Brightness enhanced DNA FIT-probes for wash-free RNA imaging in tissue

Article abstract of DOI:10.1021/ja410674h

Fluorogenic oligonucleotides enable RNA imaging in cells and tissues. A high responsiveness of fluorescence is required when unbound probes cannot be washed away. Furthermore, emission should be bright in order to enable detection against autofluorescent background. The development of fluorescence-quenched hybridization probes has led to remarkable improvement of fluorescence responsiveness. Yet, comparably little attention has been paid to the brightness of smart probes. We describe hybridization probes that combine responsiveness with a high brightness of the measured signal. The method relies upon quencher-free DNA forced intercalation (FIT)-probes, in which two (or more) intercalator dyes of the thiazole orange (TO) family serve as nucleobase surrogates. Initial experiments on multi-TO-labeled probes led to improvements of responsiveness, but self-quenching limited their brightness. To enhance both brightness and responsiveness the highly responsive TO nucleoside was combined with the highly emissive oxazolopyridine analogue JO. Single-stranded TO/JO FIT-probes are dark. In the probe-target duplex, quenching caused by torsional twisting and dye-dye contact is prevented. The TO nucleoside appears to serve as a light collector that increases the extinction coefficient and transfers excitation energy to the JO emitter. This leads to very bright JO emission upon hybridization (F/F₀ = 23, brightness = 43 mL mol^-1 cm ^-1 at λ_ex = 516 nm). TO/JO FIT-probes allowed the direct fluorescence microscopic imaging of oskar mRNA within a complex tissue. Of note, RNA imaging was feasible under wide-field excitation conditions. The described protocol enables rapid RNA imaging in tissue without the need for cutting-edge equipment, time-consuming washing, or signal amplification.

Full text of DOI:10.1021/ja410674h

Article
pubs.acs.org/JACS
Brightness Enhanced DNA FIT-Probes for Wash-Free RNA Imaging in
Tissue
Felix Hovelmann,† Imre Gaspar,‡ Anne Ephrussi,‡ and Oliver Seitz*,†
̈
†Institut fur Chemie der Humboldt-Universitat zu Berlin, 12489 Berlin, Germany
̈
̈
‡European Molecular Biology Laboratory, 69117 Heidelberg, Germany
S
* Supporting Information
ABSTRACT: Fluorogenic oligonucleotides enable RNA
imaging in cells and tissues. A high responsiveness of
fluorescence is required when unbound probes cannot be
washed away. Furthermore, emission should be bright in order
to enable detection against autofluorescent background. The
development of fluorescence-quenched hybridization probes
has led to remarkable improvement of fluorescence
responsiveness. Yet, comparably little attention has been paid
to the brightness of smart probes. We describe hybridization
probes that combine responsiveness with a high brightness of
the measured signal. The method relies upon quencher-free DNA forced intercalation (FIT)-probes, in which two (or more)
intercalator dyes of the thiazole orange (TO) family serve as nucleobase surrogates. Initial experiments on multi-TO-labeled
probes led to improvements of responsiveness, but self-quenching limited their brightness. To enhance both brightness and
responsiveness the highly responsive TO nucleoside was combined with the highly emissive oxazolopyridine analogue JO. Single-
stranded TO/JO FIT-probes are dark. In the probe−target duplex, quenching caused by torsional twisting and dye−dye contact
is prevented. The TO nucleoside appears to serve as a light collector that increases the extinction coefficient and transfers
excitation energy to the JO emitter. This leads to very bright JO emission upon hybridization (F/F0 = 23, brightness = 43 mL
mol−1 cm−1 at λex = 516 nm). TO/JO FIT-probes allowed the direct fluorescence microscopic imaging of oskar mRNA within a
complex tissue. Of note, RNA imaging was feasible under wide-field excitation conditions. The described protocol enables rapid
RNA imaging in tissue without the need for cutting-edge equipment, time-consuming washing, or signal amplification.
enhanced molecular beacon type probes. Metals,8 small
INTRODUCTION
■
molecules,9 as well as modifications of the hairpin stem10−13
Fluorescently labeled oligonucleotides are among the most
influence fluorescence quenching in the unbound state and,
important tools used to detect and localize specific nucleic acid
targets in biological fluids, cells, and tissues.1−4 Significant effort
has been invested in the development of smart probes that
therefore, allowed improvements of the achievable signal-to-
background ratio. The use of enhanced quencher dyes,14,15
nanoparticles,16−19 or graphene20−23 allowed further increases
show increases in fluorescence upon hybridization. Such
responsive probes enable applications in real-time PCR,5
fluorescent in situ hybridization (FISH), and live cell RNA
imaging6 when separation of unbound probes is undesired,
difficult, or even impossible.
in fluorescence quenching. To foster dye−dye interactions,
fluorescent dyes were incorporated as base surrogates within
the double helical stem region24−29 or coupled to peptide
nucleic acid (PNA) scaffolds.30,31 Recently, intracellular RNA
was detected by means of target-triggered fluorogenic reactions
between two reactive probes.32,33
The described efforts have led to remarkable improvement of
the fluorescence responsiveness. However, comparably little
attention has been paid to the brightness of fluorescent
signaling. The quencher moieties typically involved inevitably
reduce brightness, not only in the unbound state, but also in the
target-bound state of the probe. For envisaged applications in
wash-free FISH and live cell RNA imaging we sought
hybridization probes that combine responsiveness with the
The performance of fluorogenic hybridization probes within
cellular systems is characterized by two main features:
responsiveness and brightness of fluorescent signaling. High
responsiveness is required for discrimination of the unbound
probe, whereas brightness is critical to reliably detect the probe
signal against a given fluorescence background of the cell. The
introduction of DNA-based molecular beacons marked an
important event in nucleic acid technology.7 In the original
molecular beacon format, hairpin formation is used to bring
two terminally appended dyes into proximity. Hybridization
with the target opens the hairpin structure. The increase of the
distance between the reporter fluorophore and the quencher
dye is accompanied by enhancement of fluorescence emission.
Substantial efforts have been focused on the development of
Received: October 24, 2013
Published: December 2, 2013
© 2013 American Chemical Society
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high brightness of fluorescent signaling required to detect and
localize mRNA in autofluorescent tissues.
Our work relies upon quencher-free DNA FIT-probes, in
which an intercalator dye of the thiazole orange (TO) family
serves as a surrogate nucleobase (Figure 1A).34 Hybridization
80000 L mol−1 cm−1) and quantum yields (ϕds ≤ 0.35). We
envisaged two approaches to increase the brightness. In multi-
TO-labeled probes, the incorporation of multiple ‘TO bases’
will allow for more effective absorption of photons (Figure 1B).
The formation of the probe−target duplex will lead to an
increase of TO emission which will be accompanied by a color
change, owing to the separation of the TO units and the
accompanying disruption of excitonic interactions. There is
literature precedence for multilabeled hybridization probes, but
their brightness has not been assessed.25,43,44
Alternatively, the ‘TO base’ will be accompanied by a second
fluorescent base surrogate which shows a small bathochromic
shift so that FRET could occur along an energy gradient
(Figure 1C). This second dye should be selected to enable
overlap of the absorption spectra, in which case the ‘TO base’
could act as a light collector. We chose the JO chromophore
that is red-shifted by 10 nm.45 Owing to a large extinction
coefficient (εmax ≈ 110 000 L mol−1 cm−1) and a high quantum
yield (ϕmax ≈ 0.8), the JO dye is significantly brighter than TO,
but the responsiveness of JO-based FIT-probes is low (vide
infra). Nevertheless, for a combined TO/JO-containing FIT-
probe we predicted a high responsiveness, because contact
quenching will reduce the fluorescence of both dyes in single-
stranded probes.
Synthesis. The TO and JO dyes were introduced into DNA
by means of preformed nucleotide building blocks. Serinol-TO
and serinol-JO phosphoramidites were synthesized by adapting
a previously described method (Scheme S1).34 Oligonucleotide
synthesis was performed by applying known protocols.46
Dual-TO FIT-Probes. The incorporation of a second
fluorescence base surrogate will change the optical properties
of a FIT-probe. To rule out that the change of brightness is due
to the sequence context around the second dye, we measured
the dual label probes as well as both single label probes. The
TO-nucleotide was walked through 15 positions of a 27mer
oligonucleotide directed against a segment of neuraminidase
mRNA of H1N1-influenza (Figure 2, left panel). The positional
screen was used to characterize the optical properties as well as
the effect of TO incorporation on duplex stability (Table S1).
Particular emphasis was placed on the brightness (Br =
ε(excitation) × quantum yield) of the probe−target complex.
The fluorescence quantum yields of single stranded TO-
labeled probes were rather low (ϕss = 0.02−0.04, see also Table
S1). As previously reported, formation of the probe−target
RNA-duplexes was accompanied by a significant enhancement
of fluorescence quantum yields (up to ϕds = 0.24, Figure 2A,
see also Table S1).34 Nine of the 15 probes tested provided
useful (≥ 5-fold) enhancement of fluorescence intensity upon
hybridization. However, the assessment of responsiveness was
based on quantum yield enhancement and brightness values
(1.7−9.3) rather than on fluorescence intensity, because this
better complies with the experimental setup in fluorescence
microscopic imaging, where emitted light is measured over
wide band-pass filters.
Figure 1. Schematic representation of (A) single TO, (B) double TO,
and (C) TOJO-labeled DNA FIT-probes.
forces the dye to intercalate between predetermined base
pairs.35 The concomitant viscosity increase restricts torsions
around the methine bridge and prolongs the lifetime of the TO
excited state.36 FIT-probes are highly responsive and exhibit
marked increases in fluorescent emission upon hybridiza-
tion.31,37−42 However, the detection of RNA in autofluorescent
tissue has remained challenging (vide infra). We sought a
means to increase the brightness of fluorogenic DNA probes
without compromising their responsiveness. To achieve this
goal, we compared two approaches that involve the
introduction of and energy transfer between two or more
fluorescent base surrogates. We show that high brightness is
obtained when two different, yet spectrally overlapping ‘dye
nucleobases’ of the TO family are combined. The probes are
brighter than fluorescein-based molecular beacons and conven-
tional unquenched linear probes. The usefulness was
demonstrated in imaging of RNA within a challenging tissue.
Wash-free FISH with brightness enhanced DNA FIT-probes
enabled the localization of oskar mRNA in developing ovaries
of Drosophila melanogaster by using both narrow band and
broad band excitation as applied in confocal fluorescence and
cost-efficient wide-field epifluorescent microscopes, respec-
tively.
In the single-stranded state, the dual TO-probes showed a
rather low emission that was broad and red-shifted, from 515
nm (TO monomer emission) to up to 590 nm (Figure 2B, see
also Figure S2). The absorbance spectra revealed a pronounced
shoulder at 485 nm and in many cases the maximum was blue-
shifted from 515 nm by 30 nm. These characteristics resemble
the properties of the DNA stain TOTO and other TO dimers
known to from H-aggregates.25,47−49 Hybridization with the
RNA target disrupted the TO-TO aggregates, resulting in
RESULTS
■
Thiazole orange containing FIT-probes show remarkable
responsiveness Fds/Fss of fluorescence upon hybridization.37−41
However, the brightness of fluorescence Fds in the probe
bound state is limited by the extinction coefficient (εmax
≈
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Figure 2. (A−D) Comparison of absorbance (blue) and fluorescence emission (black) of the indicated probes before (dashed) and after (solid)
hybridization with complementary RNA (AAAUACAACGGC AUAAUAACUGAAACC). (E−H) Comparison of enhancement in fluorescence
quantum yield (ϕds/ϕss, black bars) and brightness (red bars) in double-stranded state of single TO (E), double TO (F), single JO (G), and TO-JO-
labeled probes (H). Conditions: 0.5 μM probe and 5 equiv RNA-target, when added, in PBS (100 mM NaCl, 10 mM Na2HPO4, pH 7) at 37 °C; for
single and double TO: λex = 485 nm, λem = 500−700 nm; for JO and TOJO: λex = 500 nm, λem = 510−700 nm, slitex = 5 nm, slitem = 5 nm. Scale bar
are the same for (A-D) and (E-F).
absorbance and emission spectra of isolated TO dyes.50 For
multi-TO-labeled probes, the hybridization was accompanied
by a change in emission color, which was observable even by
the naked eye (Figure S4).
The introduction of a second TO nucleotide resulted in
increased extinction coefficients. However, the quantum yields
were reduced by nearly 50% and, therefore, brightness of the
target-bound state did not improve (Figure 2B, see also Table
S1). We characterized the distance dependence of this self-
quenching effect (Figure 3, black diamonds). The local minima
in self-quenching at 4 nt and 8 nt spacing probably reflect the
orientation of the transition dipole moments.51,52 Obviously,
self-quenching was strongest when the TO units were in close
proximity and generally lower at increased distance. Self-
quenching is an effect of excitation energy homotransfer. The
Figure 3. Distance dependency of quenching (Q) in dual-TO FIT
probes (black diamonds) and TO/JO-labeled probes (blue square). Q
resulting delocalized excited states are believed to be
particularly vulnerable to the quench processes.53 Our experi-
= 1 − Fds(xy)/(Fds(x) + Fds(y)) with Fds(xy) is the emission intensity
ments showed that the distance of 13 nucleotides was not
sufficient to completely avoid self-quenching. Given the
relatively small rise per base pair (2.9 Å) of an A-type DNA-
RNA duplex, we assumed that rather long oligonucleotides
would be required to disrupt homo-FRET between the TO
dyes.52,54
at 535 nm of the double-labeled probe and Fds(x) or Fds(y) is the
intensity of corresponding single labeled probes at 535 nm upon
excitation at 485 nm.
TO unit with the lower quantum yield is limiting to the
achievable overall quantum yield, that is, dissipation of energy
would be predominantly caused by this ‘weakest piece of the
chain’. This assumption is in line with the optical properties of
The data exposed the significance of energy transfer between
the TO-moieties in multilabeled probes. We surmised that the
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a
Table 1. Comparison of TO-, JO-, and TO/JO-FIT-Probes with Molecular Beacon and Linear Probes
b
Br
probe
type
Sequence, X = TO, Y = JO
ϕss
ϕds
ϕds/ϕss
[mL mol−1 cm−1
]
TO-neu-e
TO2-neu-en
JO-neu-e
single TO
double TO
single JO
GGTTTCAGTT X TTATGCCGTTGTATTT
GGTTTCAGTT X TTATGCCG X TGTATTT
GGTTTCAGTT Y TTATGCCGTTGTATTT
GGTTTCAG X TATTAT Y CCGTTGTATTT
0.02
0.014
0.14
0.03
0.23
0.17
0.63
0.36
0.26
10.0
12.5
4.6
9.3
12.0
18.9
28.5
TOJO-neu-cj TOJO
12.1
8.7
c
c
c
MB-neu-1
molecular beacon 6FAM-CCGACTTTCAGTTATTATGC CGTTGTATTTGTCGG-
BHQ1
0.03
15.5
FAM-neu
FISH
GGTTTCAGTTATTATGCCGTTGTATTT-6FAM
0.57
0.46
0.8
20.5
a
Conditions: For TO see Figure 2; for JO and TO-JO see Figure 4. For MB-Neu-1 and FAM-Neu: 0.5 μM probe and 5 equiv RNA, when added, λex
b
c
= 485 nm, λem = 495−700 nm, in PBS at 37 °C, slitex = 5 nm, slitem = 5 nm. Brightness in target bound state. Absorbance of the quencher was not
subtracted.
probes that contain a third and a fourth TO-dye (Figure S3).
Regardless of the number of TO units involved, the TO
emission remained at similar intensity levels. Given the rather
long reach of self-quenching by energy transfer we inferred that
high brightness can only be obtained when ‘weak pieces of the
chain’ are avoided. We constructed probes in which two strong
emitters were positioned at distances with minimal self-
quenching (4 or 8 nucleotides). The best dual-TO FIT-
probe, TO2-neu-en, showed an 18-fold enhancement of
fluorescence intensity at 535 nm, as compared to 12-fold for
the best single-TO FIT-probe, TO-neu-n (Table S1).
However, the gain in brightness was only 29% (Figure 2D).
We conclude that dual TO-labeling will not provide a notable
advantage in RNA imaging experiments when emission is
measured via the commonly used wide band-pass filters.
TO/JO-Labeled Probes. The experiments on dual-TO
FIT-probes exposed self-quenching as the major factor limiting
the achievable brightness. To improve upon brightness, we
combined the responsive TO nucleotide with a highly emissive
fluorophore such as the JO nucleotide. The JO dye was placed
at 15 different positions within the 27 nt oligonucleotide
(Figure 2C and G, see also Table S2 and Figure S5). The
spectroscopic measurements confirmed that the JO-probes
have higher absorbance and provide brighter emission than the
corresponding TO-labeled probes (Figure 2E). The JO-labeled
probes afforded high quantum yields, in both the single-
stranded and the target-bound state. Thus, the fluorescence of
single JO-probes is bright (Br = 13−20 mL mol−1 cm−1) but
shows comparably modest responsiveness (ϕds/ϕss ≈ 3−4).
We next evaluated TO/JO-containing FIT-probes. Based on
our findings with dual TO-labeled probes, we combined bright
and responsive positions at various distances (2, 4, 5, 6, 7, 8,
and 10 nt). The fluorescence spectroscopic measurements
exposed remarkable performance of TO/JO-FIT-probes. The
single-stranded TO/JO-probes were dark (Figure 2D and H;
see also Figures S6 and S7). The quantum yields in absence of
target remained nearly as low as for single TO-labeled probes
(TO/JO-probes: ϕss = 0.03−0.04) and significantly lower than
the quantum yields of the corresponding JO-only probes (JO-
probes: ϕss = 0.14−0.16, Table S2). The shape and the change
of the absorbance spectra upon hybridization suggest that the
quenching of the JO emission is caused by contact quenching
and/or exciplex formation (Figure 2D, see also Figures S6 and
S7). The addition of RNA target led to remarkable florescence
enhancements that exceeded the responsiveness of the best
single TO-FIT-probes. Of note, 7 out of 8 probes provided an
enhancement in quantum yield by factor ∼10 upon duplex
formation (Figure 2H). Most importantly, the quantum yields
(ϕds = 0.27−0.36) together with the high extinction
coefficients, caused by spectral overlap, resulted in significantly
improved brightness (Br = 20−29 mL mol−1 cm−1).
To characterize distance effects in double-stranded TO/JO-
labeled probes, the intensity of fluorescence at 535 nm was
measured and compared with the sum of fluorescence
intensities of the single labeled components. The quenching
values Q = 1 − Fds(TOJO)/(Fds(TO) + Fds(JO)) are
considerably smaller than calculated for TO2-probes and
show rather small deviations from 0 (Figure 3, blue squares).
Due to the high spectral overlap it is not possible to quantify
energy transfer in TO/JO-probes by steady-state fluorescence
measurements. However, in a ‘gedankenexperiment’ two
scenarios may be distinguished: (1) At large distances and/or
poor orientation of transition dipole moments energy transfer
between TO and JO would be inefficient and the TO and JO
emission signals would be additive. This would result in bright
emission of the TO/JO-probe. (2) At shorter distance and/or
suitable orientation of the chromophores energy transfer would
become efficient. Following the gradient to longer wavelength,
the energy transfer from TO to JO should be favored, because
the spectral overlap between TO emission and JO absorption is
higher than the overlap between JO emission and TO
absorption (Figure S8). The TO dye would serve as a light
collector and the brightness of emission would, again, be high
due to the high extinction coefficient. This case may result in
emission signals that exceed the sum of the components (Q <
0) because the relatively low TO quantum yield will no longer
be limiting. Of note, six out of the eight TOJO-probes belonged
to this category (see also Figure S9). Regardless of the
mechanisms involved, we conclude that TO/JO probes
combine the best of the TO and the JO worlds and allow for
improvement of both probe responsiveness and the brightness.
Comparison of Different Fluorescent Probes. By using
conventional fluorophore−quencher systems it is difficult to
synergistically improve upon both responsiveness and bright-
ness of fluorescent signaling. The FAM-labeled oligonucleotide
FAM-neu (Table 1) may be regarded as the prototype of
unquenched hybridization probes used in many applications
including RNA fluorescent in situ hybridization (RNA FISH).
These probes show high brightness of fluorescence (Br = 20.5
mL mol−1 cm−1) but are virtually nonresponsive. This
inevitably calls for carefully optimized wash protocols in
order to remove unbound probe in imaging applications. The
optimized FAM/quencher-labeled molecular beacon probe
MB-neu-1 provided a useful responsiveness, but, as expected,
the presence of the quencher reduced the brightness in the
target bound state by 25% compared to FAM-neu. The best
single-TO FIT-probe TO-neu-e showed an even higher
responsiveness, but the comparatively low brightness (Br =
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9.3 mL mol−1 cm−1) was of concern. By contrast, a high
brightness was obtained for the JO containing FIT-probe JO-
neu-e; however, its responsiveness was rather low. It was the
combination of the TO and the JO nucleosides in TOJO-neu-
cj that allowed for significant improvement of fluorescence
signaling. This TOJO FIT-probe was even brighter than the
conventional unquenched FAM-labeled probe FAM-neu.
Imaging oskar mRNA by Wash-Free Fluorescence in
Situ Hybridization. We tested the FIT-probes within an
intact tissue − the ovaries of Drosophila melanogaster − in situ.
These organs are composed of egg-chambers at different
developmental stages that consist of an oocyte connected to its
15 sibling nurse cells, encapsulated by a single layer of follicular
epithelial cells (see Figure 4). As a target, we used the well-
characterized oskar mRNA, which localizes at the posterior pole
of oocytes from midoogenesis onward (see Figure 4).55 We
deliberately avoided detailed screening and examined 3
positions within the targeted segment (nt 2209−2229 of
CDS) for JO and TO (Figure S10). The two best TO-positions
were used for the construction of TO/JO FIT-probes (Table
2). The resulting TOJO-osk-ac and TOJO-osk-ca conferred
very bright fluorescence signals upon hybridization. The JO
labeled probes JO-osk were synthesized for comparison and
showed the expected low responsiveness and high brightness
(Br = 25−28 mL mol−1 cm−1).
The brightest representative of each class (TO-osk-a, JO-
osk-a, and TOJO-osk-ca, Figure S10) was evaluated in
confocal fluorescence microscopy imaging experiments. We
devised a simple and rapid protocol of performing fluorescent
in situ hybridization (FISH). After a short fixation with 4%
para-formaldehyde and a quick denaturation of RNA secondary
structures by heat, the probes were applied to the dissected
ovaries in presence of detergent (0.3% Tween-20) that
permeabilizes the fixed cells and allows free diffusion of the
probe into the cytoplasm. Following a 45 min long hybrid-
ization, the specimens were mounted on slides within the
hybridization solution, without any washing steps. Due to the
responsiveness of the DNA-FIT probes, oskar mRNA could be
detected in oocytes of different developmental stages (Figure
4A-C, arrows) in patterns identical to those described
previously, despite the presence of unbound probe molecules.56
To assess the visual performance of the probes, we defined the
signal-to-background ratio as the mean signal intensity at the
posterior pole from stage 9 of oogenesis onward (Figure 4A-C,
red arrows) over the mean signal measured in the surrounding
follicular epithelium (Figure 4A, blue arrowheads), which does
not express oskar mRNA. This ratio was found to be the
greatest for the dual-labeled TOJO-osk-ca, both when excited
at its maximum, 530 nm, or at 514 nm (Figure 4D).
As visual screening is usually carried out on cost-efficient
instrumentation, such as on wide-field epifluorescent micro-
scopes operated by a human observer, we decided to simulate
this environment by using dual excitation to mimic a broadband
excitation filter and a simple photomultiplier tube (PMT) with
fully open pinhole and low gain on the detector side. We found
that both egg-chamber morphology and oskar localization in
the different developmental stages was, again, best visualized by
the dual labeled TOJO-osk-ca probe (Figure 4A′). The use of
JO-osk-a yielded a strong oskar signal; however, the elevated
background both outside the egg-chamber and in the follicular
epithelium reduced the contrast necessary to allow proper
recognition of different features of the specimen (Figure 4B′).
Conversely, in the case of the single TO labeled probes, the
Figure 4. FIT probe signal in developing Drosophila oocytes. (A−C′)
Representative images of FISH performed with 0.2 μM TOJO-osk-ca
(A,A′), JO-osk-a (B,B′), or TO-osk-a (C,C′) probes. Confocal
sections (A−C) were taken exciting the probes close to their maximal
absorbance (indicated in the panels), widefield acquisition (A′−C′)
was simulated by exciting with both 510−518 and 526−534 nm laser
lines and imaging with open pinhole (7.7 Airy units). All images were
adjusted to the same linear intensity scale. White arrows mark oskar
mRNA in early oocytes. Orange arrows show central accumulation of
mRNA during stage 8 of oogenesis. Red arrows tag stage 9−10, when
oskar localizes to the posterior pole of the oocyte. The asterisk shows
regions of strong autofluorescence by yolk granules within the oocytes
(e.g., granular signal within the ooplasm in (C′). Bar is 50 μm. (D)
Signal to background ratio (SBR) of FIT probes. Mean signal intensity
was measured close to the posterior pole of stage 9−10 oocytes (red
arrows) in an area engulfing accumulating oskar and was divided by the
mean intensity measured within the surrounding follicle cells
(indicated by blue arrowheads) using different excitation wavelengths
and acquisition setup (514 nm, 530 nm, and 514 + 530 nm indicate
confocal imaging; widefield indicates simulated widefield imaging as
described above). (Mann−Whitney U test, p < 0.001, α = 0.05).
Number of oocytes (N) is indicated next to the key.
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a
Table 2. TO-, JO-, and TO/JO-Probes Targeted against oskar RNA
b
probe
Sequence, X = TO, Y = JO
TM [°C]
ϕss
ϕds
ϕds/ϕss
Br [mL mol−1 cm−1
]
TO-osk-a
TO-osk-b
TO-osk-c
TOJO-osk-ac
TOJO-osk-ca
JO-osk-a
CTCG X TTCA A TAAC T TGCAGT
CTCG T TTCA X TAAC T TGCAGT
CTCG T TTCA A TAAC X TGCAGT
CTCG X TTCAATAAC Y TGCAGT
CTCG Y TTCAATAAC X TGCAGT
CTCG Y TTCA A TAAC T TGCAGT
CTCG T TTCA Y TAAC T TGCAGT
CTCG T TTCA A TAAC Y TGCAGT
56
55
55
47
51
54
56
56
0.04
0.07
0.02
0.05
0.06
0.26
0.53
0.27
0.31
0.18
0.16
0.30
0.42
0.77
0.79
8.7
2.7
7.5
6.2
7.6
3.0
1.5
2.8
14.3
8.3
6.4
24.1
29.1
28.3
26.1
25.2
JO-osk-b
JO-osk-c
0.77
a
b
Conditions: For TO-labeled probes see Figure 2; for JO and TOJO-labeled probes see Figure 4. Brightness in target bound state.
reduced relative strength of the specific oskar signal caused a
similar insufficiency of visibility (Figure 4C′).
that has to be considered when multiple base surrogates are
incorporated within the target recognition sequence concerns
the stability of the resulting probe−target complexes. It has
been shown that the stability loss per base surrogate increases
as the number of base replacements is increased.43,64 In
addition, our work on multi-TO-labeled probes suggests that
the considerable extent of self-quenching limits the achievable
brightness.
The work described herein shows that a combination of two
different, yet spectrally overlapping, fluorophores can provide
increases in brightness. The TO/JO FIT-probes contain two
different fluorescent base surrogates. Torsional twisting around
the methine bridges and intramolecular dye−dye interactions
deplete the dyes excited states in the absence of target. Duplex
formation hinders torsional twisting and contact-mediated
energy transfer between the dyes. Additionally, the TO
nucleoside can transfer energy to the JO emitter, as indicated
by the broad excitation spectra (Figure S9). While JO itself is
highly fluorescent, TO can serve as a light collector. This leads
to very bright emission signals.
Taken together, we have found that the DNA-FIT probes −
especially the TO/JO labeled ones − are well-suited to perform
rapid (∼1.5 h) FISH in complex tissues and allow sufficient
visibility of the target even on non-cutting-edge equipment.
Although conventional RNA probes which contain multiple
DIG-labeled uracil nucleotides and multiple signal amplification
steps, based on peroxidase-coupled anit-DIG antibodies,
resulted in much brighter specimen with higher contrast
(Figure S11), this standard technique of RNA visualization is
very demanding of labor and reagents, rendering its use
cumbersome and expensive in medium- and large-scale screens.
The strongly reduced requirements of DNA-FIT probe based
FISH for labor (∼1.5 h versus the ∼1.5 days for conventional
FISH) and equipment while preserving sensitivity may turn this
method a viable alternative in RNA localization screens of any
scale.
DISCUSSION
■
The TO/JO FIT-probes were brighter and showed higher
enhancements in fluorescence quantum yield upon hybrid-
ization than sequence-analogous FAM/BHQ-labeled molecular
beacons.65 However, the environmental responsiveness of TO
emission could be a reason for concern. The positional
screening within a sequence directed against mRNA coding
for neuraminidase of H1N1 influenza showed useful properties
for > 50% of the tested probes. Two out of three positions
evaluated for oskar FIT-probes proved to be useful. This
resembles the efforts needed to optimize molecular beacons
wherein the length and sequence of the stem structure must be
adjusted to prevent undesired opening and, yet, permit high
signal gain upon hybridization with the target (Figure S12).
The brightness and the mechanism of fluorescence signaling
by TO/JO−DNA FIT-probes is different from other thiazole
orange-containing probes. The so-called Light-Up probes
contain a TO dye appended to one of the terminal ends of
PNA.66 Their brightness is limited due to the use of a single
TO unit. In the ECHO probes, TO dimers connect via a long
linkage to sequence-internal nucleobases.67 The TO dyes form
an H-aggregate in the single strand that is disrupted upon
hybridization and the accompanying intercalation. ECHO
probes optimized for avoidance of intra- and intermolecule
dimerization show high responsiveness which enabled the
detection of abundant targets by RNA-FISH.68,69 While
brightness has not been characterized, published data suggests
that the brightness of the TO-dimer-labeled ECHO-probes is
comparable to analogous probes containing a single TO
dye.49,70 A recent report described RNA duplexes in which one
strand contained a TO-unit, while the other strand featured a
The dynamic regulation of mRNA localization appears to be a
widespread phenomenon that applies to a large fraction of
exported transcripts.57 Studying mRNA localization in the past
two decades involved the extensive use of visual screening
techniques,57−59 which are generally very tedious, time-
consuming applications and often are based on the localization
of RNA binding proteins rather than the mRNA itself.59
Fluorescent in situ hybridization is probably the most
frequently used method for the detection and localization of
specific RNA targets. However, the need for intensive washing
and signal amplification is costly over time. It was our aim to
explore whether FIT-probes with high responsiveness and
brightness might enable rapid fluorescence microscopic analysis
of cells and tissues by wash-free FISH. A variety of fluorescent
oligonucleotide probes provide a turn-on of fluorescence upon
hybridization. Recent contributions successfully applied in the
analysis of intracellular targets include binary linear FRET
probes60 and various forms of DNA and PNA molecular
beacons.6,41,44,61,62 An exquisite responsiveness has been
reported for improved molecular beacons.8−31 However, most
probes required the involvement of a nonfluorescent quencher,
which limits the achievable brightness because quenching still
occurs after the hairpin has been opened. The quencher has
been omitted in wavelength-shifting beacons,63 but remaining
energy transfer in the target-bound state affects their
responsiveness. In-stem beacons,26−29 excimer-controlled bea-
cons,24 and, recently, multilabeled linear beacons43 rely upon
intimate contact between fluorescent base surrogates. Though
remarkably responsive in vitro, the brightness of these
multilabeled probes has not been assessed yet. One drawback
19030
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Journal of the American Chemical Society
Article
thiazole red or Cy3 unit.71 Energy transfer between the two
dyes was used to follow the integrity of the RNA duplex in a
cellular context. The signaling intensity measured after
occurrence of the biological event is, again, limited by the
emission of a single TO-like dye.
ASSOCIATED CONTENT
* Supporting Information
Experimental and characterization data, NMR spectra of
compounds, fluorescence and absorbance spectra of probes.
This material is available free of charge via the Internet at
http://pubs.acs.org.
■
S
The design criteria for bright and responsive hybridization
probes that we have described involve the use of a dye pair that
(a) senses local changes of the structure, (b) enables contact-
mediated quenching in the single-stranded state, (c) provides
overlapping absorption spectra, and (d) includes a bright
acceptor that enables efficient FRET after probe hybridization.
In support of this guideline, many criteria have been met in
TO/ICC-containing PNA FIT-probes, which have been
reported to provide bright fluorescence upon hybridization.31
However, the terminally appended ICC dye does not have the
ability to sense structural changes in the environment. This
probably explains why TO/JO−DNA FIT-probes afford a
higher responsiveness than sequence analogous TO/ICC-PNA
FIT-probes (Figure S13).
AUTHOR INFORMATION
Corresponding Author
*E-mail: oliver.seitz@chemie.hu-berlin.de
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was financially supported by the Deutsche
Forschungsgemeinschaft and by a Kekule-
der Chemischen Industrie for Felix Hovelmann, and by an
EMBL Interdisciplinary Postdoctoral Fellowship (EIPOD) to
Imre Gaspar.
■
́
fellowship of Fonds
̈
One could surmise that the introduction of two JO dyes
could also lead to bright and responsive probes. Though the
dual JO-probes may show a higher responsiveness than single
JO-probes, we expect little increase of brightness, due to self-
quenching.
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