 ns.28 Each of the other nine dyes studied
here is structurally very similar to YO-PRO-I, and therefore
each of them is also likely to have only one low-energy
electronic transition.
A priori two processes come to mind that could cause base
content to produce differential emission enhancements for
J. Phys. Chem., Vol. 99, No. 51, 1995 17937
Monomethine Cyanine Dyes
R n
POPO-I: X = O , R = M e , n = Z .
R = -(CHz),Nt(CH,),(CHz),N'02(CH,),-
Po-PRO-1: X = 0.R = Me,n = 1, R = -(CH2)3N'(CH,),
Analogl: X=O,R=Et, n = l . R = E t
BOBO-1: X = S. R = M e . n = 2,
R' .-(CH~)~N'(CHI)Z(CH~)~N"CH,)I(CH~)~.
BO-PRO-1: X = S. R =Me,n = 1, R = -(CHz)3N'(CH3)3
Analog2 X=S.R=Et. n = l , R=Et
R
YOYO-1: X =O, R = Me,n =2.
R = -(CHz),N'(CH,)z(CHz),N*(CH,),(CH,),-
YO-PRO-1: X = 0.R =Me, n = 1, R' = -(CH2),N+(CH,),
Analog3: X=O.R=Et. n = l , R-Et
TOTO.1: X =S. R =Me, n =2.
R = -(CH~)3N'(CH3)z(CHz)~Ni(~~)z(CH2),-
TO.PR0-1: X = S, R = Me, n = 1. R = -(CH,),N+(CH,),
Analog4 X = S , R=Et, n = I , R = E t
Figure 1. Structural drawing of four monomethine cyanine dyes as
(1) dicationic monomers, (2) quadruply cationic bichromophores, and
(3) monocationic analogs of the DNA-staining dyes.
sequences could in principle produce different types of binding
sites than dGdC sequences. If one type of site provided greater
immobilization toward torsion about the central methine bridge,
it could thereby produce a greater emission enhancement. The
second process that could differentially alter emission enhance-
ments for cyanine dyes bound to DNA duplexes is excited-
state ET quenching by DNA n ~ c l e o s i d e s . ~Guanosine
~~*~ is the
easiest nucleoside to oxidize, while thymidine and cytidine are
the easiest nucleosides to reduce. Adenosine is difficult to
oxidize and is also the nucleoside least easy to r e d u ~ e . ~ OThis
-~~
paper also reports electrochemical measurements of the oxida-
tion and reduction potentials of six cyanine dyes that model all
10 of the dyes studied here. These redox potentials are used to
estimate the free energy for both reductive dye* quenching by
dG, AG"(dG'+/dye'-), and oxidative dye* quenching by dT and
dC, AG"(dye'+/dT-) and AG"(dye'+/dC'-), respectively.
Because the structurally related dyes studied here are all likely
to have a single lowest energy excited singlet state as has been
proven for YO-PRO-1 and YOYO-1,28they can all be expected
to have emission quantum yields, Qem,that are proportional
to their emission lifetimes, z, according to eq 1, where &ad is
the radiative (or natural) lifetime. For YO-PRO-1 and YOYO-1
this has been demonstrated to the true in both organic solvents
and when the dyes are intercalated into ds DNA.28 Equation 1
means minimally that if one dye is placed on several types of
ds DNA and both z and Qem are measured for each type of
DNA, the calculated value of rrad should be the same in all cases.
To the extent the oscillator strengths of the lowest energy excited
states of the different dyes are the same, eq 1 will also mean
that ?&d will be the same for all of the dyes on all types of ds
DNA. In fact the shapes of the absorbance bands of the lowest
energy excited states for all of the dyes studied here are
17938 J. Phys. Chem., Vol. 99, No. 51, I995
7
Trimethine Cyanine Dyes
- and stored in a desiccator. For emission lifetime and quantum
I
1 .
YOYO-3: X = 0. R = Me,n =2,
R =- ~ C H ~ ~ ~ N ' ~ C H ~ ~ ~ ~ ~ H , ~ , N " C H I ) I ~ ~ ~
Analog 6: X = 0,R =Et, n=l, R' = -CH(CH,)z
Figure 2. Structural drawing of two trimethine cyanine (carbocyanine)
dyes as (1) quadruply cationic bichromophores and ( 2 ) monocationic
analogs of the DNA-staining dyes.
remarkably similar.*' One qualification to this last statement
is that in water but not in the presence of ds DNA or alcohol
bichromophoric dyes such as YOYO-1 form intramolecular
dimers and show exciton splitting in their lowest energy
electronic absorbance bands.28 However, in the presence of ds
DNA YOYO-1 is a bisintercalator with a ground-state absor-
bance band similar to that of the monomer YO-PRO-1 bound
to ds DNA.33 Thus the constancy of experimental q a d values
on several kinds of ds DNA for the each of the 10 dyes studied
here can provide an important verification of the accuracy of z
and CP,, measurements.
This work compares @ ,,, z, and q a d data for 10 different
cyanine dyes in the presence of three different types of ds DNA
and CP,, data for the same dyes in the absence of ds DNA to
leam some of the answers to the following questions: (1) What
kinds of emission decay kinetics are present for these different
dyes on different types of ds DNA? (2) How do the emission
enhancements (or emission quantum yields) vary for the
different dyes on different types of ds DNA? (3) IS there
evidence that any of the DNA-bound dyes have their emission
lifetimes shortened due to ET quenching by DNA bases? (4)
Does changing from one dye to another when studying ds DNA
mean that very different emission characteristics and binding
modes will occur for the different dyes? (5) Are there important
differences in the emission properties of these dyes when they
are bound to long segments of ds DNA, as found for samples
of CT-DNA, versus when they are bound to much shorter
duplexes such as (dAdT),o and (dGdC)6? (6) Are there major
differences between monomeric and bichromophoric forms of
these dyes when they are bound to different types of ds DNA?
(7) How invariant is &ad among these different dyes? (8) What
free (or unbound) dye lifetimes are consistent with the measured
emission enhancements and calculated '&ad values?
Experimental Section
Materials. For redox studies six different cyanine dyes
(analogs 1-6 shown in Figures 1 and 2) analogous to the
Molecular Probes cyanine dyes studied were synthesized in the
Dye Research Laboratory of Eastman Kodak Co. Spectrograde
acetonitrile (CH3CN, Kodak Laboratory Chemicals) was dried
over 4 A molecular sieves. Tetrabutylammonium tetrafluo-
roborate (TBABF4, Kodak Laboratory Chemicals) was recrys-
tallized three times from ethanouwater, vacuum dried at 50 "C,
Netzel et al.
yield studies, commercial samples of the Molecular Probes
cyanine dyes were used as supplied by the manufacturer in either
200 pL or 1 mL vials with 1 mM dye dissolved in 1:4 DMSO:
water solutions. 18.2 MQ water for buffer solutions was
supplied by a Milli-Q water purification and deionization system
with a 0.22 p m final filter. The Molecular Probes cyanine dyes
were studied spectroscopically both as dicationic monomers and
as quadruply cationic bichromophores (see Figures 1 and 2).
Electrochemical studies were carried out only on the monoca-
tionic analogues of the Molecular Probes dyes.
DNA Duplex Preparation and Characterization. Calf
thymus DNA was purchased from Sigma Chemical Co. (St.
Louis, MO). Prior to use, the solid CT-DNA was hydrated in
phosphate buffer pH 7.0 (prepared as described below) for 3
days in a refrigerator. To examine the sequence dependence
of cyanine dye emission lifetimes, both (dAdT)lo and (dGdC)6
duplexes were synthesized at Operon, Inc., and purified with a
Pharmacia LCC-500 FPLC system using ProRPC 15 pm silica
reverse-phase c8 columns, HR10/10. Solution A was 10 mM
in triethyl ammonium acetate (TEAA) pH 7.0; solution B was
1:l by volume acetonitri1e:TEAA. The elution rate was 2.8
A h i n , and the 0-50% B gradient was run in 30 min.
(dAdT)lo oliogomers separated as a single peak as determined
by their absorbance at 254 nm, but (dGdC)6-oligomers were
isolated as two peaks. Reinjecting sample from each of the
two peaks again gave the same two peaks. dGdC self-com-
plementary base-pairing interactions were so strong that both
single-strand and duplex forms were in equilibrium under these
chromatographic conditions. In the middle of this work, solution
B was changed to 70% acetonitrile and 30% TEAA. With this
more strongly denaturing eluent, the (dGdC)6 oligomers also
separated as a single peak.
(dAdT)lo and (dGdC)6 duplexes were suspended in aqueous
phosphate buffer pH 7.0 with 0.1 M NaCl and 1 mM Na2EDTA
(disodium ethylenediaminetetraacetic acid) at 2-3 pM duplex
concentration. Under these conditions the T, of (dAdT)lo
duplexes was 48 "C and the melting curve (absorbance at 260
nm versus temperature) was well fit by a two-state thermody-
namic T, is the temperature at which 50% of a ds
DNA sample is melted into random coils or single strands (ss).
The T, for (dGdC)6 duplexes was higher than this. Similar
sample conditions as described for the T, experiments were
also used for the fluorescence lifetime measurements.
Preparation of Dydds DNA Samples for Emission Life-
time Measurements. The dye:DN&,,l,, concentration ratio
for (dAdT)lo and (dGdC)6 samples was ca. 1:l with ca. 3-5
p M dye; this corresponded to a dye:DNAbasepa,r (DNAb,) ratio
of 1:20 and 1:12, respectively. DNA molar extinction coef-
ficients at 260 nm, also respectively, were taken as equal to
6600 and 8400 M-I cm-I per nucleotide phosphate.36 The dye:
DNAb, ratio for CT-DNA samples was 1:10; the CT-DNA molar
extinction coefficient at 260 nm was taken to be 6600 M-' cm-I
per nucleotide pho~phate?~ DNA-staining experiments reported
in the literature have used dye:DNAb, ratios as large as 1:5
without adverse consequences.6 For POPO-1 and YOYO-1 on
(dAdT)lo duplexes, both 1:20 and 1:lO dye:DNAb, loadings
were examined, and the same distribution of emission lifetimes
was observed in each case. All dyeDNA samples were made
freshly each day in 7.5 mM phosphate buffer pH 7.0 with 0.1
M NaCl and 1 mM NaZEDTA and used for no more than two
lifetime measurements. When not in use for either deaeration
or spectroscopic measurements, the samples were stored on ice
in the dark or in a refrigerator. The usual protocol involved
mixing equal volumes of equal-concentration dye and ds DNA
Cyanine Dyes Bound to Double-Strand DNA
samples in plastic cuvettes, equilibrating the mixture for 90 min,
transferring the equilibrated mixture to quartz cuvettes, and
deaerating by bubbling with water-saturated argon for 20 min.
Finally, comparison of UV/vis absorbance spectra taken before
and after laser kinetics measurements showed only modest dye
absorbance losses. A number of samples were also allowed to
equilibrate in plastic cuvettes over night in the refrigerator. No
differences were found for the emission kinetics of these samples
versus similar samples equilibrated for 90 min.
Electrochemical Measurements. Electrochemical experi-
ments were performed using Princeton Applied Research Corp.
(PAR) 173 potentiostat in conjunction with PAR Models 175
universal programmer, PAR 179 digital coulometer, and 124
A lock-in amplifier. A Hewlett-Packard Model 239A low-
distortion oscillator was used in ac measurements. Formal
oxidation and reduction potentials for dyes were obtained via
phase-selective second-harmonic ac voltametry (quadrature
component) at an applied frequency of 400 Hz (16 mV peak-
to-peak signal) as described p r e v i ~ u s l y . ~Solutions
~ for ac
voltametric examination were prepared in CH3CN and contained
0.1 M TBABF4 and c a 5 x M dye. All solutions were
degassed with argon prior to use. The working electrode was
a Pt disk (ca 0.02 cm2) that was polished with 1 mm diamond
paste (Buhler Metadi), rinsed with water, and dried before each
experiment. Current-voltage curves were recorded on a
Hewlett-Packard Model 7045A X - Y recorder. All potentials
were measured versus the NaC1-saturated calomel electrode
(SCE) at 22 "C.
Absorbance and Fluorescence Quantum Yield Measure-
ments. Absorbance spectra were recorded on either an IBM
Instruments 9420 or a Perkin-Elmer Lambda-6 spectrophotom-
eter. Extinction coefficient measurements were made in
polystyrene cuvettes to counteract surface adsorption of the
polycationic cyanine dyes from aqueous solutions. Molar
extinction coefficients (cmax)for the free dyes were determined
by dilution of a DMSO (dimethyl sulfoxide) or DMF (dimeth-
ylformamide) stock solution with a gravimetrically determined
concentration into 10 mM TRIS, 50 mM NaCl, 1 mM EDTA,
pH 7.4 and recording the maximum absorbance. Serial dilutions
were used to confirm adherence to the Beer-Lambert law.
Extinction coefficients for the CT-DNA-bound dyes were
determined by addition to 2.5 mL of 4 pM dye solution in pH
7.4 TRIS to yield a molar ratio of approximately 50 CT-DNA
base pairs per dye molecule (the CT-DNA concentration was
calculated from the 260-nm absorbance, A260, of the stock
solution). The dye/CT-DNA complex was allowed to equili-
brate for at least 90 min before recording the absorbance
spectrum. The change in the maximum absorbance relative to
the aqueous free dye, after correction for dilution due to DNA
addition was converted to give Emax for the complex.
Fluorescence excitation and emission spectra for dye/CT-
DNA samples were recorded on either a PTI Alphascan or an
SLM-5OOC spectrofluorometer. Solutions for fluorescence
measurements typically contained 0.4 pM dye in 10 mM TRIS,
50 mM NaCl, 1 mM EDTA, pH 7.4 with the addition of 20
pM (base pairs) CT-DNA, equilibrated as above. Fluorescence
quantum yields (aem) for these samples were determined relative
to fluorescein 0.1 M NaOH (ae,,, = 0.92) by integration of
corrected emission spectra.38 Appropriately oriented polarizers
were used to eliminate the possible effects of non-isotropic
fluorescence from the dye/CT-DNA complexes. The fluores-
cence quantum yield enhancement on DNA binding was
determined by measuring and integrating the respective fluo-
rescence emission spectra with excitation at the isosbestic point
between the correspondingabsorbance spectra. Careful subtrac-
J. Phys. Chem., Vol. 99, No. 51, I995 17939
tion of background signals measured using a dye-free blank was
necessary for accurate quantitation of the very weak emission
for the free dye. In some cases, a titration was performed in
which the fluorescence of a 0.4 pM aqueous dye solution was
enhanced by seqential addition of very small amounts of DNA
(1-5 nM base pairs). The resulting linear increase in fluores-
cence was plotted and extrapolated to zero DNA concentration
to obtain a more accurate estimate of the fluorescence emission
of the free dye.
Fluorescence emission spectra and emission quantum yields
for dye/(dAdT)lo and dye/(dGdC)6 samples were recorded on
an SLM-8000C spectrofluorometer relative to the same dye on
CT-DNA. Solutions were made to have the same c a 0.05
absorbance at each dye's absorbance maximum aqd were excited
ca. 15-nm blue of the absorbance maximum with a 1-nm
bandwidth. As above appropriately oriented polarizers were
used to eliminate the possible effects of nonisotropic fluores-
cence. The emission spectra were then corrected for the
wavelength-dependent response of the spectrofluorometer,
converted to quanta units from intensity units, and integrated.
The relative emission quantum yields were calculated as
described by Demas and C r o ~ b y . Errors ~ ~ in the emission
quantum yield measurements are c a f 5 % for the dye/ds DNA
complexes and c a f 1 0 % for the free dyes. These percent errors
sum to produce c a f 1 5 % error in the emission enhancement
factors (see Table 4).
Fluorescence Lifetime Measurements. All fluorescence
decays were recorded on a Tektronix SCDlOOO transient
digitizer (50.35 ns rise time calculated from the bandwidth,
I 1 2 0 ps rise time for a step input 0.5 times the vertical range)
and wavelength resolved with a 0.1-m double monochromator
(Instruments SA, model DH10) in additive dispersion. 2-mm
slits were used producing an 8-nm bandpass. The 1200-grooves/
mm holographic gratings were blazed at 450 nm. After passing
through the monochromator, the emission was detected with a
Hamamatsu 1564U microchannel plate (200 ps rise time). The
excitation and emission beams were oriented at 90" with respect
to each other with the Glan-Thompson emission polarizer set
at 54.7" ("magic angle") with respect the vertical excitation-
polarization to eliminate rotational diffusion artifacts in the
emission lifetime measurement^.^^ Emission for all lifetime
measurements was excited at 355 nm with the third harmonic
of an active-passive mode-locked Nd3+NAG laser manufac-
tured by Continuum, Inc. Typically 35-pJ excitation pulses of
c a 25-ps duration were collimated into a 3-mm diameter beam
and passed through a second Glan-Thompson polarizer before
entering the sample cuvette. Photon Technology, Inc., software
was modified by the manufacturer to process 1000 data points
per decay curve and was used to deconvolute the instrument
response from the emission decay to yield exponential lifetime
fits to the emission decay data. Emission lifetime tests were
carried out on commercial samples of anthracene (Aldrich,
99+%) and 1-aminoanthracene (Aldrich, 99+%) which were
dissolved in cyclohexane and degassed in 0-ring-sealed optical
cells with three FPT cycles on a vacuum line (2 x Torr).
Recorded emission decays for these samples were fit with single
exponential lifetimes of 5.1 and 22.5 ns, respectively, for
anthracene and 1-aminoanthracene, These lifetimes agreed well
with their respective literature values of 4.9 and 22.8 ns4'
The overall temporal resolution of the emission kinetics
system is generally near 0.2 ns; however, in ideal circumstances
it can be as good as c a 50 ps after deconvolution. A detailed
description of the lifetime fitting procedure used here is
presented in a recent paper by Netzel et al.42 for nine sets of
17940 J. Phys. Chem., Vol. 99, No. 51, 1995
Emission Decay Kinetics for
POPO-1 Bound to (dAdT),, ."
looool M
*oool II \
I-Lam0 I
0 10 20 30 40 50
-8.00,....,....,....,....,....~
0 10 20 30 40 50
Time (ns)
Figure 3. Emission kinetics plots for a mixture of bichromophore
POPO-1 and (dAdT),o duplex, 1 2 0 dye:DNAb, ratio with 3.0pM dye,
in phosphate buffer pH 7.0 with 0.1 M NaCl and 1 mM NaZEDTA:
(top) plots of the experimental emission decay (decay), the 355-nm
excitation-pulse instrument response (lamp), and an iterative reconvo-
+
lution fit to the biexponential equation, A I exp(-f/rl) A2 exp(-r/rz)
+ A3, versus time (see Table 2 for the amplitude (An)and lifetime (r,J
parameters for this dye/ds DNA system); (bottom) plot of the difference
between the calculated fit and the experimental data divided by the
analog noise of the detection system versus time. The reduced chi-
square statistic, x:, for this fit is 1.4.
emission decays on four timescales (20, 50, 100, and 500 ns)
and includes the following: the equations used; plots of residual
differences between experimental emission decays and calcu-
lated multi-exponential curves; linear and logarithmic plots of
emission decays, lamp decays and exponential curves; as well
as specific x? values (the reduced chi-square statistic) for the
plotted exponential curves. Values of X? for emission lifetime
fits for the dyelds DNA complexes studied here generally ranged
from ca. 1-8. Figure 3 presents plots of emission decay
kinetics, an iterative reconvolution fit to this decay data, and
the residual differences for this fit for the POPO-1 bichro-
mophore bound to the (dAdT)lo duplex. The :X statistic for
this fit is 1.4.
In general, the accuracy to which a lifetime component can
be determined is proportional to the relative emission area of
that component. For that reason relative area data are presented
with the lifetime values. On the other hand, relative emission
amplitudes are proportional to the number of emitting species
with the corresponding lifetime; thus these data are also given.
The combination of finite detector response time and small
relative emission areas (1-3%) for a number of the subnano-
second lifetime components presented in this work causes such
values to be highly uncertain. Emission lifetime components
2 1 ns generally also have significant relative emission areas
and are consequently much more reliable. However, whenever
two emission lifetimes are less than a factor of 2 different for
either bi- or triexponential decay processes, relative amplitude
errors of f 2 0 % and lifetime errors of f10-20% are
Typical errors for emission lifetimes that can be fit with only a
single exponential are 2-4% for lifetimes 5 10 ns and 1-2%
for lifetimes greater than 10 ns.
Emission kinetics were analyzed with the following criteria
in mind: (1) reproducibility of a given measurement and (2)
Netzel et al.
TABLE 1: Redox Potentials and Electronic Absorbance
Maxima of Cyanine Dyes
dye analog
1
2
i,,,"E0(dyeq2+/dye+),V
(nm)
415b
445
1.10b
1.04
E"(dye+/dye'), V
- 1.70b
- 1.so
3 416 1.14 -1.30
4 502 1.07 -1.23
5 520 0.68 -1.39
6 592 0.12 - 1.08
a In MeOH. Estimated value.
consistency of lifetime components found by fitting data from
several time ranges. In addition to these criteria, Ockham's
razor was used to demand a significant improvement in x?
before an additional lifetime component was added. Generally,
this was at least a 0.5-1.0 lowering of x?. We did, however,
find many times that the number of required lifetimes was
robust. That is, an attempt to add another lifetime just repeated
a previous lifetime component or an attempt to remove a lifetime
component gave a very much larger X? value.
The general fitting procedure began by insuring that each
emission lifetime was recorded in a sufficiently wide time
window so that its emission decayed into the noise (typically
f 4 - 8 counts compared to 10 000-12 000 counts in the signal's
peak after background subtraction). These decay data were fit
first, and their longest lifetime component was then used as a
fixed lifetime in fits of data taken on finer time scales with
narrower time windows. The finer time scales allowed better
resolution of the faster decay components. The finest time scale
used in this work was 20 ns, corresponding to one time point
every 20 ps. Each kinetics trace on each time scale recorded
1000 data points; data analyses used all 1000 points; and all
data curves which were fit were themselves the result of
averaging 1000 photoexcitation events as well as 1000 back-
ground events and subtracting the latter from the former.
Results and Discussion
Redox Potentials for Six Unsymmetical Cyanine Dyes.
Table 1 presents measured and estimated redox values for six
monocationic cyanine-dye analogues (dye+) of the Molecular
Probes DNA-binding dyes. One-electron reduction potentials
E"(dyeo2+/dye+)and E"(dye+/dye') were measured at a Pt
electrode in CH3CN/TBABF4 by the method of phase-sensitive
second-harmonic ac ~ o l t a m e t r y . ~ ~
Free Energies for Excited-State Electron Transfer Quench-
ing. The above electrochemical data on the oxidation and
reduction of cyanine dyes can be combined with similar redox
data for nucleosides and the energies of the first excited single
states, Eo,o(dye*),of the Molecular Probes ds DNA-binding dyes
to estimate the free energies for excited-state electron-transfer
quenching, AG"(ET), of the ds DNA-bound dyes according to
eq 2,44whereE" is a reduction potential, D is an electron donor,
A is an electron acceptor, and w(r) is a coulombic interaction
term between oxidized donor and reduced acceptor which
represents free energy due to separating the ionic products a
distance r relative to each other, w(m) = 0.45,46Generally in
very polar media the coulombic term is less than ca 0.1 eV
and will be neglected here.45,47-50
Depending on the redox properties of a given nucleoside, a
ds DNA-bound cyanine dye can be either reduced or oxidized
when in its lowest energy excited state. The easiest nucleoside
Cyanine Dyes Bound to Double-Strand DNA J. Phys. Chem., Vol. 99,No. 51, 1995 17941
TABLE 2: Cyanine Bichromophore Excited State Energieg To the extent electronic couplings and reorganization
and ET Quenching Free Energid energies47,48.54-58do not vary, AGO values from Table 2 should
cyanine Eo,o(dye"+*), AG'(dG'+/ AGo(dye*f(n+l)/dC(H') provide reasonable estimates of the relative ET quenching rates
dye eV dye'+'""), eVc or dT/dT(H)'), eVC for the 10 dyes studied here. Analysis of this data yields four
POPO- 1 2.79 -0.26 -0.24 predictions of ET quenching rate differences for different sets
BOBO-1 2.63 -0.30 -0.14 of dyes. (1) Quenching rates for monomethine cyanine dyes
YOYO-1 2.48 -0.35 +0.11 bound to (dGdC)6 duplexes should exceed those of the trime-
TOTO- 1 2.37 -0.31 +0.15 thine dyes on the same duplexes. (2) Quenching rates for
POPO-3 2.25 -0.03 -0.12
YOYO-3 2.00 -0.09 4-0.17 monomethine dyes on (dGdC)6 duplexes should exceed those
of the same dyes on (dAdT)lo duplexes. (3) ET quenching for
The excited-state energies of the monomeric cyanine dyes are nearly three,of the cyanine dyes, YOYO-1, TOTO-1, and YOYO-3,
identical to those of their corresponding bichromophoric forms. In
the case of ET between dG and dye"+*, the products are dG'+ and is not spontaneous in (dAdT)lo duplexes. (4)For the case of
dye'+"+'); for ET between dC or dT and dye"+*, the nucleoside is YOYO-1 and TOTO-1, ET quenching on (dGdC)6 duplexes is
reduced (and most likely protonated) and the dye is oxidized, dye'+'"+'). very favorable, AGO = -0.35 and -0.31 eV, respectively, while
For monomeric dyes n = 2 and for bichromophoric dyes n = 4. dG, on (dAdT)lo duplexes it is not likely to occur, AGO = +0.11
dC, and dT are respectively, 2'-deoxyguanosine, 2'-deoxycytidine, and and +0.15 eV, respectively.
2'-deoxy thymidine,
The assumption that electronic couplings and reorganization
to oxidize is guanosine with a reduction potential for guanosine energies are similar for a series of similar D/A molecules held
monophosphate cation (GMP+/GMP) estimated to be 0.83 V together by similar electrostatic and hydrophobic interactions
(versus SCE).3',32.5' The easiest nucleosides to reduce are as is the case here is reasonable. However, whether or not
thymidine and cytidine with similar reduction potentials of possible ET quenching processes are in fact likely to be observed
-1.45 V (versus SCE).31.32%51 However, because of the ready for any of the cyanine dye/ds DNA complexes is not known
protonation of reduced cytosine in a DNA duplex by its base- due to lack of information on the complexes' geometries,
paired partner guanosine, it is estimated that in ds DNA electronic couplings, and reorganization e n e r g i e ~ . ~ It
~ ,is~ ~ - ~ ~
protonated reduced cytosine, dC(H)', is c a 200 mV easier to known that YO-PRO-1 and YOYO-1 are, respectively, mono-
form than is dT-.3'932 Contrary to this opinion, bimolecular and bisintercalators in ds DNA.33 This makes it very likely
ET quenching of photoexcited tetrahydroxytetrahydrobenzo[a]- that all of the quinoline dyes, YO and TO types, are also
pyrene (BPT*) by dC and dT is found to require a polar protic intercalators. To a first approximation, the deactivating torsion
solvent, because it does not occur in the polar organic solvent about the central methine bridge is likely to be more restricted
DMSO.52 This latter result implies that in water the products for intercalated dyes than for surface-associatedor groove-bound
of the reductions of dC and dT are already the protonated dyes. With this in mind, it is interesting to see whether the
reduced species, respectively, dC(H)' and dT(H)'. If this is true, emission lifetimes are significantly longer for the YO and TO
the reduction potential of dC will be the same in water and in types of dyes than for the PO and BO types. These latter dyes
ds DNA.53 These nucleoside redox values are combined in Table contain smaller pyridinium aromatic rings in place of the larger
2 with the excited state energies of ds DNA-bound cyanine dyes quinolinium ones in the former dyes.
to yield estimates of the free energies of dye*/ds DNA ET Cyanine Bichromophore Fluorescence Lifetimes When
quenching processes. Bound to ds DNA. Fluorescence lifetimes are measured for

TABLE 3: Fluorescence Lifetimes (nanoseconds) of Bichromophore Dyes Bound to DNA Duplexee

duplex POPO- 1 BOBO-1 YOYO-1 TOTO- 1
(dAdT)io [0.31] 0.43 (5%) [OS91 0.21 (15%) [0.41] 0.21 (6%) [OS11 0.17 (9%)
[0.69] 3.68 (95%) [0.36] 1.40 (59%) [0.35] 1.33 (31%) [0.33] 1.39 (44%)
[0.05]4.22 (26%) [0.24] 3.85 (63%) [0.16] 2.98 (47%)
av t 2.67 0.84 1.48 1.02
(dGdC)6 [0.39] 0.42 (7%) [0.60] 0.63 (32%) [0.24] 1.06 (6%) [0.41] 0.21 (4%)
[0.61] 3.43 (93%) [0.40] 2.05 (68%) [0.76] 5.08 (94%) [0.29] 2.35 (34%)
[0.30] 4.20 (62%)
av t 2.26 1.12 4.12 2.03
CT-DNA [OS81 0.19 (7%) [0.57] 0.16 (12%) [0.42] 0.22 (4%) [0.35] 0.33 (6%)
[0.24] 2.75 (41%) [0.33] 1.18 (51%) [0.40] 2.89 (56%) [0.18] 1.74 (16%)
[O. 181 4.80 (52%) [0.10] 2.80 (37%) [0.18] 4.53 (40%) [0.47] 3.25 (78%)
av t 1.63 0.76 2.06 1.96
duplex POPO-3 YOYO-3
(dAdT)io C0.6710.48 (23%) [0.65] 0.45 (30%)
[0.33] 3.34 (77%) [0.35] 1.98 (70%)
av t 1.42 0.98
(dGdCh [0.77] 0.42 (25%) [0.74] 0.46 (35%)
[0.23] 4.10 (75%) [0.26] 2.45 (65%)
av t 1.27 0.97
CT-DNA [0.41] 0.56 (11%) [0.51] 0.090 (6%)
[OS91 3.25 (89%) [0.26] 0.83 (27%)
[0.23] 2.27 (67%)
av t 2.15 0.78
a Relative amplitudes in the emission decay fits are given as decimal fractions in brackets before the lifetimes; relative emission areas are given
as percentages in parentheses after the lifetimes. Average emission lifetimes are calculated as the sum of the products of the emission lifetimes and
their corresponding relative amplitudes.
17942 J. Phys. Chem., Vol. 99, No. 51, 1995
six cyanine dyes as bichromophores and four cyanine dyes as
monomers when bound to (dAdT)lo, (dGdC)6, and CT-DNA
duplexes. The monomeric dyes each have two positive charges
favoring strong binding to negatively charged DNA duplexes,
association constants c a lo6M-1.27 The bichromophoric dyes
consist of two monomeric dyes of the same type joined either
at their pyridinium or quinolinium nitrogens (see Figures 1 and
2) with 11-atom linking chains, -(CH2)3-Nf(CH3)2-(CH2)3-
N+(CH&-(CH2)3-; these quadruply cationic dyes bind ds
DNA c a 100-fold more strongly than the corresponding
monomeric dyes.27 Table 3 lists the fluorescence lifetimes for
six such bichromophores. To compare the dyes on different
types of ds DNA, average lifetimes are calculated by summing
the products of the emission lifetimes and their corresponding
relative emission amplitudes for each dye/ds DNA system.
Several observations can be made about the lifetimes in Table
3. First, all of the bichromophores show either bi- or triexpo-
nential decay kinetics reflecting multiple modes of bichro-
mophore/ds DNA association for each dye/ds DNA system.
Second for each of the three types of ds DNA, the thiazole form
of a bichromophore on a given type of duplex has a shorter
average emission lifetime than does the corresponding oxazole
form. Sometimes, as for BOBO-1 and POPO-1, the lifetime
differences are striking.
A third observation is that there is no correlation of the length
of a dye’s emission lifetime with the AGO values in Table 2 for
various ET quenching reactions involving DNA bases (shorter
lifetimes should correspond to more negative AGO values). To
focus this point, one can refer specifically to the four predictions
noted above concerning differences in ET quenching rates for
different groups of dyes. (1) Quenching rates for monomethine
cyanine dyes bound to the (dGdC)6 duplex do not exceed those
of the trimethine dyes on the same duplex. (2) Quenching rates
for monomethine dyes on (dGdC)6 do not exceed those of the
same dyes on (dAdT)lo. (3) The three cyanine dyes, YOYO-
1, TOTO-1, and YOYO-3, for which ET quenching is not
spontaneous on (dAdT)lo have very short average emission
lifetimes on this duplex. (4) For the case of YOYO-1 and
TOTO-1 for which ET quenching is very favorable on (dGdC)6
and unfavorable on (dAdT)lo, the average emission lifetimes
on the former are c a 2-fold longer than on the latter.
A fourth observation is that three of the monomethine
bichromophores (all except POPO- 1) have shorter average
emission lifetimes on (dAdT)lo than on (dGdC)6; the increase
in average lifetime on switching from (dAdT)lo to (dGdC)6 is
particularly large for the quinolinium dyes, YOYO-1 and TOTO-
1. The two trimethine dyes have similar average emission
lifetimes on both of the short duplexes. Importantly, all of the
average emission lifetimes of the bichromophoric dyes on CT-
DNA are in the same range as those of the corresponding dyes
on the short duplexes. However, the average lifetimes on CT-
DNA do not appear to be related simply to either of the
corresponding average lifetimes on the short duplexes.
Bichromophore Aggregation Effects. In methanol the
POPO- 1 and BOBO- 1 bichromophores showed similar UV/vis
spectra consisting of featureless, asymmetric absorbance bands,
respectively, at 423 and 452 nm. Also in methanol YOYO-1
and TOTO-1 showed similar UV/vis spectra consisting of
asymmetric absorbance bands, respectively, at 48 1 and 507 nm,
but with weak vibronic shoulders on their blue edges. POPO-
1, BOBO-1, and TOTO-1 were also examined in pH 7.0
phosphate buffer solution in the 1- 16 pM concentration range.
In contrast to their spectra in methanol, they each showed two
overlapping absorbance bands with the higher energy one 20-
40% stronger than the lower energy one. Equally interesting
Netzel et al.
Normalized Absorbance Spectra
For TOTO-1
i
400 440 480 520 560 600
Wavelength (nm)
Figure 4. Normalized absorbance spectra for TOTO-1 in four different
solutions: (1) methanol (MeOH), 1.9 p M dye; (2) phosphate buffer as
in Figure 3, 7.4 p M dye; (3) 1:20 dye:DNAb, mixture with (dAdT)lo
duplex in phosphate buffer, 2.5 p M dye; (4) 1:12 dye:DNAb, mixture
with (dGdC)6 duplex in phosphate buffer, 2.5 p M dye.
Normalized Absorbance Spectra
For TO-PRO.1
0 0.80 -
3
-e
2 0.60 -
n
<
400 440 480 520 560 600
Wavelength (nm)
Figure 5. Normalized absorbance spectra for TO-PRO-I in MeOH
and aqueous phosphate buffer pH 7.0 (made as described in Figure 3),
3.0 p M dye in each solution.
was the observation that the absorbances of both of these bands
varied linearly with concentration. Figure 4 shows normalized
absorbance spectra for TOTO-1 in methanol, in buffer, and
bound to (dAdT)lo and (dGdC)6 duplexes in buffer. Figure 5
shows normalized absorbance spectra for TO-PRO-1, the
corresponding monomeric dye (see Figure l), in methanol and
in buffer. These data support the conclusions that in buffer
solution (1) cyanine bichromophores self-associate to form
intramolecular dimers (referred to here as “dimers”) and (2)
intermolecular aggregates of cyanine dimers are not present at
concentrationsless than 16 pM.67368Previous work on YOYO-1
in aqueous buffer has similarly concluded that this bichro-
mophore also forms internal dimers in buffer solution.28 There
the altered absorbance spectrum of YOYO-1 in buffer versus
in alcohol was ascribed to exciton coupling between the dimer’s
chromophores. Additionally, the absorbance spectrum of
YOYO-1 in buffer was shown to depend on the concentration
of added salt, consistent with a salt-dependent shift in the
internal-dimedopen-configuration equilibrium.28
A related observation is that the absorbance spectra of the
six cyanine bichromophores studied here have nearly identical
bandshapes whether bound to CT-DNA, (dAdT)lo,or (dGdC)6
duplexes (see Figure 4). Also the band shapes for these
bichromophore/ds DNA complexes look like red-shifted mon-
Cyanine Dyes Bound to Double-Strand DNA J. Phys. Chem., Vol. 99, No. 51, 1995 17943
TABLE 4: Photophysical Properties of DNA-Binding Bichromophoric Cyanine Dyes
Cyanine BichromoDhores Bound to DNA Duplexes
~~~ ~~
0.70 1
bichromophore POPO- 1 BOBO- 1 YOYO- 1 TOTO- 1
@eml(dAdTjlola
@‘em[(dGdCj6?
@.em(CT-DNA?
0.56
0.44
0.60
0.14
0.19
0.22
0.43
0.64
0.52
0.16
0.39
0.34
./
enhancement 800 970 460 1400
(CT-DNAY
rrad(CT-DNA),d ns 2.7 3.5 4.0 5.7
Qem(dimer)’ 7.5 x 10-4 2.3 x 10-4 11 x 10-4 2.4 x 10-4
c,,,(dimer)f 122700 130700 109900 100700
c,a,(CT-DNA)g 92 400 113 600 98 900 117 OOO
bichromophore POPO-3 YOYO-3 0.001 /
2.0
0.0 3.0 4.0
1.0 5.0
@em[(dAdT)lo]’ 0.5 1 0.18 Lifetime (ns)
Qem[(dGdC)# 0.38 0.16
@.,,(CT-DNA)b 0.46 0.15 Figure 6. Plot of emission quantum yield versus average emission
enhancement 190 460 lifetime for 16 bichromophoric dye/ds DNA systems (solid circles).
(CT-DNAY The solid line represents a least-squares fit of the plotted data to eq 1;
rrad(CT-DNA),dns4.7 5.2 the correlation coefficient (R) of the fit is 0.88, and the reciprocal of
@.,,(dimer)’ 24 x 10-4 3.3 x 10-4 the slope is 5.1 ns.
c,,,(dimer)f 129 900 h
E,,,(CT-DNA)~ 146 400 167 000 re~pectively.~ The value reported here for TOTO-1 of 1400 is
in reasonable agreement with this report. However, there is a
Emission quantum yield (&5%)determined relative to the same large discrepancy between the 460 result here for YOYO-1 and
bichromophore on CT-DNA. bEmission quantum yield (f5%) of a
bichromophore when bound to CT-DNA determined relative to that of the earlier report. This discrepancy shows the difficulty
fluorescein in 0.1 M NaOH (aem = 0.92). Ratio ( f l 5 % ) of the of accurately determining the relative quantum yields of the
emission quantum yield in aqueous buffer pH 7.4 of a bichromophore free dyes. Because their emissions are very weak, an extrapola-
when bound to CT-DNA to that when it is a free dimer in the same tion procedure (see above) was used where necessary in these
solution. Radiative lifetime on CT-DNA equal to the average t(CT- studies.
DNA) from Table 3 divided by @,,(CT-DNA) in this table. e Qem(CT- The quantum yield data in Table 4 confirm a previous
DNA) divided by enhancement (CT-DNA).f Molar extinction coeffi- conclusion based on average emission lifetimes; namely, for a
cient (M-’ cm-’) at the absorbance maximum of the dye in 10 mM
Tris buffer with 1 mM EDTA and 50 mM NaCl at pH 7.4. g Same given type of ds DNA, monomethine thiazole dyes are poorer
buffer conditions as in footnote f but with the addition of CT-DNA emitters than the corresponding oxazole dyes. Similarly the
with a molar dye:DNAbp ratio of 1:50 or smaller. Value not pyridinium dye YOYO-3 has both lower emission quantum
determinable due to dye aggregation in buffer solution. yields and lower average emission lifetimes on the three types
of ds DNA than does the quinolinium dye POPO-3. In general,
omeric absorbances as seen in methanol and do not show similar correlated variations of emission quantum yield and
double-banded structure as do the dimers in buffer. Thus the average emission lifetime are found as ds DNA is varied for a
UV/vis spectra of the bichromophore/ds DNA complexes are given dye or dye is varied for a given ds DNA duplex. A way
consistent with each end of the bichromophore binding sepa- of testing the consistency of the emission data in Tables 3 and
rately to the DNA duplex. Thus the physical and spectroscopic 4 is to plot measured quantum yields versus average lifetimes
properties of cyanine bichromophores bound to ds DNA should as in done in Figure 6 and fit this data to eq 1. The correlation
be similar to those of the corresponding monomeric cyanine coefficient (R) for the fit is 0.88, and the resulting radiative
dyes. These conclusions agree with the report that YOYO-1 lifetime is 5.1 ns (inverse slope). In Figure 6 data from 16 out
binds as bisintercalator to linear coliphage T2 DNA (164 kbp of 18 possible dye/ds DNA systems are used. Data for POPO-1
ds DNA) at dye:DNAb, ratios as large as 1:4.33 At higher dye on CT-DNA (see Table 4) and POPO-3 on (dAdT)lo were not
loadings (larger dye:DNAb, ratios) extemal binding becomes used because they give uncharacteristically low radiative rates
n ~ t i c e a b l e .In
~ ~this work, dye:DNAbpratios were carefully kept of 2.7 and 2.8 ns, respectively. The t-statistical analysis at the
no larger than 1:10. 99% confidence level for the same 16 dye/ds DNA systems
Emission Quantum Yields, Emission Enhancements, and plotted in Figure 6 gives a mean radiative lifetime of 5.1 f.0.8
Radiative Lifetimes for Bichromophoric Cyanine Dyes ns.
Bound to ds DNA. Emission quantum yields are presented in Since bichromophores bound to ds DNA act as linked pairs
Table 4 for the six bichromophoresdiscussed above when bound of independent monomeric dyes, one can compare monomeric
to (dAdT),o, (dGdC)6, and CT-DNA duplexes as well as when
free in buffer solution. For all three types of ds DNA, the bound
bichromophores fluoresced much more strongly than did the
-
and bichromophoric radiative rates. Integration of the area under
the So SIabsorbance band for YO-PRO-1 on CT-DNA was
reported to yield a theoretical radiative lifetime of 5 ns.28
free dimers. From a broad perspective, the quantum yields for Similarly, use of eq 1 and measurement of z and a,, for this
each dye on all three types of ds DNA are similar. The dye on CT-DNA yielded a radiative lifetime of 6.0 m2*Given
exception is TOTO-1 where the quantum yield on (dAdT)lo is the difficulties of accurately determining both lifetimes and
about one-half that on the other two duplexes. The average relative emission amplitudes for multiexponential decay pro-
emission lifetime data for this dye in Table 3 c o n f m this result. c e s ~ e s ?the
~ intemal consistency of the data in Tables 3 and 4
From a detailed perspective, the emission quantum yield of a and the agreement between radiative lifetimes measured here
given dye can generally be 30-70% higher on some forms of and those determined elsewhere is gratifying.
ds DNA than on others. Importantly as will be pointed out Fluorescence Lifetimes of Monomeric Cyanine Dyes When
later, there is no clear dominance of one of the three types of Bound to ds DNA. Table 5 lists fluorescence lifetimes for four
ds DNA for producing the greatest emission quantum yield. monomeric monomethine cyanine dyes, PO-PRO-1, BO-PRO-
There is a prior report of emission enhancementsfor YOYO-1 1, YO-PRO-1, and TO-PRO-1, when bound to (dAdT)lo,
and TOTO-1 when bound to CT-DNA of 3200 and 1100, (dGdC)6, and CT-DNA duplexes. Each of these monomeric
17944 J. Phys. Chem., Vol. 99, No. 51, 1995 Netzel et al.

TABLE 5: Fluorescence Lifetimes (nanoseconds) of Monomeric Dves Bound to DNA Duulexee

~

duplex PO-PRO- 1 BO-PRO-1 YO-PRO- 1 TO-PRO- 1

(dAdT)io [0.46] 0.21 (2%) [0.41] 0.44 (7%) [0.44] 0.66 (14%) [0.34] 0.41 (1 1%)
[0.54] 3.65 (98%) [0.59] 2.50 (93%) [0.56] 3.34 (86%) [0.66] 1.77 (89%)
av t 2.05 1.66 2.16 1.31
(dGdC)6 [0.52] 0.09 (3%) [0.61] 0.17 (20%) [0.32] 1S O (16%) [0.40] 1.36 (24%)
[0.48] 2.85 (97%) [0.39] 1.09 (80%) [0.68] 3.99 (848) [0.60] 3.02 (76%)
av t 1.43 0.53 3.19 2.36
CT-DNA C0.311 0.36 (4%) [0.36] 0.11 ( 5 % ) [0.26] 0.29 (4%) [0.44] 0.23 (8%)
[0.69] 3.42 (96%) [0.28] 0.66 (20%) [0.25] 1.74 (19%) [0.27] 1.57 (32%)
[0.36] 1.92 (75%) [0.49] 3.49 (77%) [0.29] 2.76 (60%)
av t 2.47 0.92 2.22 1.33
a The same layout conventions for the data are used as in Table 3.

dyes has two positive charges favoring binding to negatively TABLE 6: Photophysical Properties of DNA-Binding
charged DNA duplexes. Cyanine Monomeric Dye#
A phase-modulation based emission lifetime measurement for monomeric dye PO-PRO-1 BO-PRO-1 YO-PRO- 1 TO-PRO-1
YO-PRO-I bound to CT-DNA has been reported in a different @m[(dAdT)~ol 0.36 0.16 0.28 0.13
buffer system from that used here.28 There a 50 mh4 Tris, 50 @,,[(dGdC)s] 0.093 0.037 0.41 0.23
mM boric acid, and 1.25 mh4 Na2EDTA buffer was used, while @em(CT-DNA) 0.39 0.16 0.44 0.25
in Table 5 a 7.5 mh4 phosphate buffer pH 7.0, 1 mM Na2EDTA, enhancement 460 890 920 1800
and 100 mh4 NaCl buffer was used. Addition of salt increases (CT-DNA)
r,,d(CT-DNA), ns 6.3 5.8 5 .O 5.3
duplex stability which is a consideration for the short duplexes @,,(free dye) 8.5 x 1.8 x 4.8 x 10-4 1.4 x 10-4
studied here but can also reduce the affinity of cyanine dyes t(free dye),b ps 5 1 2 0.7
for ds DNA. Nevertheless, the published biexponential emission cmax(freedye) 75 500 76 200 66 000 68 500
lifetimes of 1.65 and 3.63 ns (each component having 50% €ma,(CT-DNA) 50 100 58 100 52 000 62 800
relative amplitude) agree well with the 1.74 and 3.49 ns a See Table 4 for definitions of the photophysical properties listed
components for this dye bound to CT-DNA in Table 5. The here. Lifetime of the free dye in buffer solution equal to the average
major difference between this work and that reported in the lifetime on CT-DNA divided by enhancement(CT-DNA), assumes
literature is that here a better fit to the emission decay is s,,d(CT-DNA) = trad(freedye).
produced with a triexponential rather than a biexponential
function. This has the effect of reducing the average emission aggregation in the 1- 12 pM concentration range. Additionally
lifetime in this work to 2.2 ns compared to 2.6 ns in the each of these dyes obeyed the Beer-Lambert law precisely in
literature; the corresponding radiative lifetimes for these two phosphate buffer solution in this same concentration range. Thus
cases are then 5.0 and 6.0 ns, respectively. Biexponential comparison of emission properties between DNA-bound and
functions provide good fits to the emission decay data for this free dye in buffer is not complicated for these monomeric dyes
dye for the two short duplexes; also, the two lifetimes found by exciton splitting of their lowest-electronic ground-state
for this dye bound to the (dGdC)6 duplex,l.50 and 3.99 ns, are absorbance bands as is the case for the bichromophoric dyes.28
similar to those found in the phase-modulation experiment. The absence of aggregation effects for the monomers in buffer
As is the case for the bichromophoric dyes, all of the means that the paucity of their emission when free in solution
monomeric cyanine dyes in Table 5 show bi- and triexponential is an intrinsic photophysical property.
decay kinetics reflecting multiple modes of dyeDNA association Table 6 shows a similar correlation of the variation of average
for each dye/ds DNA system. Similarly, the same pattem is emission lifetime and emission quantum yield for the monomeric
present among the average lifetimes of the monomers as is also dyes bound to ds DNA as for the bichromophoric dyes bound
present for the bichromophores; namely, for a given dye and to ds DNA. In particular, the thiazole containing monomers,
duplex the thiazole form of the dye has a shorter average BO-PRO-1 and TO-PRO-1, show a large emission quantum
emission lifetime than does the corresponding oxazole form. yield change in opposite directions as did their average lifetimes
Also for these monomers as for the bichromophores, there is on switching from (dAdT)lo to (dGdC)6 duplexes, respectively,
no correlation of length of emission lifetime with estimated AGO a 77% decrease and a 77% increase. Also, the thiazole dyes,
for possible ET fluorescence quenching reactions with DNA BO-PRO-I and TO-PRO-I have lower emission quantum yields
bases. Finally, the two thiazole monomers, BO-PRO-1 and TO- on all three types of ds DNA than do the corresponding oxazole
PRO-1, show large average emission lifetime differences on dyes, PO-PRO-I and YO-PRO-I. Importantly the pyridinium
switching from (dAdT)lo to (dGdC)c duplexes, but in the and quinolinium monomers in Table 6 show distinct differences
opposite direction from each other, respectively, a 68% decrease in emission yields for the two short duplexes: the pyridinium
and an 80% increase, dyes (PO-PRO-1 and BO-PRO-1) have much higher yields on
Fluorescence Quantum Yields, Emission Enhancements, (dAdT)lo duplexes, and the quinolinium dyes (YO-PRO-1 and
Radiative Lifetimes, and Free Monomer Lifetimes. Table 6 TO-PRO1) have higher yields on (dGdC)6 ones. In fact the
presents fluorescence quantum yields and emission enhance- above noted large quantum yield change for BO-PRO-1 and
ments for the four monomethine monomers whose lifetimes TO-PRO-1 on switching from (dAdT)lo to (dGdC)6 duplexes
were presented in Table 5. In all cases the ds DNA-bound can now be associated with the fact that these two dyes are in
monomeric dyes fluoresced much more strongly than did the different chromophore classes. The average emission lifetimes
free monomers with emission enhancements ranging from 500 for the monomeric dyes in Table 5 show a similar but less
to 2000. The absorbance spectra of each of these four dyes, pronounced propensity for the pyridinium dyes to have longer
PO-PRO-1, BO-PRO-1, YO-PRO-1, and TO-PRO-1, were also average lifetimes on (dAdT)lo duplexes and the quinolinium
compared in MeOH and phosphate buffer solutions. Both dyes to have longer average lifetimes on the (dGdC)6 ones.
solutions gave very similar spectra without indication of Aspects of this same pattem of pyridiniudquinolinium duplex
Cyanine Dyes Bound to Double-Strand DNA
specificity for enhanced emission are also present in the quantum
yield data for the bichromophores in Table 4. For the
pyridinium dyes, there is a muted preference for (dAdT)lo
duplexes on the part of POPO-1 and POPO-3. For BOBO-1
this is not true, but all of the quantum yield values for this dye
are low and much the same on all three types of ds DNA. For
the quinolinhm dyes, a clear preference for (dGdC)6 duplexes
on the part of YOYO-1 and TOTO-1 is present. This is not
true of YOYO-3 whose quantum yields are low and also much
the same on all three types of ds DNA.
The average lifetime data from Table 5 can be combined with
the emission quantum yield data in Table 6 to produce radiative
lifetimes for the monomeric dyes on ds DNA. These values
for CT-DNA are explicitly shown in Table 6. The average
radiative lifetime for the four monomers bound to CT-DNA is
5.6 ns. Statistical analysis (upper and lower limit t-statistics)
shows the errors of this average value to be f l . O and f 1 . 9 ns,
respectively, at the 95 and 99% confidence levels. This result
agrees very well with that found earlier for the bichromophores
on all three types of ds DNA, 5.1 f 0.8 ns, and with those
published in the literature for YO-PRO-1 bound to CT-DNA,
5 ns (theoretical) and 6.0 ns (experimental).
Since the radiative rates of the monomeric dyes are expected
to be the same whether they are free in buffer solution or bound
to ds DNA, and the emission enhancement is the ratio of the
emission quantum yields for a dye bound to ds DNA to that
when it is free in solution, it can be shown using eq 1 that an
estimate of the lifetime of a free monomeric dye in buffer,
z(free dye), can be obtained by dividing its average emission
lifetime when bound to CT-DNA by its emission enhancement
factor. These values are presented in Table 6 and range from
1 to 5 ps. This means that the emission detection system used
here is largely blind to free dye emission. Additionally, while
full intercalation can restrict torsion about a cyanine dye’s central
methine bridge sufficiently to produce dye*/ds DNA complexes
with lifetimes as long as 3-5 ns (see especially the data for
YO-PRO-1 and YOYO-1 bound to CT-DNA in Tables 3 and
5 ) , a wide range of lifetimes between those of a free dye and
those of a fully intercalated dye are possible for cyanine dye*/
ds DNA complexes. Intermediate emission lifetimes between
these two extremes likely correspond to varying degrees of
restricted torsion about the central methine bridge. This
situation can prove experimentally difficult if a dye upon binding
to ds DNA produces a significant fraction of dyelds DNA
conformers with ultrashort (less than 0.2 ns) lifetimes. In such
a case the detection equipment used here would underestimate
the relative amplitude of the ultrashort component and as a result
overestimate the dye’s average emission lifetime. This appears
to be the case for PO-PRO-1 and BO-PRO-1 bound to (dGdC)6
duplexes, because their calculated radiative lifetimes are,
respectively, 15.4 and 14.3 ns.
In view of the good agreement between experimental radiative
rates for bichromophoric dyes on all ds DNA and for monomeric
dyes on CT-DNA, it is surprising to find poor agreement with
these values for the radiative rates of monomeric dyes on short
duplexes. Using the data in Tables 5 and 6, radiative rates for
the monomers on the short duplexes are found to range from
5.7 to 15.4 ns. Removing the two very high values noted above
from further consideration, leaves 6 dyelds DNA systems in
this class and yields an average radiative rate of 8.6 ns.
Statistical analysis (upper and lower limit t-statistics) shows the
errors for this estimate of the average radiative lifetime to be
f 2 . 2 and f 3 . 4 ns, respectively, at the 95 and 99% confidence
levels. One result is immediately apparent: the radiative
lifetime data for the monomers on short duplexes is much more
J. Phys. Chem., Vol. 99, No. 51, 1995 17945
Monomeric Cyanine Dyes
Bound to DNA Duplexes
0.50 7 / /
4
.a o.loy,*,
, T I , , ,
Short Duplexes
0.00
0.0 1 .o 2.0 3.0 4.0
Lifetime (ns)
Figure 7. Plot of emission quantum yield versus average emission
lifetime for 10 monomeric dye/ds DNA systems presented as two sets
of data: (open circles) CT-DNA and (solid circles) short duplexes. The
two solid lines represent least-squares fits to each group of data. The
correlation coefficients (R) for the two sets of data are 0.95 (open
circles) and 0.83 (solid circles); the reciprocals of the slopes are 5.6 ns
(open circles) and 8.0 ns (solid circles).
scattered than that for bichromophores on all ds DNA and for
monomers on CT-DNA (see Table 6). Use of single-limit
t-statistics at the 99% confidence level shows that the highest
average radiative lifetime for the bichromophores is 5.8 ns and
the lowest average radiative lifetime for the monomers on short
duplexes in 6.0 ns. Thus despite the increased scatter in the
monomer data, it is possible to say that the average radiative
lifetime of these monomers is greater than that of the bichro-
mophores. Since this is not true for the same monomers on
CT-DNA, it appears that most of the average emission lifetimes
for the monomer dyes on short duplexes are systematically
biased toward long lifetimes. This is plausible if the monomeric
dye/ds DNA complexes have a larger fraction of conformers
with ultrafast emission decays than the bichromophoric ones.
Figure 7 presents a plot of emission quantum yields versus
average emission lifetimes for 10 monomeric dye/ds DNA
systems. The data are plotted as two groups, CT-DNA duplexes
in one and short duplexes in the other, and each group is fit to
eq 1. The CT-DNA data yield a correlation coefficient (R) of
0.95, while data for the short duplexes yield an R of 0.83. The
corresponding radiative lifetimes obtained from the reciprocals
of the slopes are, respectively, 5.6 and 8.0 ns. As concluded
from statistical analysis, the average emission lifetimes for the
monomeric dyes bound to short ds DNA are biased toward long
lifetimes. Clearly, this is not the case for the same dyes bound
to CT-DNA.
The monomeric dyes differ from the bichromophoric ones
in three major ways. First, the monomeric dyes show a much
clearer ds DNA specificity with respect to emission yield than
do the bichromophores. In particular, pyridinium dyes have
higher emission quantum yields on (dAdT)lo duplexes and
quinolinium dyes have higher yields on (dGdC)6 duplexes.
Importantly, the emission quantum yields for each dye on CT-
DNA correspond very well with the higher of the short duplex
yields. Second, the correlation of emission quantum yields with
average emission lifetimes is poorest for the monomeric dyes
on short duplexes ( R = 0.83), compared to either bichro-
mophoric dyes (R = 0.88) or monomeric dyes on CT-DNA ( R
= 0.95). Bichromophores do not show any differences with
respect to type of ds DNA in the correlation of emission yields
with average emission lifetimes. Third, the average radiative
lifetime for the monomeric dyes on short ds DNA (8.6 ns) is
larger than that found for either the bichromophores (5.1 ns) or
monomers on CT-DNA (5.6 ns). Statistical analysis at the 99%
confidence level shows that the average radiative lifetime of
17946 J. Phys. Chem., Vol. 99, No. 51, 1995
the monomers on CT-DNA can not be lower than 6.0 ns, while
the average for the bichromophoric dyes cannot be higher than
5.8 ns.
All three of the above differences between monomeric dyes
and bichromophoric dyes are in accord with a lower ds DNA
association constant for the monomers (-lo6 M-I) than for the
bichromophores (-los M-’).27.33 In particular, weaker binding
dyes are likely to be more sensitive to ds DNA structural
differences (Le., have more stringent binding conditions) than
stronger binding ones. It is also likely that weaker binding
cyanine dyes will have a larger fraction of dye/ds DNA
conformers with weakly restricted torsion about the central
methine bridge. This occurrence would lead directly to longer
“apparent” average emission lifetimes (due to detector limita-
tions as explained earlier) and to greater scatter in the radiative
lifetime data for a series of dyes on different ds DNA samples.
One surprising result in light of this is that the average
radiative lifetime of the monomeric dyes when bound to CT-
DNA agrees very well with that of the bichromophoric dyes.
This suggests that the distribution of lifetimes for the monomeric
dyes bound to short duplexes has a greater population density
in the (0.2 ns time region than the distribution of lifetimes for
these same dyes bound to CT-DNA. Since the end regions of
a duplex, where there is less stability than in central regions,
are more important in short duplexes than in long ones, this is
plausible. Additionally, since the emission quantum yield of
each of the monomeric dyes when bound to CT-DNA matches
so well the higher of the short-duplex quantum yields, it is highly
likely that pyridinium monomers favor binding to AT-rich
regions and quinolinium monomers favor binding to GC-rich
regions in CT-DNA. Support for the conclusion that these
cyanine dyes are base-content sensitive regarding emission yield
can also be obtained by considering the ds DNA dependence
of the emission quantum yields for bichromophores. While the
propensity of pyridinium bichromophores for higher emission
yields at AT-rich regions is much less than that of pyridinium
monomers, the propensity of quinolinium bichromophores for
higher emission yields at GC-rich regions is comparable to that
of quinolinium monomers: on switching from (dAdT)lo to
(dGdC)6 duplexes the emission quantum yield increases for
YOYO-1 and TOTO-1 are, respectively, 49 and 140%; for the
corresponding monomers YO-PRO-1 and TO-PRO-1 they are,
respectively, 46 and 77%. Bichromophoric dyes appear to bind
sufficiently strongly to ds DNA that both their emission lifetime
and their quantum yield data are independent of the length of
DNA duplex used. One final note of caution is, however, in
order. Since the emission quantum yields of the bichromophoric
dyes bound to CT-DNA do not clearly match the higher of the
short duplex emission yields, it is not likely that these dyes have
a significant propensity for binding to AT- versus GC-rich
regions. The less stringent binding conditions resulting from
bichromophores having larger ds DNA association constants
than monomers work against base-content selectivity for bichro-
mophore binding and emission yield. TOTO-1 may be an
exception, however, because it has a 2-fold emission yield
increase on switching form (dAdT)lo to (dGdC)6 duplexes, and
the emission yield on CT-DNA (0.34) matches that on (dGdC)6
duplexes (0.39).
Conclusions
All of the 10 monomeric and bichromophoric cyanine dyes
investigated here exhibit either bi- or triexponential emission
decay kinetics reflecting different dye/ds DNA modes of
binding. Complicated emission decay pattems are reasonable
for these dye/ds DNA complexes, because of the simple exited-
Netzel et al.
state lifetime control mechanism operative in cyanine dyes. In
particular, restriction of torsion about the central methine bridge
in these dyes produces both longer excited state lifetimes and
increased emission quantum yield^.'^,^^^^^ Thus it is very likely
that cyanine dyes can bind to DNA duplexes in a variety of
ways so as to produce complexes with a variety of degrees of
torsional restriction. In keeping with this interpretation, the
average radiative lifetime for 16 bichromophore/ds DNA
systems is measured to be 5.1 f 0.8 ns (99% confidence level)
which agrees very well with previously reported radiative
lifetimes for one of the same ‘dyes, YO-PRO-1, bound to CT-
DNA: 5 ns (theoretical) and 6 ns (experimental).28 A similar
average radiative lifetime of 5.6 ns was found for monomeric
cyanine dyes bound to CT-DNA duplexes. Assuming that free
monomeric dyes have the same radiative lifetimes as when they
are bound to ds DNA, the emission enhancements measured
here imply that free monomeric dyes have emission lifetimes
of 1-5 ps in aqueous buffer solutions. At the other extreme,
the longest emission lifetimes for YOYO-1 and YO-PRO-1,
which are known to intercalate into CT-DNA duplexes,33are
found to be 3-5 ns in this work. Clearly a wide range of
emission lifetimes is possible for cyanine dye/ds DNA com-
plexes consistent with varying degrees of restricted torsion about
their central methine bridge.
Although binding-induced torsional restriction is responsible
for the large emission enhancements of these dyes (up to 1800),
it is also possible that there could be competing ET quenching
processes. In this case, significant base-content sensitivity of
the emission enhancements might be produced. Careful scrutiny
of the lengths of average emission lifetime for these 10 dyes
on (dAdT)lo and (dGdC)6 duplexes finds that they do not vary
as expected if ET emission quenching were an important
process. Predictions of the relative rates of ET quenching of
excited dye emission by the four DNA nucleosides are based
on estimates of the free energy of such reactions using redox
data for cyanine dye analogs of the DNA-staining dyes.
The above noted pattem of longer average emission lifetimes
for oxazole dyes than for thiazole dyes for both monomeric and
bichromophoric forms is reflected (as shown in Tables 4 and
6) in oxazole dyes having a greater emission quantum yields
than thiazole dyes with no exceptions on all three kinds of ds
DNA. Despite the lower emission yields of the thiazole dyes,
however, they always produce larger emission enhancements
on CT-DNA. These observations suggest that the thiazole to
oxazole switch (which is structurally minor, one atom in
monomers and two atoms in bichromophores) changes funda-
mental photophysical properties of the dye but is benign in terms
of the different types of ds DNA. Users of these dyes can thus
choose an oxazole if they need maximum fluorescence from a
DNA stain or a thiazole if they require minimum background
emission from unbound stain.
There are also differences in emission quantum yield between
the pyridinium and quinolinium dyes when bound to (dAdT)lo
and (dGdC)6 duplexes. These differences are very distinct for
the monomeric dyes where pyridinium dyes have 4-fold greater
emission yields on (dAdT)lo duplexes and quinolinium dyes
have 2-fold greater emission yields on (dGdC)6 duplexes. A
2-fold quantum yield increase on switching from (dAdT)lo to
(dGdC), duplexes is also present for the quinolinium bichro-
mophore, TOTO-I. Very importantly, for this bichromophore
and the four monomers the emission quantum yield on CT-
DNA matches very well the higher of the short duplex quantum
yields. This suggests, but does not prove, that the pyridinium
and quinolinium cyanine dyes selectively associate with AT-
and GC-rich regions, respectively, when bound to CT-DNA.
Cyanine Dyes Bound to Double-Strand DNA
For the other five bichromophores studied, there is little
difference in emission quantum yield between the two types of
short duplexes. Additionally, the emission yields of these
bichromophores when bound to CT-DNA do not clearly
correspond to either of the short duplex emission yields. Thus,
for these dyes there is no clear evidence that they exhibit base-
content selectivity with respect to emission yield.
Base-content binding selectivity and hence emission yield
selectivity for the monomeric dyes and the general absence of
base-content emission yield selectivity for the bichromophoric
dyes is consistent with a lower ds DNA association constant
(-lo6 M-I) for the monomers than for the bichromophores
(-lo8 M-').*' Because of their lower binding strengths,
monomers are more likely to be sensitive to the structure of ds
DNA than bichromophores. Surrounding media might also
affect the binding selectivity of cyanine dyes. In particular,
high salt conditions weaken the binding of cationic dyes to ds
DNA and thus might enhance their base-content selectivities
for binding and emission yield. This is mostly likely to occur
for cyanine monomers. In contrast, low salt conditions strengthen
the binding of cationic dyes to ds DNA and thus might further
reduce the already low level of base-content emission yield
selectivity found here for the bichromophoric dyes.
Acknowledgment. This work was supported by the United
States Department of Energy, Office of Health and Environment,
Radiological and Chemical Physics Research Division (Grant
No. DE-FG05-03ER61604). T.L.N. acknowledges helpful
conversations with Drs. David Wilson and Alex Siemiarczuk.
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