Synthesis of new fluorogenic cyanine dyes and incorporation into RNA fluoromodules

Article abstract of DOI:10.1021/ol702920e

A new fluorogenic cyanine dye was synthesized and found to have low fluorescence quantum yield in fluid solution and in the presence of double-stranded DNA but 80-fold enhanced fluorescence in viscous glycerol solution. An RNA aptamer selected for binding to the new dye exhibits K _d = 87 nM and 60-fold fluorescence enhancement. The dye-aptamer pair is a fluoromodule that can be incorporated into fluorescent sensors and labels.

Full text of DOI:10.1021/ol702920e

ORGANIC
LETTERS
2008
Vol. 10, No. 8
1561-1564
Synthesis of New Fluorogenic Cyanine
Dyes and Incorporation into RNA
Fluoromodules
Tudor P. Constantin,† Gloria L. Silva,† Kelly L. Robertson,† Tamara P. Hamilton,†
Kaitlin Fague,† Alan S. Waggoner,‡ and Bruce A. Armitage*,†,‡
Department of Chemistry and Molecular Biosensor and Imaging Center, Carnegie
Mellon UniVersity, 4400 Fifth AVenue, Pittsburgh, PennsylVania 15213-3890
army@cmu.edu
Received January 29, 2008
ABSTRACT
A new fluorogenic cyanine dye was synthesized and found to have low fluorescence quantum yield in fluid solution and in the presence of
double-stranded DNA but 80-fold enhanced fluorescence in viscous glycerol solution. An RNA aptamer selected for binding to the new dye
exhibits Kd
)
87 nM and 60-fold fluorescence enhancement. The dye−aptamer pair is a fluoromodule that can be incorporated into fluorescent
sensors and labels.
Fluorogenic cyanine dyes such as thiazole orange (TO, Figure
1), oxazole yellow (YO), and their derivatives are widely
as signaling elements in sensors, where they are covalently
attached to recognition moieties such as nucleic acid oligo-
mers2 or peptides.3 Enhanced fluorescence from the dye
signals binding of the sensor to its target.
The fluorogenic behavior of unsymmetrical cyanines such
as TO and YO arises from restriction of excited-state twisting
about the central methine bridge separating the two hetero-
cycles,4 which normally leads to rapid nonradiative deactiva-
(1) (a) Lee, L. G.; Chen, C.; Liu, L. A. Cytometry 1986, 7, 508-517.
(b) Rye, H. S.; Yue, S.; Wemmer, D. E.; Quesada, M. A.; Haugland, R. P.;
Mathies, R. A.; Glazer, A. N. Nucleic Acids Res. 1992, 20, 2803-2812.
(c) Nygren, J.; Svanvik, N.; Kubista, M. Biopolymers 1998, 46, 39-51.
(2) (a) Ishiguro, R.; Saitoh, J.; Yawata, H.; Otsuka, M.; Inoue, T.; Sugiura,
Y. Nucleic Acids Res. 1996, 24, 4992-4997. (b) Seitz, O.; Bergmann, F.;
Heindl, D. Angew. Chem., Int. Ed. 1999, 38, 2203-2206. (c) Svanvik, N.;
Westman, G.; Wang, D.; Kubista, M. Anal. Biochem. 2000, 281, 26-35.
(3) (a) Babendure, J.; Liddell, P. A.; Bash, R.; LoVullo, D.; Schiefer, T.
K.; Williams, M.; Daniel, D. C.; Thompson, M.; Taguchi, A. K. W.; Lohr,
D.; Woodbury, N. W. Anal. Biochem. 2003, 317, 1-11. (b) Carreon, J. R.;
Mahon, K. P., Jr.; Kelley, S. O. Org. Lett. 2004, 6, 517-519.
Figure 1. Fluorogenic cyanine dyes.
used for fluorescence imaging and detection of DNA and
RNA.1 These dyes have also found numerous applications
† Department of Chemistry.
‡ Molecular Biosensor and Imaging Center.
10.1021/ol702920e CCC: $40.75
© 2008 American Chemical Society
Published on Web 03/14/2008

tion to the ground state.5 Viscous solvents or restrictive local
environments (such as a DNA intercalation site) inhibit this
motion and lead to significantly enhanced (i.e., 101-103-
fold) fluorescence quantum yields (φf).
DIR has intense absorbance in the red region of the visible
spectrum with λmax ) 602 nm and ꢀmax ) 134 000 M-1 cm-1
in methanol. The dye also exhibits the expected fluorogenic
behavior: fluorescence is very low in aqueous buffer but
increases ca. 80-fold in a solution of 90% glycerol in water
(Figure 2). Meanwhile, in the presence of double-stranded
While most applications for fluorogenic cyanines have
been for in vitro analysis and detection, we are interested in
developing a catalog of intracellular labels and sensors. These
fluoromodules would consist of specific dye-RNA or dye-
protein partners where the RNA or protein apomodule is
genetically encoded and expressed, whereas the dye is
delivered exogenously into the cell. The obvious complica-
tion in using an unsymmetrical cyanine as the dye component
is the strong tendency of these dyes to bind nonspecifically
to cellular DNA and RNA, which would compete with the
specific RNA or protein apomodule. Herein we report the
design and synthesis of a new fluorogenic cyanine dye with
significantly reduced affinity for double-stranded DNA and
nonspecific RNA. This dye was then used to select high
affinity fluorescence-activating RNA aptamers from a com-
binatorial library.
The target dye, dimethylindole red (DIR), is shown in
Figure 1. Two features were intended to suppress nonspecific
DNA binding: the bulky dimethylindole heterocycle should
hinder intercalation between π-stacked base pairs in DNA
or RNA, while the anionic propylsulfonate substituent on
the quinoline ring system introduces nonspecific electrostatic
repulsion from polyanionic nucleic acids. While the same
repulsion would reduce binding affinity for a specific RNA
partner, the ability to select high affinity RNA aptamers for
negatively charged small molecules such as GTP indicated
that the electrostatic repulsion could be compensated by other
specific binding interactions.6
Figure 2. Fluorescence emission spectra recorded for DIR (left)
and TO (right) in aqueous buffer, calf thymus DNA, and 90%
glycerol. [Dye] ) 1.0 µM, [DNA] ) 100 µM base pairs. Buffer
and DNA spectra overlap for DIR.
DNA, the fluorescence of DIR increases only 2-fold. For
comparison, Figure 2 also shows the same experiments done
with TO. The fluorescence for this dye is significantly
enhanced both by glycerol and DNA, further verifying that
the structural elements designed into DIR successfully
suppress nonspecific binding to DNA.
The synthesis of DIR is shown in Scheme 1. Alkylation
We next used an affinity chromatography-based in vitro
selection method to obtain RNA aptamers for DIR.7 A biotin-
conjugated analogue of DIR was synthesized as described
in the Supporting Information and immobilized on a column
packed with streptavidin-agarose beads. A na¨ıve RNA pool
containing ∼1014 unique sequences was obtained by in vitro
transcription of the corresponding DNA pool. The pool was
designed to contain a short internal self-complementary
sequence that is expected to fold into a hairpin structure. A
similar library was used by Davis and Szostak to obtain high-
affinity aptamers for GTP.6
Scheme 1. Synthesis of Dimethylindole Red (DIR)
The RNA pool was subjected to multiple rounds of affinity
selection and amplification by reverse-transcription PCR. The
ability of the enriched pool obtained after each selection-
amplification cycle to enhance the fluorescence of DIR was
tested. Fluorescence enhancement first appeared in round 9,
and selection was continued through round 15, at which point
individual aptamers were isolated by cloning into E. coli.
A total of 32 RNA aptamers were screened for DIR
fluorescence enhancement. As shown in Figure 3, six of these
aptamers yielded greater than 5-fold enhancement, including
three that enhance DIR fluorescence more than 20-fold. Thus,
while the selection was based solely on affinity for DIR, a
significant percentage of the aptamers sufficiently restrict
of 4-methylquinoline with propanesultone yielded inner salt
1, which was subsequently converted to the reactive hemi-
dye 2 by condensation with N,N-diphenylformamidine. The
dye was formed by reaction of 2 with 1-methyl-2,3,3-
trimethyl-3H-indolium bromide under basic conditions.
(4) Silva, G. L.; Ediz, V.; Yaron, D.; Armitage, B. A. J. Am. Chem.
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11616-11622.
(7) Ellington, A. D.; Szostak, J. W. Nature 1990, 346, 818-822.
Org. Lett., Vol. 10, No. 8, 2008
1562

Figure 3. DIR fluorescence enhancement by RNA aptamers
obtained from in vitro selection. [DIR] ) [RNA] ) 200 nM.
Figure 5. Fluorescence titration experiment for determining KD
of DIR-aptamer interaction. [DIR] ) 100 nM. Data points and
error bars represent mean and standard deviation from three separate
trials. Red line is fit to 1:1 binding model.
the conformational mobility of DIR to generate large fluoro-
genic effects.
One of the aptamers (DIR-Apt1) was chosen for further
study. This aptamer strongly enhances DIR fluorescence,
whereas the starting RNA pool has no effect on dye fluor-
escence (Figure 4). Two experiments were performed to
spite of the anionic sulfonate group and the lack of strong
hydrogen-bonding groups, a high-affinity RNA aptamer was
readily selected for binding to DIR.
There are two other examples in the literature of RNA
fluoromodules. In one case, an RNA aptamer for malachite
green binds with KD ) 117 nM and enhances fluorescence
more than 1000-fold.9 While the fluorescence enhancement
is greater than for the DIR fluoromodule reported here, the
cyanine dyes are much better suited to obtaining a catalog
of variable-color fluoromodules than are the triphenylmethyl
dyes represented by malachite green. Meanwhile, Sparano
and Koide selected RNA aptamers for a dimethylaniline
(DMA) derivative.10 Attachment of the DMA group to
fluorescein led to substantial quenching of the dye fluores-
cence, most likely because of electron transfer from DMA
to the dye. Addition of the DMA-binding RNA aptamer
restored the fluorescence, although micromolar concentra-
tions of the aptamer were required. This strategy is potentially
generalizable to any dye that can be quenched by the DMA
group, provided the necessary dye-DMA conjugate can be
prepared.
Most recently, Sando and co-workers reported selection
of DNA aptamers for derivatives of Hoechst 33258 that were
engineered to have low binding to double-stranded DNA.11
While the DNA aptamers cannot be expressed in cells, it is
reasonable to expect that genetically encodable RNA aptam-
ers could be selected for the same dye. The main advantage
of our fluorogenic cyanines over Hoechst analogues is the
broader wavelength range that is accessible to the cyanines.
The fluoromodule described here should find applications
as a signaling component for sensors. For example, the DIR-
Apt1 aptamer could be fused with another aptamer that binds
Figure 4. Fluorescence enhancement of DIR by DIR-Apt1 and
na¨ıve RNA pool. Samples contained 200 nM DIR and were excited
at 602 nm.
verify the specificity of binding. First, the fluorescence en-
hancement of DIR-Apt1 was unaffected by a 10-fold excess
of the starting RNA pool (data not shown). Second, addition
of an RNA aptamer selected for binding to the structurally
unrelated dye malachite green8 enhances DIR fluorescence
less than 2-fold (Figure S1, Supporting Information).
A continuous-variations experiment verified that DIR-Apt1
forms a 1:1 complex with DIR (Figure S2, Supporting
Information). Fluorescence titration experiments were then
used to determine the dissociation constant for the dye-
aptamer complex: KD ) 86 ( 17 nM (Figure 5). Thus, in
(9) Babendure, J. R.; Adams, S. R.; Tsien, R. Y. J. Am. Chem. Soc.
2003, 125, 14716-14717.
(10) (a) Sparano, B. A.; Koide, K. J. Am. Chem. Soc. 2005, 127, 14954-
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1803.
Org. Lett., Vol. 10, No. 8, 2008
1563

to a separate target in order to create allosteric bifunctional
aptamers that bind DIR and, therefore, “light up” only when
the target molecule is bound.12 Alternatively, genetic encod-
ing of aptamers has been used to regulate translation of
mRNA through reversible binding of a small molecule to
its cognate aptamer that is transcribed along with the RNA
of interest.13 Similarly, the DIR-binding aptamer could be
genetically encoded and expressed inside cells not only for
controlling translation but alternatively for detecting tran-
scription. In addition, DIR should be readily applied to
selection of protein apomodules based on single-chain
antibody fragments using the approach described recently
by our center.14
design of the fluorogenic dye. For years, unsymmetrical
cyanines have been designed to bind to DNA through
nonspecific intercalation. Incorporating sterically bulky and/
or anionic substituents (as in DIR) yields dyes that retain
fluorogenic behavior in response to conformational restriction
while suppressing nonspecific binding to DNA and RNA.
Using these strategies together with established variations
in heterocycle structure and polymethine bridge length will
produce a family of dyes spanning the visible and near-IR
regions of the spectrum. Combining rational design and
synthesis of these dyes with the power of in vitro selection
should ultimately yield a catalog of dye-RNA fluoromodules
for applications in fluorescence sensing and labeling.
In conclusion, we report the design and synthesis of a new
fluorogenic dye, dimethylindole red, and selection of high-
affinity RNA aptamers that activate the fluorescence of the
dye. The fluorogenic behavior of the dye is likely due to
torsional restriction of the excited state when in a viscous
solvent or when confined by a tightly binding, specific RNA
aptamer. While other fluorescence-activating aptamers have
been reported, the novelty of our approach lies in the rational
Acknowledgment. We thank the NIH (1 1U54RR022241-
01) for support of this research and Dr. Brigitte Schmidt
(Molecular Biosensor and Imaging Center) for helpful
discussions. We also acknowledge the CMU Center for
Molecular Analysis, supported by NSF Grant No. DBI-
9729351 and the NMR facility, funded in part by NSF Grant
No. CHE-9808188.
(12) Stojanovic, M. N.; Kolpashchikov, D. M. J. Am. Chem. Soc. 2004,
126, 9266-9270.
(13) (a) Werstruck, G.; Green, M. R. Science 1998, 282, 296-298. (b)
Suess, B.; Fink, B.; Berens, C.; Stentz, R.; Hillen, W. Nucleic Acids Res.
2004, 32, 1610-1614. (c) Mandal, M.; Breaker, R. R. Nat. ReV. Mol. Cell
Biol. 2004, 5, 451-463. (d) Desai, S.; Gallivan, J. P. J. Am. Chem. Soc.
2004, 126, 13247-13254.
(14) Szent-Gyorgi, C.; Schmidt, B. A.; Creeger, Y.; Fisher, G. W.; Zakel,
K. L.; Adler, S.; Fitzpatrick, J. A. J.; Woolford, C. A.; Yan, Q.; Vasilev,
K. V.; Berget, P. B.; Bruchez, M. P.; Jarvik, J. W.; Waggoner, A. S. Nature
Biotechnol. 2007, 26, 235-240.
Supporting Information Available: Synthetic details and
spectral data for DIR and DIR-biotin conjugate; competition
experiments with malachite green (MG) and MG-binding
aptamer; and continuous-variations experiment to determine
binding stoichiometry for DIR and DIR-Apt1. This material
is available free of charge via the Internet at http://pubs.acs.org.
OL702920E
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Org. Lett., Vol. 10, No. 8, 2008