A "clickable" styryl dye for fluorescent DNA labeling by excitonic and energy transfer interactions

Article abstract of DOI:10.1002/chem.201102622

In conclusion, we have shown that the CyIQ dye represents a promising covalently attached fluorescent label for DNA, since it shows good brightness and excellent photostability. In particular, the photochemical stability of CyIQ is significantly enhanced compared to the routinely applied fluoresceine. The dye is "clickable", that means it can be incorporated postsynthetically and therefore easily into oligonucleotides by using the cycloaddition between azides and acetylenes. In this study, the "click" reaction was performed representatively to 2'-propargylated uridines in DNA. This structural approach has the advantage that the preference for pairing between the modified uridine and adenine in the counterstrand is potentially maintained and thereby the DNA duplex conformation stays stable.[9a] One could envision that this experimentally simple labeling method can be extended to other acetylene-containing building blocks in RNA and DNA, which are either commercially or synthetically available. Beside the application as single internal DNA marker, the CyIQ label can be combined with itself and TR, all chromophors adjacent to each other as intra-strand fluorophore pairs. Excitonic interactions between the CyIQ dyes and energy transfer interactions between CyIQ and thiazole red provide interesting alternatives for fluorescence readouts for DNA hybridization, which are either fluorescence enhancement or fluorescence color change, respectively.

Full text of DOI:10.1002/chem.201102622

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DOI: 10.1002/chem.201102622
A “Clickable” Styryl Dye for Fluorescent DNA Labeling by Excitonic and
Energy Transfer Interactions
Moritz M. Rubner, Carolin Holzhauser, Peggy R. Bohlꢀnder, and
Hans-Achim Wagenknecht*[a]
Fluorescent bioanalytics and fluorescent cell imaging of
nucleic acids demands the design and synthesis of new dyes
that exhibit potentially interesting optical properties particu-
larly in combination with other dyes.[1] Dual covalent label-
ing of nucleic acids has turned out to be very useful for cre-
ating tailor-made fluorescent probes for such applications.[2]
For instance, hybridization-sensitive probes can be created
by two covalently attached thiazole orange (TO) fluoro-
phores that interact excitonically only in the single strand.[3]
On the other hand, observable fluorescence color changes
can be achieved by applying an energy transfer that occurs
between two adjacent fluorescent labels that are forced into
close proximity by the surrounding DNA architecture.[4]
With respect to the preparation of such dual labeled oligo-
nucleotides recent developments of the “click”-type Huis-
gen–Sharpless–Meldal cycloaddition for nucleic acids offers
great advantages since (unexpectedly) complicated and
Scheme 1. Synthesis of the CyIQ azide 7: a) dioxane, 1018C, 19 h; 67%;
b) piperidine, EtOH, reflux, 24 h; 87%; c) CBr4, Ph3P, dichloromethane,
room temperature, 18 h; 70%; d) NaN3, DMF, room temperature 18 h;
65%.
time-consuming syntheses of DNA building blocks for po-
tentially interesting dyes can be avoided.[5]
The cyanine-styryl-type dyes represent very promising
candidates for nucleic acids based primarily on their recent-
ly reported property as bright noncovalent binders to
RNA.[6] Herein, we present the synthesis of a “clickable”
CyIQ (cyanine indole quinoline) fluorophore that can be
easily attached covalently to any desired 2’-position in nu-
cleic acids. Moreover, the optical properties of the CyIQ
dye can be tuned by excitonic interaction with a second ad-
jacent label, or by energy transfer processes with thiazole
red (TR)[7] as a second fluorescence base surrogate.
The CyIQ dye consists of a quinoline part that is conju-
gated with an indole by a styryl-type bridge. The dye can be
prepared in a four-step synthesis including the introduction
of the “clickable” azide functionality (Scheme 1). It starts
with commercially available 2-methyl quinoline (1) that is
alkylated with 3-iodopropanole (2) as the short linker be-
tween the dye and the azide group. Condensation of the
methyl group of 3 in the presence of piperidine yields dye 5.
In the next steps, an Appel reaction leads to iodide 6, and
the azide is introduced by nucleophilic substitution to yield
7, ready for postsynthesis DNA modification.
Due to its positive charge the CyIQ dye 5 is sufficiently
water soluble for titration experiments with a random-se-
quence double-stranded DNA (Figure 1). The absorption
spectra of this titration reveal significant changes; especially
the absorption shift from 465 nm (without DNA) to 472 nm
(with DNA) indicates excitonic dye–dye interactions of 5 (in
the absence of DNA), which are interrupted by the interac-
tion of the dye with an increasing amount of DNA. These
dye–dye interactions cause
a
significant fluorescence
quenching of 5 in aqueous solution without DNA. With in-
creasing amounts of DNA the fluorescence intensity of the
dye is recovered and finally enhanced to the 17-fold when
the saturation plateau is reached. This fluorescence en-
hancement is typical for cyanine dyes and higher than stan-
dard ethidium bromide; with the latter dye a tenfold in-
crease was observed in similar titrations.[8]
[a] Dipl.-Ing. M. M. Rubner, Dipl.-Chem. C. Holzhauser,
Dipl.-Chem. P. R. Bohlꢀnder, Prof. H.-A. Wagenknecht
Karlsruhe Institute for Technology, Institute for Organic Chemistry
Fritz-Haber-Weg 6, 76131 Karlsruhe (Germany)
Fax : (+49)721-608-44825
With CyIQ azide 7 in hand, we performed “click”-type re-
actions according to our published protocols.[9] The dye was
attached to 2’-propargylated uridines in oligonucleotides in
E-mail: Wagenknecht@kit.edu
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201102622.
Chem. Eur. J. 2012, 18, 1299 – 1302
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1299

dyes are combined in the same strand adjacent to each
other. Finally, in DNA3 the CyIQ dye is combined with the
red emitter TR, again in the same strand. The latter dye was
incorporated into oligonucleotides as a DNA base substitu-
tion by using the corresponding synthetic phosporamidite as
a DNA building block.[7]
The CyIQ dye as a single modification in oligonucleotides
displays solid optical properties that make this dye a suita-
ble candidate for fluorescent bioanalytics and imaging.
CyIQ exhibits an absorption maximum at 495 nm that lies in
an excitation range comparable to well-known dyes, such as
thiazole orange, BODIPY, fluoresceine and Cy3. The fluo-
rescence at 565 nm occurs with a quantum yield of 0.07 in
the duplex; together with an extinction coefficient of
41500mÀ1 cmÀ1 a brightness of 2900mÀ1 cmÀ1 is achieved.
These values are comparable to single thiazole orange labels
in DNA and therefore show the applicability of CyIQ for
bioanalytical applications. The melting temperatures (Tm,
see Table S1 in the Supporting Information) reveal repre-
sentatively for two duplexes that there is no significant de-
crease of thermal stability as a result of a single CyIQ 2’-
modification: DNA4 (bearing the dye in a T–A environ-
ment) has a Tm of 62.08C, which is only 0.58C lower com-
pared to a completely unmodified one (T instead of U),[9a]
and DNA7 (bearing the dye in a G–C environment) has a
Tm of 69.78C, which is even 1.78C higher than a comparable
unmodified duplex (see the Supporting Information).[9a]
The photostability of fluorescent dyes, however, is an in-
creasingly critical parameter for applications especially with
respect to fluorescent cell imaging. Hence, we tested the
photochemical stability of CyIQ as a single modification in
double stranded DNA in comparison with the standard dyes
thiazole orange, fluoresceine, BODIPY and Cy3, which
absorb in a similar wavelength range. The irradiation was
performed with a 75 W Xe arc lamp and a <305 nm cut-off
filter. The photobleaching was followed by UV/Vis absorp-
tion spectroscopy and fluorescence spectroscopy at the dye-
typical wavelengths between 490 and 520 nm. The results
show that the CyIQ dye is significantly more stable com-
pared to thiazole orange, BODIPY and fluoresceine
(Figure 2). Extensive photobleaching to approximately 10%
remaining fluorescence intensity was observed with
BODIPY as an internal DNA modification[11] in less than
10 min, and with thiazole orange[7] and fluoresceine in less
than 2 h. Remarkably, after 2 h irradiation of CyIQ-modi-
fied DNA1 approximately 75% fluorescence was main-
tained. Extensive photobleaching of the CyIQ dye in DNA
was observed only after irradiation for a significantly pro-
longed time (more than 15 h). Only the Cy3 dye was more
stable.
Figure 1. UV/Vis absorption spectra (left) and fluorescence spectra
(right) of the titration of 5 with DNA. Solid line: free dye (24.0 mm) with-
out DNA; gray line: intervening titration steps; dotted line: DNA
(13.2 mm) with 5 (24.0 mm), at the beginning of saturation (50 mm Na-Pi-
buffer; pH 7; lexc =495 nm).
60–70% yield. The corresponding phosphoramidite is com-
mercially available. This structural approach has the major
advantage that it places the CyIQ dye into close proximity
of the DNA duplex while potentially keeping the Watson–
Crick-type base pairing of the uridine at the site of modifi-
cation intact.[10] DNA1 represents a reference duplex that
bears a single CyIQ modification in the middle (Scheme 2).
We studied the optical properties of the single CyIQ modifi-
cation also in different base pair environments (see DNA4–
DNA7 in the Supporting Information). In DNA2, two CyIQ
Nevertheless, together with the above-mentioned fluores-
cence properties it becomes clear that CyIQ represents an
important alternative to the routinely applied dyes thiazole
orange, BODIPY and, most importantly, fluoresceine as an
internal and covalent label for DNA. In contrast to the
more photostable Cy3 dye it is important to point out, that
the 2’-modification with the CyIQ dye has the advantage of
Scheme 2. Sequences of modified DNA1–DNA3.
1300
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Chem. Eur. J. 2012, 18, 1299 – 1302

A “Clickable” Styryl Dye for DNA Labeling
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Figure 2. Photobleaching of different dyes covalently attached to DNA
(2.5 mm in 10 mm Na-Pi-buffer, pH 7.0, 250 mm NaCl) carried out with a
75 W Xe arc lamp and a 305 nm cut-off filter (for the sequences of
DNA8–DNA12, see the Supporting Information). Double stranded
~
&
*
DNA9 (BODIPY; ), DNA10 (fluorescein; ); DNA1 (CyIQ, );
!
^
DNA8 (TO; ); DNA12 (Cy3, ).
maintaining Watson–Crick base pairing (based on similar
experiments published recently)[9] and thereby keeps the
DNA duplex intact.
In another set of experiments, the CyIQ dye was com-
bined with itself or thiazole red as second adjacent fluoro-
phore in DNA2 and DNA3, respectively (Figure 3). The ab-
sorption spectrum of single stranded DNA2 bearing the in-
trastrand CyIQ–CyIQ fluorophore pair exhibited a signifi-
cant hypsochromic shift to 463 nm. Based on the absorption
changes that were observed in titration experiments with
the CyIQ dye 5 (Figure 1) this result supports the idea that
the CyIQ dye undergoes excitonic interactions. Typically,
such H aggregates of DNA-interacting dyes exhibit signifi-
cant fluorescence quenching,[12] that is, in fact, observed for
single-stranded DNA2. The fact that the fluorescence is not
quenched quantitatively can be attributed probably to the
conformational dynamics of DNA as a biopolymer and the
presence of conformational subensembles with unstacked
CyIQ dyes. Nevertheless, the excitonic interactions of the
stacked ensemble can be interrupted by annealing DNA2
with the corresponding complementary counterstrand. As a
result an approximately sevenfold fluorescence enhance-
ment is observed from the doubly CyIQ-labeled single to
the double strand. Accordingly, the absorption maximum is
shifted back to 495 nm.
On the other hand, the fluorescence of CyIQ as a cova-
lently attached label in DNA shows a pretty good overlap
with the absorption of TR as a base substitution in DNA.[7]
Hence, it is reasonable to assume that energy transfer might
take place between the two dyes. Indeed, if the doubly la-
beled single strand DNA3 is excited at the CyIQ-typical
wavelength 495 nm a significant amount of the thiazole red
fluorescence at approximately 660 nm is obtained. Interest-
ingly, this energy transfer process is blocked nearly com-
pletely when single-stranded DNA3 is annealed with the
Figure 3. UV/Vis absorption (left) and fluorescence (right) of DNA1
(top), DNA2 (middle) and DNA3 (bottom); solid red lines represent
single strands, dotted blue lines are double strands, each 2.5 mm in 10 mm
Na-Pi-buffer, pH 7.0, 250 mm NaCl, lexc =495 nm.
corresponding counterstrand. Accordingly, the red fluores-
cence vanishes and turns into a green color.
In conclusion, we have shown that the CyIQ dye repre-
sents a promising covalently attached fluorescent label for
DNA, since it shows good brightness and excellent photosta-
bility. In particular, the photochemical stability of CyIQ is
significantly enhanced compared to the routinely applied
fluoresceine. The dye is “clickable”, that means it can be in-
corporated postsynthetically and therefore easily into oligo-
nucleotides by using the cycloaddition between azides and
acetylenes. In this study, the “click” reaction was performed
representatively to 2’-propargylated uridines in DNA. This
structural approach has the advantage that the preference
for pairing between the modified uridine and adenine in the
counterstrand is potentially maintained and thereby the
DNA duplex conformation stays stable.[9a] One could envi-
sion that this experimentally simple labeling method can be
extended to other acetylene-containing building blocks in
RNA and DNA, which are either commercially or syntheti-
cally available. Beside the application as single internal
DNA marker, the CyIQ label can be combined with itself
and TR, all chromophors adjacent to each other as intra-
Chem. Eur. J. 2012, 18, 1299 – 1302
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1301

H.-A. Wagenknecht et al.
Angew. Chem. 2009, 121, 6602–6606; Angew. Chem. Int. Ed. 2009,
48, 6480–6484.
strand fluorophore pairs. Excitonic interactions between the
CyIQ dyes and energy transfer interactions between CyIQ
and thiazole red provide interesting alternatives for fluores-
cence readouts for DNA hybridization, which are either
fluorescence enhancement or fluorescence color change, re-
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Keywords: click chemistry
transfer · exciton
· cyanine · DNA · energy
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