Polyfluorophore labels on DNA: Dramatic sequence dependence of quenching

Article abstract of DOI:10.1002/chem.200901607

We describe studies carried out in the DNA context to test how a common fluorescence quencher, dabcyl, interacts with oligodeoxynucleoside fluorophores (ODFs) - a system of stacked, electronically interacting fluorophores built on a DNA scaffold. We teste

Full text of DOI:10.1002/chem.200901607

FULL PAPER
DOI: 10.1002/chem.200901607
Polyfluorophore Labels on DNA:
Dramatic Sequence Dependence of Quenching
Yin Nah Teo,[a] James N. Wilson,[a, b] and Eric T. Kool*[a]
Abstract: We describe studies carried
out in the DNA context to test how a
common fluorescence quencher,
dabcyl, interacts with oligodeoxynu-
cleoside fluorophores (ODFs)—a
system of stacked, electronically inter-
acting fluorophores built on a DNA
scaffold. We tested twenty different tet-
rameric ODF sequences containing
varied combinations and orderings of
pyrene (Y), benzopyrene (B), perylene
(E), dimethylaminostilbene (D), and
spacer (S) monomers conjugated to the
3’ end of a DNA oligomer. Hybridiza-
tion of this probe sequence to a dabcyl-
labeled complementary strand resulted
in strong quenching of fluorescence in
85% of the twenty ODF sequences.
The high efficiency of quenching was
also established by their large Stern–
Volmer constants (KSV) of between
2.1ꢀ104 and 4.3ꢀ105 mꢀ1, measured
with a free dabcyl quencher. Interest-
ingly, quenching of ODFs displayed
strong sequence dependence. This was
particularly evident in anagrams of
ODF sequences; for example, the se-
quence BYDS had a KSV that was ap-
proximately two orders of magnitude
greater than that of BSDY, which has
the same dye composition. Other ana-
grams, for example EDSY and ESYD,
also displayed different responses upon
quenching by dabcyl. Analysis of spec-
tra showed that apparent excimer and
exciplex
emission
bands
were
quenched with much greater efficiency
compared to monomer emission bands
by at least an order of magnitude. This
suggests an important role played by
delocalized excited states of the p stack
of fluorophores in the amplified
quenching of fluorescence.
Keywords: DNA
·
excimer
·
exciplex · fluorescence quenching ·
fluorescent probes
Introduction
leads to the removal of the quencher either by physical sep-
aration or by enzymatic or chemical reaction and fluores-
cence is restored, signaling the biomolecular interaction.
Fluorescence signal readouts in such approaches depend
not only on the photophysical properties of fluorophores,
but also on the efficiency of the interaction with quenchers.
The search for better quenchers has brought about the de-
velopment of multiquencher designs[9] and the use of novel
materials, such as carbon nanotubes[10] and gold,[11] as
quenchers. Another approach to higher-efficiency quenching
involves harnessing the intrinsic properties of multichromo-
phoric systems to produce amplified quenching. An impor-
tant example of this is found in luminescent conjugated
polymers, some of which have demonstrated a remarkable
degree of enhanced fluorescence quenching. Small-molecule
quenchers, such as methyl viologen, had been shown to effi-
ciently quench the fluorescence of conjugated polymers with
exceptionally high Stern–Volmer quenching constants.[12]
Hyper-efficient quenching of cationic conjugated polymers
by gold nanoparticles has also been observed.[13] Such en-
hanced quenching phenomena have been used in DNA de-
tection assays[14,15] and in enzyme detection, such as with
proteases.[16]
Fluorescence quenching-based strategies have become a
highly useful tool for interrogating biological systems.
Quencher–fluorophore pairs are in common use for studying
protein interaction and recognition, and have been extreme-
ly valuable in DNA sequence reporting.[1] Various fluoro-
genic oligonucleotide probe formats have been developed,
such as molecular beacons,[2] Scorpion primers,[3] Taqman
probes[4–6] and QUAL probes.[7,8] These methods rely on the
efficient quenching of the fluorophore at close proximity to
the quencher either through collisional quenching and/or
Fçrster energy transfer mechanisms. DNA hybridization
[a] Y. N. Teo, Dr. J. N. Wilson, Prof. Dr. E. T. Kool
Department of Chemistry, Stanford University
Stanford, CA 94305-5080 (USA)
Fax : (+1)650-725-0259
E-mail: kool@stanford.edu
[b] Dr. J. N. Wilson
Current address: Department of Chemistry
University of Miami, Coral Gables, FL 33146 (USA)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200901607.
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Conjugated polymers can exhibit such high sensitivity to
quenching due to their multichromophoric structures.[17] The
exceptional amplification of quenching is due to the efficient
migration of the exciton in the extended p conjugation
along the polymer backbone.[18] Thus when the quencher is
brought in close proximity with the conjugated polymer
through electrostatic interactions, “superquenching” occurs
through a combination of high mobility of the exciton and
favorable noncovalent association between the fluorescent
polymer and the quencher.
While such polymeric systems can be effective in biomo-
lecular detection assays, it would be useful in many cases if
similar enhanced quenching properties were available in
small, discrete molecules. This could allow for conjugation
to biomolecules (e.g., antibodies and DNA) and other small
molecules, and would enable many specific assays and
better controlled interactions. Moreover, small molecules
can be simpler to characterize, and their properties, such as
solubility, cell permeability and photophysical characteris-
tics, can be relatively straightforward to vary and control.
In recent years, the deoxyribose backbone of DNA has
been employed as a scaffold for the design of multichromo-
phoric systems. Base substitutions have been made with
phenanthrenes and pyrenes,[19,20] clusters of methyl red and
naphthyl red dyes[21,22] and bipyridyl and biphenyl moiet-
ies.[23] Pyrenes have also been covalently attached to the
5’ position of uridine to form regular helical p arrays along
the major groove of the DNA duplex.[24,25] Besides DNA,
multilabeling of other nucleic acid architectures, for exam-
ple, RNA[26] and LNA,[27] have also been carried out. Nonco-
valent assembly of chromophores on the DNA structure has
also been observed with intercalating dyes.[28]
lived, particularly prone to this quenching, or are other
ODF sequences and types of excited states viable as well?
Second, is methyl viologen unique as a highly effective
quenching partner because of its dicationic, flat aromatic
structure, or can other common quenchers exhibit such ef-
fects? Third, can ODFs be conjugated to a biomolecule, for
example, DNA, and still retain their ability to be quenched
by common quenchers?
Here, we investigate these issues by conjugating a variety
of ODFs to a 14-mer oligonucleotide strand. The effect of
conjugation to DNA on the quenching properties is investi-
gated, as is the effect of varied chromophore sequence and
composition. We also report on the use of dabcyl, a
common quencher in DNA fluorogenic probes, as an effi-
cient quencher of an assortment of sequences of ODFs. We
find that the susceptibility of ODFs to efficient quenching
depends upon the formation of delocalized excited states,
such as excimers or exciplexes. As a result, quenching effi-
ciency is highly dependent not only on ODF composition,
but also on sequence, as demonstrated by a number of ODF
sequence anagrams that have very different properties. The
results underscore that polyfluorophore systems can behave
as cooperative units in the excited state, and that close inter-
actions of neighboring chromophores can have a strong in-
fluence on their properties.
Results
A representative set of twenty tetrameric sequences of
ODFs was chosen from a larger random library for this
study; for hybridization studies, they were appended to the
3’ terminus of a 14-mer probe DNA sequence (Table 1). The
Our laboratory has developed a novel system of discrete
fluorophores built on the deoxyribose–phosphate backbone,
called oligodeoxyfluorosides (ODFs), in which consecutive
aromatic fluorophores, such as pyrene, perylene and benzo-
pyrene, replace the natural DNA bases.[29,30] This DNA-like
structure allows aromatic fluorophores to interact electroni-
cally in the ground state through p stacking, and results in
multiple forms of energy and excitation transfer in the excit-
ed state. Excimers and exciplexes are commonly observed
and result in large Stokes’ shifts of more than 200 nm.[31] By
varying the length, monomers and sequence, highly diverse
and tunable properties result; for example, sets of dyes with
multiple emission colors have been identified that can be ex-
cited at one excitation wavelength.[32] In addition to their
useful photophysical properties, ODFs have the advantages
of being readily synthesized in any sequence on an automat-
ed DNA synthesizer; moreover, the anionic DNA backbone
renders them water soluble.
Table 1. DNA sequences used in this study.
Strand ID
Sequence[a]
P1–P20
D1
5’-TCC ACA GAA ACA TA–XXXX-3’
3’-AGG TGT CTT TGT AT-5’
Q1
3’-AGG TGT CTT TGT AT–dabcyl-5’
[a] XXXX denotes the sequence of ODF; refer to Table 2 for ODF se-
quences.
ODF sequences were chosen to represent multiple random
chromophore compositions, as well as varied sequences of
the same compositions. The ODF labels were synthesized
from five monomers, in which the base-replacement fluoro-
phore was a benzopyrene (B), pyrene (Y), perylene (E), di-
methylaminostilbene (D) or an abasic spacer tetrahydrofur-
an moiety (S). a-Glycosidic anomers, which can stack with
one another in a DNA context,[31] were used for synthesis
convenience in this study. Figure 1 shows the chemical struc-
tures of these monomeric fluorosides and Figure 2 shows the
structures of two representative ODF labels. Note that se-
quences of ODFs are listed in 5’ to 3’ order in analogy to
DNA.
We previously reported the highly efficient quenching
properties of oligomeric pyrene excimers (not conjugated to
DNA) using methyl viologen as a free quencher.[33] The
quenching efficiency, with Stern–Volmer constants up to
4.7ꢀ106 mꢀ1 was comparable to those observed in conjugated
polymers. This observation raised a number of questions.
For example, are pyrene excimers, which are especially long
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Sequence-Dependent Quenching of Polyfluorophores
FULL PAPER
crease in peak/valley ratio, as reported previously.[34] How-
ever, emission spectra of the ODF labels were in nearly all
cases quite different from the combined monomer emis-
sions. All cases except one (P6) showed broad bands be-
tween 450–600 nm; this is consistent with excited state
dimer (exciplex and excimer) states (see the Supporting In-
formation). The maxima of these bands varied from 460 to
530 nm, and some cases showed multiple maxima (as many
as three) in this range. The one exception (P6, sequence
BSDY) is a blue–violet fluorophore with no longer-wave-
length bands.
In general, the twenty DNA-conjugated ODFs were effi-
cient fluorophores, with quantum yields for emission as high
as 44% (Table 2). The quantum yields varied with chromo-
phore composition and sequence; for example, P7 (BYDS)
gave a lower quantum yield of 4.5%, while the sequence P8
(EDSY), which is different by only one monomer, has a
quantum yield of 44.3%. An even more striking comparison
was observed between P7 (BYDS, Ffl =4.5%) and its se-
quence-rearranged anagram, P6
Figure 1. Structures of monomeric deoxyfluorosides perylene (E), benzo-
pyrene (B), dimethylaminostilbene (D), pyrene (Y) and spacer (S).
(BSDY, Ffl =42.8%), in which
quantum efficiencies varied
strongly without a change in
composition.
In addition to quantum effi-
ciency, wavelengths of emission
varied substantially with se-
quence as well (Table 2), an
effect documented in an earlier
set of pyrene–perylene ODFs[34]
as well as more recently in a li-
brary of unconjugated tetramer
ODFs.[32] Examples in the pres-
ent study include P1 (labeled
with DEBY), which is a whitish
multiemissive fluorophore, as
compared with its anagram P3
(DYBE), a green emitter. A
second comparison is sequence
P8 (labeled with EDSY, a blue–
violet dye) versus sequence P9
(ESYD, a green–yellow fluoro-
phore).
Figure 2. Structures of two representative oligodeoxyfluorosides from the twenty in this study. These are at-
tached to the 3’ end of a DNA probe sequence (R).
Dabcyl quenching of ODF
Fluorescence properties of ODFs: Absorption and emission
strands upon hybridization:
spectra of the twenty ODF labels conjugated to single-
stranded DNA were measured in pH 7.0 hybridization
buffer (see the Experimental Section). Lists of the absorp-
tion and emission bands are given in Table 2, and emission
spectra are shown in the Supporting Information. Quantum
yields for fluorescence were measured in the same buffer;
these are also given in Table 2. In general, the absorption
spectra were simple combinations of the added spectra of
the monomer components consistent with ground-state
stacking of the chromophores (data not shown), although in
many cases there was moderate line broadening and a de-
Dabcyl (4-((4’-dimethylamino)phenylazo)benzoic acid) is a
common quencher widely used in a variety of biomolecular
applications[35] and nucleic acid probes.[36–38] Thus we chose
to explore its quenching effects on ODFs upon hybridiza-
tion, to explore the utility of varied ODF–dabcyl fluoro-
phore quencher pairs as reporters of DNA hybridization.
Dabcyl was attached to the 5’ end of the complementary
DNA strand (Q1; Table 1); thus upon hybridization to each
of the 3’-ODF-labeled strands (P1–P20), dabcyl was brought
into proximity with the various labels. The fluorescence
spectrum of each of the ODFs was measured before and
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E. T. Kool et al.
Table 2. Sequences and photophysical data for twenty composition- and sequence-varied ODFs appended to
Three ODF labels showed
poor quenching by dabcyl on
the complementary strand.
Both sequence P6 (BSDY) and
P8 (EDSY) exhibited predomi-
nantly the pyrene monomer
emission, which showed almost
no quenching. Probe P14
(EEDE) showed 51% quench-
ing at the emission wavelengths
between 450 and 500 nm.
the 3’ end of the DNA probe sequence.[a]
[b]
[b]
Strand
ID
ODF sequence
(5’!3’)
lmaxabs
lmaxem
Fem
Qeff KSV [mꢀ1
[%] exciplex
]
KSV [mꢀ1
]
G
[nm]
[nm]
monomer
P1
P2
P3
P4
P5
P6
P7
P8
D
E
D
B
E
B
B
E
E
D
B
E
E
E
E
E
E
E
Y
Y
E
D
Y
E
Y
S
Y
D
S
B
B
B
B
B
D
D
S
Y
E
S
B
S
Y
Y
E
Y
E
Y
S
Y
D
Y
S
257, 347, 399, 448 375, 395, 464, 489 0.229 81
1.3ꢀ105
2.9ꢀ105
2.5ꢀ105
5.7ꢀ104
1.4ꢀ105
n.a.[d]
neg.[c]
258, 349, 403, 452 461, 490
256, 349, 403, 442 462, 492
258, 347, 401, 450 492
257, 347, 401, 451 465, 493
264, 326, 342, 401 375, 395
258, 346, 349, 402 375, 416, 467
264, 342, 451
257, 346, 349, 451 460, 488
257, 343, 349, 451 375, 463, 490
261, 349, 403
258, 402, 451
257, 355, 450
254, 355, 449
256, 347, 451
257, 347, 451
256, 347, 451
257, 346, 451
254, 349, 441
264, 332, 350
0.188 87
0.130 77
0.194 85
0.092 84
0.428
0.045 73
0.443
3.5ꢀ104
neg.[c]
neg.[c]
neg.[c]
7
8.3ꢀ102
neg.[c]
4.9ꢀ104
4.3ꢀ105
3.5ꢀ105
1.9ꢀ105
2.5ꢀ104
1.5ꢀ105
1.5ꢀ105
8.4ꢀ104
2.1ꢀ104
6.2ꢀ104
2.3ꢀ105
7.4ꢀ104
1.1ꢀ105
n.a.[d]
375, 395, 459
1
4.3ꢀ104
neg.[c]
P9
0.191 84
0.303 74
P10
P11
P12
P13
P14
P15
P16
P17
P18
P19
P20
S
Y
S
2.5ꢀ104
8.0ꢀ103
2.0ꢀ104
neg.[c]
375, 395, 416, 470 0.205 85
Hybridization of ODF-labeled
strands with DNA alone: To
evaluate whether quenching of
the fluorescence of ODFs was
due to dabcyl and not just the
complementary DNA strand,
all twenty ODF-labeled sequen-
ces were hybridized to the natu-
ral complementary oligomer,
D1 (Table 1), which lacked the
quencher group. The effects of
S
S
470, 500
461, 490
374, 454, 478
464, 490
490
375, 395, 494, 530 0.148 75
461, 490
462, 489
378, 395, 490
0.219 88
0.161 86
0.062 51
0.130 88
0.141 77
D
E
Y
E
S
Y
Y
S
D
Y
Y
E
S
E
E
Y
Y
S
1.5ꢀ103
neg.[c]
neg.[c]
neg.[c]
0.421 92
0.110 77
0.025 86
9.1ꢀ103
4.4ꢀ104
6.9ꢀ103
E
S
E
Y
[a] See the Experimental Section for conditions. [b] KSV values measured with free dabcylamide quencher (1)
in water. [c] neg.: cases in which KSV was zero or slightly negative; see the Supporting Information for plots.
[d] n.a.: cases in which no long-wavelength (e.g., excimer or exciplex) band was present.
after the addition of Q1. The percentage quenching of fluo-
rescence was determined by the relative fluorescence inten-
sities at the maximum emission wavelength before and after
the addition of Q1. We used a one molar excess equivalent
to ensure that all ODF-labeled DNAs were hybridized with
a quencher.
The experiments revealed that efficient quenching by
dabcyl occurred in 17 of the 20 ODFs tested (Table 2).
These sequences had more than 70% of their fluorescence
quenched upon the addition of the dabcyl-conjugated com-
plement Q1. Two instances of this effect are shown in
Figure 3 with labeled probes P4 (BEBY) and P15 (EYYE),
which yielded 85 and 88% quenching, respectively.
Interestingly, inspection of emission spectra before and
after hybridization with Q1 (see the Supporting Information
and Figure 3) revealed that quenching was nearly always
much more efficient with longer wavelength apparent exci-
plex emission bands compared to the monomer emission
band in cases when both were present. The majority of la-
beled sequences showed this effect; some examples include
dramatic monomer versus excimer differences, such as in se-
quences P4 (BEBY; Figure 3a), P1 (DEBY), P7 (BYDS),
and P10 (DSEY), all of which showed strong quenching of
long-wavelength bands between 450–600 nm, but essentially
no quenching of shorter-wavelength bands. Several other
cases showed similarly strong quenching of long-wavelength
bands (see, for example, Figure 3b) but had very little in the
way of short-wavelength emission for comparison, apparent-
ly due to highly efficient excimer–exciplex formation with
Figure 3. Fluorescence emission spectral changes upon hybridization of
ODF-labeled probes: a) P4, and b) P15 with dabcyl-conjugated comple-
ment, Q1 (lex =330 nm).
nearly complete lack of monomer emission.
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Sequence-Dependent Quenching of Polyfluorophores
FULL PAPER
hybridization to D1 varied from enhancement of fluores-
cence to a small degree of quenching, and several of the
ODFs displayed no change at all (see spectra in the Sup-
porting Information). The two largest changes in ODF emis-
sion were with sequences P16 (EEYY) and P3 (DYBE).
The fluorescence of EEYY was quenched by 33% (see the
Supporting Information), while the fluorescence of DYBE
was enhanced by 47%. Most others exhibited less than 20%
change in emission. Taken together, the effects of the com-
plementary DNA strand alone were generally small; thus in
the majority of cases, the quenching effects observed in the
dabcyl hybridization experiments came entirely (or nearly
so) from the presence of the dabcyl group on the opposite
DNA strand.
104 mꢀ1, typically one order of magnitude less efficient than
that of the excimer–exciplex band in a given sequence. Half
(10 of 20) of the ODF sequences tested did not show any
quenching of the emission of the monomer band. Some se-
quences even showed an enhancement of emission; for ex-
ample, sequence P5 (EYBE) gave a 55% increase in mono-
mer emission with the addition of 5 mm dabcylamide, while
at the same time the exciplex emission was decreased by
39%. The formation of the excimer or exciplex band was
dependent on buffer conditions, as observed in the case of
P20 (YSSY), in which the excimer band was missing under
the conditions used for determining KSV values.
Notably, quenching of ODFs was highly sequence depen-
dent even when monomer composition was the same. This
could be readily observed in anagrams of ODF sequences.
For example, the quenching was two orders of magnitude
higher in P7 (BYDS), with KSV of 4.9ꢀ104 mꢀ1, than with P6
(BSDY), for which the KSV was 8.3ꢀ102 mꢀ1. As with the
above experiments with dabcyl-conjugated complementary
DNA, this correlated with the presence or absence of long
wavelength exciplex bands, as the exciplex band (463 nm)
was large in P7 (BYDS), but no exciplex band was present
in P6 (BSDY). In a second comparison, P19 (YYEE) and
P15 (EYYE) showed considerably better excimer–exciplex
quenching in the former as compared with the latter. Inter-
estingly, the monomer bands also showed large differences,
with P19 yielding strong quenching (KSV =4.4ꢀ104 mꢀ1) for
the pyrene–perylene monomer emissions, while P15 showed
no quenching of these bands. A similar effect was seen in
anagrams P9 and P10 (Table 2 and the Supporting Informa-
tion), as well as in P2 as compared with its anagrams P1 and
P3.
Examination of data for anagrams P8, P9 and P10 showed
cases of strong sequence dependence as well. With D and S
between E and Y, there was less excited state delocalization
as seen by the much smaller exciplex band in P8 (EDSY)
compared to P9 (ESYD) and P10 (DSEY). The dominant
emission band in P8 (EDSY) was the monomer emission
band at 375 and 395 nm while the dominant band in P9
(ESYD) and P10 (DSEY) was the exciplex band at 460 and
490 nm. The exciplex emission band was better quenched by
an order of magnitude greater than the quenching of the
monomeric emission band. Fluorescence emission spectra of
P9 and P10 in the quenching experiments are shown in
Figure 4, together with their respective Stern–Volmer plots.
The sequence dependence of quenching as observed in
anagrams of ODFs could be observed in hybridization ex-
periments carried out in cuvettes. Changes were apparent by
visual inspection as seen in Figure 5. P8 and P9 are sequence
anagrams containing the same constituents as discussed ear-
lier. Quenching of P8 (EDSY) by Q1 produced a color
change from blue to violet, consistent with loss of the long
wavelength emission upon quenching. P9 (ESYD), which
possessed a more yellowish tone due to a dominant emission
at 450–550 nm, yielded a different color change upon
quenching.
Stern–Volmer relation: To quantify the intrinsic sensitivity
of DNA-conjugated ODFs to dabcyl, a small water soluble
dabcyl-amide compound (1) was synthesized from the free
acid of dabcyl through its acid chloride derivative. Synthesis
details can be found in the Supporting Information.
Quenching efficiency was quantified by using the Stern–
Volmer relation:
I0
I
¼ KSV½Qꢁ þ 1
where I0 and I are the steady state fluorescence intensities
in the absence and presence of quencher 1. The Stern–
Volmer constant (KSV) was derived from the slope of linear
fits to the data from each complex constrained to intersect
at y=1. The Stern–Volmer constants of all ODFs attached
to DNA are given in Table 2.
The data show that the Stern–Volmer constants for the
ODFs are large. Interestingly, in all cases in which it was
present, the apparent exciplex emission bands were much
more efficiently quenched than the monomer emission.
More specifically, the long-wavelength, broad bands (~430–
600 nm) were strongly quenched by increasing amounts of
dabcylamide, while the monomer emission bands typical of
B, Y, D monomers (330–440 nm) were only slightly affected
or were unaffected. Table 2 shows KSV values listed for both
sets of bands. The KSV values of the entire set of long-wave-
length bands ranged from 2.1ꢀ104 mꢀ1 in P15 (EYYE) to
4.3ꢀ105 mꢀ1 in P8 (EDSY). Monomeric emission bands, in
contrast to this, gave smaller KSV values of 8.3ꢀ102 to 4.4ꢀ
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E. T. Kool et al.
Figure 4. Sequence dependence of fluorescence and of quenching in ODF fluorophores that have the same dye composition but with different ordering.
Shown are the fluorescence emission spectra of: a) P8 (sequence EDSY), b) P9 (ESYD), and c) P10 (DSEY) with varied amounts of quencher 1 added.
Their respective Stern–Volmer plots for short- and long-wavelength emission bands are shown below the respective emission spectrum; note the large re-
sponse of long-wavelength bands as compared with short-wavelength bands. Additional examples are available in the Supporting Information file.
Discussion
In our study, a common quencher, dabcyl, was tested for its
effectiveness in quenching the fluorescence of various multi-
chromophoric ODF sequences. Large Stern–Volmer con-
stants were found with a free quencher, demonstrating
strong inherent ability of the ODFs to be quenched. For a
DNA-conjugated dabcyl moiety, the results have shown that
quenching is effective in 85% of the twenty ODFs tested. In
all cases, the long wavelength emission band, which was ap-
parently due to exciplex or excimer emission, was quenched
more efficiently than the monomeric emission band.
Contact quenching is likely to play a central role in this
quenching effect since the quencher, dabcyl, was in close
proximity to ODFs upon hybridization and the lifetimes of
many ODFs are long (in the 6–120 ns range).[34] In addition,
the absorption spectrum of dabcyl (lmaxabs =468 nm) over-
laps with the emission spectra of many of the ODFs; thus
Figure 5. Varied fluorescence and quenching in anagrams of ODF-labeled
oligonucleotide strands in buffer, observed by visual inspection. Images
of buffered solutions of: a) P8 (EDSY), and b) P9 (ESYD), before (left)
ments of KSV has only one cationic charge compared to the
and after (right) hybridization with Q1. Cuvettes were placed on a UV
energy transfer might also play a role in the quenching
effect. The dabcyl-amide quencher (1) used in our measure-
dicationic methyl viologen used in an earlier study of the
quenching of nonconjugated ODFs.[33] Thus static quenching
by the formation of an electrostatically favorable complex
between ODF and the quencher is likely to be less efficient
when using dabcyl. However, the Stern–Volmer constants
transilluminator (354 nm).
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Sequence-Dependent Quenching of Polyfluorophores
FULL PAPER
were still comparable in both quenchers, with KSV values be-
tween 104 and 105 mꢀ1 for tetramer-length ODFs. This estab-
lishes that multiple excimer and exciplex species (beyond
pyrene alone[33]) can be highly efficiently quenched. Since
the quenching with DNA-conjugated dabcyl was not as effi-
cient as with the free quencher, we surmise that constraints
on dye location and motion due to the tether could have
had a negative effect; future tests with different tether
lengths and structures can shed light on this.
Recently, the quenching properties of a DNA–multichro-
mophore system upon hybridization was also studied by
Hꢂner and co-workers, who effectively quenched pyrene ex-
cimer fluorescence by a non-nucleosidic perylene diimide
moiety upon DNA duplex formation.[41] Both the pyrene
and perylene diimide non-nucleosidic base surrogates were
incorporated in the middle of respective DNA strands, and
DNA hybridization brought them into contact; this resulted
in efficient quenching of the pyrene excimer emission. Al-
though that work did not test other emissive dyes, it does
confirm that pyrene excimers can be effectively quenched
by hybridization in a geometry different from the current
one.
The finding of efficient quenching of different excimers
and exciplexes beyond pyrene excimer alone is likely to
have useful applications. For example, it might be possible
to generate multiple differently colored quencher–dye pairs
for probing applications, with the added advantage of being
excited with the same single excitation wavelength. This
multicolor property in ODFs allows for direct visualization
of multiple species, even in moving systems, without the use
of multiple filter sets and camera exposures.[32] The ease of
synthesis of combinatorial libraries of these ODFs suggests
some simple future strategies for screening of various
common quenchers–dye pairs that might be more effective
than the current ones.
We hypothesize that the efficient inherent quenchability
of ODFs is due to the presence of delocalized excited states,
which increases the likelihood of productive contact with
the quencher. In addition, we proposed recently[33] that if ex-
cimer–exciplex states exist in longer oligomers, the exciton
might migrate by dynamic alignment of the adjacent chro-
mophores. Thus the effects of delocalization and mobility of
the excited state might be similar in ODFs and conjugated
polymers. Association or collision with a quencher anywhere
in the delocalized domain rapidly brings about quenching,
as the exciton migrates rapidly to the site of the quencher.
In the ODF system, this mobile exciton mechanism explains
our observation of enhanced quenching of exciplex bands
over monomer emission. ODF molecules that have no exci-
plex emission, and thus do not have this delocalization
mechanism available, are poorly quenched. For example, the
sequence BSDY shows only emissions similar to those of the
pyrene and benzopyrene component monomers, and its KSV
value with dabcyl was 8.3ꢀ102 mꢀ1. Other sequences with
monomer bands had KSV values between 1.5ꢀ103 and 4.4ꢀ
104 mꢀ1, similar to values for classical monomeric fluoro-
phores, such as fluorescein, which we found to have a KSV
value of 3.1ꢀ103 mꢀ1 (see the Supporting Information).
When conjugated to a 14-mer oligonucleotide at the 5’ end,
fluorescein had a higher KSV value of 1.6ꢀ104 mꢀ1; this sug-
gests that the polyanionic DNA might increase affinity of
the quencher for the fluorophore-tagged molecule as a
whole. A similar effect is seen in quenching of conjugated
anionic polymers by methyl viologen,[12,39] which binds to
the polymers and thus favors contact quenching. However,
there is an additional favorable effect of the delocalized
state in the current ODFs; for example, the sequence
EDBY shows clear long wavelength exciplex-like bands
(461, 490 nm) and exhibits KSV =2.9ꢀ105 mꢀ1.
Additional studies would be useful to shed light on the
quenching mechanisms of dabcyl, methyl viologen and other
quenchers in polychromophore systems. Moreover, further
studies into the mechanisms of excited state mobility would
be valuable, both for lending basic insight, and also for
design of better fluorescent labels, probes and reporters in
the future.
Experimental Section
Fluoroside phosphoramidites (E, Y, D, S, B): Syntheses of the monomer
1’-a-2’-deoxyriboside 5’-dimethoxytrityl-3’-phosphoramidite derivatives of
pyrene (Y), perylene (E), benzopyrene (B) and dimethylaminostilbene
(D) were carried out as previously described.[29,30,42] The abasic tetrahy-
drofuran spacer S was obtained commercially (dSpacer CE phosphorami-
dite from Glen Research Corporation).
Synthesis of ODF-labeled oligodeoxynucleotides: Oligodeoxynucleotides
were synthesized by using an Applied Biosystems 394 DNA/RNA synthe-
sizer on a 1 mmole scale and possessed a 3’-phosphate group. Coupling
employed standard b-cyanoethyl phosphoramidite chemistry, but with ex-
tended coupling time (600 s) for fluoroside phosphoramidites. ODFs
were synthesized directly on the CPG beads and the DNA sequence was
then built on its 5’ end. All oligomers were deprotected by using the ul-
tramild deprotection method with potassium carbonate solution (0.05m;
558C, 4 h), then purified by reverse-phase HPLC. The recovered material
was quantified by absorbance at 260 nm with molar extinction coeffi-
cients determined by the nearest-neighbor method. Molar extinction co-
efficients for ODFs were estimated by adding the measured value of the
molar extinction coefficient of the fluorosides (at 260 nm) to the calculat-
ed value for the natural DNA fragments. ODF-labeled oligomers were
characterized by MALDI-TOF mass spectrometry (data are given in the
Supporting Information). Dabcyl was purchased as the 5’-dabcyl phos-
phoramidite derivative from Glen Research Corporation and attached to
Our hypothesis suggests that ODF sequences that have
more efficient delocalization, which might be judged by
higher excimer–monomer emission ratios, should be better
quenched in general than those with poor delocalization. In
addition, ODF structures of increasingly greater length
should further enhance quenching efficiency as well. In the
present cases we limited length to four, but recent studies
with multipyrene ODFs with lengths of up to eight did sup-
port this prediction.[33] In the future, it will be of great inter-
est to study the detailed mechanism of exciton mobility in
this and other p stacked excimer–exciplex systems.[28,40]
Time-resolved fluorescence and absorption studies would no
doubt lend useful insights into this.
Chem. Eur. J. 2009, 15, 11551 – 11558
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E. T. Kool et al.
the 5’ end of the complementary DNA sequence by using the manufac-
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R
F
n2
nR2
AR
A
F
FR
R
¼
ꢂ
ꢂ
FR
where F and FR are the quantum yields of the unknown and reference
compounds, respectively; n and nR are the refractive indices of water and
cyclohexane, respectively; A and AR denote the absorbances at 330 nm
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Received: June 12, 2009
Published online: September 24, 2009
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Chem. Eur. J. 2009, 15, 11551 – 11558