Polarity Sensitive Bioorthogonally Applicable Far-Red Emitting Labels for Postsynthetic Nucleic Acid Labeling by Copper-Catalyzed and Copper-Free Cycloaddition

Article abstract of DOI:10.1021/acs.bioconjchem.5b00557

Two series of new, water-soluble, membrane-permeable, far-red/NIR emitting benzothiazolium-based fluorescent labels with large Stokes' shifts were synthesized that can be conjugated to alkyne-modified biomolecules through their azide moiety via azide-alky

Full text of DOI:10.1021/acs.bioconjchem.5b00557

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Article
Polarity sensitive bioorthogonally applicable far-red
emitting labels for postsynthetic nucleic acid labeling
by copper-catalyzed and copper-free cycloaddition
Ádám Eördögh, Jeannine Steinmeyer, Krisana Peewasan,
Ute Schepers, Hans-Achim Wagenknecht, and Peter Kele
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00557 • Publication Date (Web): 20 Jan 2016
Downloaded from http://pubs.acs.org on January 26, 2016
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Polarity sensitive bioorthogonally applicable farꢀred
emitting labels for postsynthetic nucleic acid labeling
by copperꢀcatalyzed and copperꢀfree cycloaddition
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Ádám Eördögh,§,a Jeannine Steinmeyer,§,b Krisana Peewasan,b Ute Schepers,c HansꢀAchim
Wagenknecht*,b and Péter Kele*,a
a
Chemical Biology Research Group, Institute of Organic Chemistry, Research Centre for Natural Sciences,
Hungarian Academy of Sciences. Magyar tudósok krt. 2, Hꢀ1117, Budapest, Hungary. Eꢀmail:
kele.peter@ttk.mta.hu
b Institute of Organic Chemistry, Karlsruhe Institute of Technology (KIT), FritzꢀHaberꢀWeg 6, 76131 Karlsruhe,
Germany. Eꢀmail: Wagenknecht@kit.edu
c Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), H.ꢀv.ꢀHelmholtzꢀPlatz 1, 76344
EggensteinꢀLeopoldshafen, Germany.
§ These authors contributed equally to the work
ABSTRACT
Two series of new, water soluble, membraneꢀpermeable, farꢀred / NIR emitting
benzothiazoliumꢀbased fluorescent labels with large Stokes’ shifts were synthesized that can
be conjugated to alkyneꢀmodified biomolecules through their azide moiety via azideꢀalkyne
cycloaddition. We have used these azide bearing labels to make fluorescent DNA constructs
using copperꢀcatalyzed “click” reaction. All dyes showed good or remarkable fluorescence
intensity enhancement upon conjugation to DNAs. We also investigated the possibility to
incorporate the benzocyclooctyne motif through rigid (ethnynyl) or flexible (ethyl) linkers
into the DNA, thus enable copperꢀfree labeling schemes. We observed that there is a marked
difference between the two linkers applied in terms of optical properties of the labeled
oligonucleotides. We have also tested the in vivo labeling potential of these newly synthesized
dyes on HeLa cells previously transfected with cyclooctynylated DNA. Confocal fluorescent
images showed that the dyes are all able to cross the membrane and suitable for backgroundꢀ
fluorescence free fluorescent tagging of nucleic acids. Moreover, we have observed different
accumulation of the two dye series in the endosomal particles, or in the nuclei, respectively.
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INTRODUCTION
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Molecular biology methods aiming at observing and studying biochemical processes or
biomolecules in their natural environment have benefited greatly from fluorescenceꢀbased
imaging techniques. Detection of the emitted light offers a highly sensitive and relatively
cheap means of tracking with excellent temporal/spatial resolution.1,2 The use of various
fluorescent labels in combination with other detection channels further increases the range of
imaging methods.3,4 There are several ways to selectively tag the ensued biological entities:
fluorescently labeled antibodies usually offer highly selective and sensitive staining of the
biomolecule of interest, however, due to the considerable size of antibodies their use is
limited to extracellular structures or fixed cells.1 Genetically encodable fluorescent proteins
(FPs) can, in theory, be used to tag any protein of interest in various colors. Multiꢀcolor FPꢀ
based molecular biology techniques that emerged in the last decades are well credited as they
opened a new era in molecular biology related imaging techniques.1,5 However, the use of FPs
is limited to the labeling of biomolecules that are directly encoded in the genome i.e. proteins.
Postꢀtranslational modifications of proteins is also hard to follow with FPs, although recent
efforts are quite promising in this regard.6 Besides, the larger size of GFP or its variants often
influences the native function of the proteins studied. A third way for the selective installation
of fluorescent tags onto biomolecules became possible with the introduction of bioorthogonal
manipulation methods.7,8,9,10 These chemical reactions between nonꢀnative functions ensure
exclusive relationship between appropriately modified biomolecules and labels. Parallel
evolution of genetic code expansion techniques enabled the researchers to selectively modify
virtually any proteins with bioorthogonalized, nonꢀcanonical amino acids.11,12 Moreover,
bioorthogonal labeling is not limited to proteins. By means of metabolic incorporation of nonꢀ
natural building blocks (nucleotides, lipids, carbohydrates) or chemical targeting of unique
motifs (rare amino acids, or short, specific tags), enables labeling of biopolymers, lipids and
small metabolites as well.13,14,15 Bioorthogonal labeling schemes usually follow a sequential,
twoꢀstep routine. Implementation of the first bioorthogonal function into the biomolecule of
interest is followed by fluorescent tagging with a small, synthetic label harboring the
complementary cognate function.16,17 Photophysical properties, reactivity, membraneꢀ
permeability of these small labels can be synthetically tailored. Most biomolecular tagging by
bioorthogonal means deal with the fluorescent modification of proteins. This is mainly due to
the profound biological importance of this group, but also due to genetic code expansion
based advanced technologies. Other biomolecules were also manipulated bioorthogonally
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with fluorescent labels, yet, bioorthogonal modification of other biomolecules remains
somewhat less investigated. Especially bioorthogonal labeling for fluorescent nucleic acid
modifications, and corresponding applications with DNA and RNA in living cells have only
been rarely realized18 since this inꢀvivoꢀchemistry is just developing.19 This must be a major
goal for the actual research as it will provide the important chemicalꢀbiological impact for
fluorescent nucleic acid probes.
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Detection of a specific fluorescent signal is usually compromised by the appearance of nonꢀ
specific signals when using small synthetic labels. On the one hand autofluorescence of
naturally occurring chromophores can be circumvented by the use of farꢀred, near infrared
(NIR) emitting fluorophores with large Stokes’ shift.5 On the other hand, to diminish
background fluorescence caused by nonꢀspecific sticking of labels to nonꢀpolar surfaces is
more challenging. One solution to overcome background fluorescence is to use soꢀcalled
fluorogenic tags, whose fluorescence is turned on upon the specific chemical transformation
during conjugation.20,21,22,23 We have recently demonstrated that such probes are suitable to
diminish background fluorescence.22,23 Although some methods exist that can facilitate the
design of such fluorogenic tags by theoretical prediction methods, the real behavior of such
fluorogens, is rather empirical. There is another option that mimics chemically triggered
fluorogenicity. That is to use fluorescent labels with very low quantum yields, whose
fluorescence is largely dependent on the microenvironment.24,25 Recently, we have reported
on the synthesis and polarity dependent behavior of a set of NIR emitting dimethine dyes with
large Stokes’ shifts in connection with nucleic acid labeling.26 Following these observations
we have designed a new set of polarity sensitive labels and investigated their applicability in
nucleic acid labeling studies.
RESULTS AND DISCUSSION
In the design process we aimed at developing bioorthogonally conjugatable, polarity sensitive
labels with polar characteristics in order to minimize the possibility of nonꢀspecific sticking to
hydrophobic surfaces. As a starting point, we used frameworks (Figure 1) that we formerly
found amenable of conjugation mediated fluorescence turn on.26,27 It was hypothesized that
replacement of the lepidinium core of dyes 1a,b with more polar ethylꢀbenzothiazolium unit
(2a, 2b) would substantially increase polarity.
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Figure 1. Bioorthogonally applicable, NIR emitting labels with large Stokes’ shifts.26,27
Furthermore, in order to increase membraneꢀpermeability for future applications we also
aimed at investigating the effect of a more polar a propylsulfonyl pendant (3a, 3b). These
latter dyes are zwitterionic, with a net 0 charge, features that most membraneꢀpermeable dyes
share. Thus, we set forth synthetic routes in order to access labels 2 and 3 (Figure 2). It was
also hoped, that this latter modification will shift excitation and emission maxima towards the
farꢀred end or NIR regime of the spectrum.
Figure 2. Proposed target structures 2aꢀ3b.
We have started with 2ꢀmethylꢀ6ꢀnitrobenzo[d]thiazole that was obtained from commercially
available 2ꢀmethylbenzo[d]thiazole upon standard nitration reaction. Alkylation of 4 with
ethyl iodide or 1,3ꢀpropanesultone afforded compounds 5, 6 in medium to good yields. Base
catalyzed condensation of 5 and 6 with 3ꢀformylcoumarin or 2ꢀformylbenzofuran yielded 7
and 8 (Scheme 1).
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Scheme1. Synthesis of nitroꢀprecursors 7 and 8. a) EtI, 190 °C, 20 min or 1,3ꢀpropanesultone, 100 °C,
30 min; b) 3ꢀformylcoumarin or 2ꢀformylbenzofuran, piperidine (cat.), EtOH, reflux, 5 h.
Reduction of the nitro group was effected by treatment with tinꢀchloride to allow the
corresponding amines
9
and 10. All amines were subjected to
a
standard
diazotization/azidation sequence using sodium nitrite and sodium azide to furnish target
compounds 2 and 3 in good yields (Scheme 2). Compounds 2a, b were converted to their
respective hexafluorophosphate salts by treatment with excessive amount of NH4PF6 and
subsequent precipitation from aqueous solutions.
Scheme 2. Synthesis of target compounds 2, 3. a) SnCl2.2H2O, ccHCl, 22 °C, 3 or 5 h; b) NaNO2/HCl,
22 °C, 15 min then NaN3, in 1:1 HCl, 22°C, 1.5 h or 4.5 h
Since in biomolecular conjugation schemes the azide moiety is to be converted to the
corresponding triazole derivative we have made the respective model triazoles (11 and 12) in
Cu(I) catalyzed azideꢀalkyne cycloaddition reactions with Nꢀ(propꢀ2ꢀynꢀ1ꢀyl)pivalamide.
In order to assess the spectroscopic characteristics the azides and triazoles we have acquired
absorption, emission and excitation spectra and determined molar absorptivities and quantum
yields for each compound.
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Figure 3. Model triazoles 11, 12.
As expected, all dyes and their triazolic congeners were excitable in the visible region of the
spectrum with emission maxima in the farꢀred, near infrared region with notably large Stokes’
shifts. Water solubility of the dyes improved remarkably with respect to their lepidiniumꢀ
based predecessors. Further photophysical characterization of the azides and their triazolic
products revealed that the brightness of the respective triazoles is lower than that of the parent
azides. This was mainly attributed to the notably lower molar absorptivities of the triazoles,
while quantum efficiencies remained practically unchanged (Table 1).
Table 1. Summary of optical properties, of azides and triazoles 2ꢀ3 and 11ꢀ12
Compounds
2a
11a
3a
12a
2b
11b
3b
12b
λabs PBS [nm]
εabs PBS [×104 Mꢀ1cmꢀ1]
543
6.86
533
550
548
567
579
612
591
598
1.96 4.52 1.54 4.00 2.99 3.97 1.02
λexc PBS [nm]
556
664
550
653
556
655
606
604
718
600
715
593
714
λem PBS [nm]
656
718
1.7
φ
(%)a
6.36
5.07 5.32 5.32
1.37 1.64 1.19
λabs DMSO [nm]
εabs DMSO [×104 Mꢀ1cmꢀ1]
563
6.59
558
579 565 567
604
616 606 613
1.65 3.85 1.57 5.13 2.93 4.00 1.17
λexc DMSO [nm]
566
667
556
676
560
682
604
604
602
610
λem DMSO [nm]
661
724
9.7
729
9.7
729
10.1
731
9.7
φ
(%)a
32.0
36.0 27.0 28.0
a relative to Rhodamine 101 and cresyl violet for the a and b series, respectively
In order to evaluate the fluorescence properties of the newly synthesized dyes in conjugates
we attached them covalently to DNA single and double strands. The oligonucleotides were
automatically synthesized using standard conditions on solid phase. Dyes 2aꢀ3b were
postsynthetically incorporated according to our published protocol using copper(I)ꢀcatalyzed
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cycloaddition.28 The bioorthogonally reactive DNA building block of 2’ꢀpropargylꢀmodified
uridine is commercially available. The resulting singleꢀstranded DNA1aꢀDNA3b were
identified by MALDI mass spectrometry and quantified by UV/vis absorption spectroscopy.
The double stranded DNA4ꢀDNA9 were annealed using the corresponding unmodified
counterstrands by heating to 90 °C for 10 min, followed by slow cooling to room temperature.
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5' -CGC-GAG-TTA- X -AT-TAA-TAG-CC- 3'
O
O
NH
NH
N
O
N
O
DNA
R-N3
Cu(I)
X =
DNA
R
N
N
O
O
O
O
ascorbate
TBTA
O
O
O
O
N
DNA
DNA
1a: R=
1b: R=
N
N
Et2N
O
O
O
Et2N
DNA1a
DNA1b
2a: R=
2b: R=
S
S
O
Et2N
N
N
PF6
PF6
O
O
Et2N
DNA2b
DNA2a
3a: R=
3b: R=
S
N
S
O
Et2N
N
O
O
Et2N
SO3
SO3
DNA3a
DNA3b
5' -CGC-GAG-TTA- -AT-TAA-TAG-CC- 3'
3' -GCG-CTC-AAT- -TA-ATT-ATC-GG- 5'
-3b-
-A-
-3a-
-A-
DNA9
-2b- DNA8
DNA7
-2a-
DNA6
-1b-
DNA5
-1a-
-A-
DNA4
-A-
-A-
-A-
Figure 4. Sequences of DNA1aꢀDNA9 and structure of 2’ꢀmodifications.
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As mentioned before the background fluorescence of the dyes without DNA is very low. By
contrast, covalent linkage of the bioorthogonally reactive dye azides to DNA resulted in
remarkable fluorescence intensity increase at maxima of the triazolic products. Figure 5
representatively shows the difference between the emission intensity of the dye 2a without
DNA, and as part of singleꢀstranded DNA2a and doubleꢀstranded DNA6. This observation is
typical for cyanineꢀstyryl dyes. In solution without interaction with DNA, the two
heterocycles have the possibility to rotate around their methine bridge giving rise to nonꢀ
radiative relaxation pathways. In the case of covalent linkage to DNA this radiationless
pathways to dissipate excitation energy gets lost and fluorescence becomes the primary
relaxation process. These interactions slightly change from single to double strands as the
absorption maximum shifts from 591 nm to 578 nm and the emission maximum shifts from
651 nm to 640 nm. Concomitantly, the quantum yield increases further from 55% in the single
strand to 70% upon anellation with the counterstrand.
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0,15
2a
DNA2a
DNA6
4
3
2
1
0
2a
DNA2a
DNA6
0,10
0,05
0,00
400
500
λ/nm
600
700
650 700 750 800 850
λ/nm
Figure 5. Comparison of the absorption (left) and fluorescence (right) of DNA2a, DNA6 and dye 2a
Table 2 summarizes all optical properties of the synthesized dyeꢀoligonucleotide conjugates.
The quantum yields of the singleꢀstranded DNA2b and DNA3b and corresponding double
strands DNA7 and DNA9 are comparable. Obviously, the influence of the negative charge on
dye 3b is negligible. Contrary to this, incorporation of coumarin derivatives 2a and 3a into the
nucleic acid strands yields a significant increase of the quantum yield upon anellation to up to
70% in DNA6. There seems to be a major change of interactions between DNA and the
covalently attached dye associated to the coumarin motif since dyes 2a and 3a induce
destabilization of the corresponding DNA double strands by 4 °C whereas dyes 2b and 3b
yield a stabilization by 6 °C, each compared to unmodified DNA double strand as reference.
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Table 2. Summary of optical properties, quantum yield (ϕF) and melting temperatures of DNA1aꢀ
DNA13.
6
7
8
9
DNA
λmax,abs [nm] λmax,em [nm]
ϕF [%]
16.0
1.8
Tm [°C]
585
649
591
669
597
679
562
644
578
646
585
659
612
658
657
654
703
792
651
731
657
737
695
795
640
721
646
725
730
730
722
721
ꢀ
DNA1a
DNA1b
DNA2a
DNA2b
DNA3a
DNA3b
DNA4
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ꢀ
55.1
8.1
ꢀ
ꢀ
42.3
8.0
ꢀ
ꢀ
17.5
0.7
67
66
60
71
61
71
ꢀ
DNA5
70.3
14.4
58.0
13.0
8.7
DNA6
DNA7
DNA8
DNA9
DNA10b
DNA11b
DNA12
DNA13
8.6
ꢀ
9.4
54
53
13.2
Despite the broad applicability of the copper(I)ꢀmediated cycloaddition chemistry between
azides and ethynyl groups, as demonstrated above, the use of metal catalysts, especially the
cytotoxic copper ions, bears significant risks for live cell imaging. Extensive purification by
HPLC or the use of additives (such as EDTA) is needed to provide real copperꢀfree
fluorescent DNA probes. Among the copperꢀfree alternatives19, the strainꢀpromoted
cycloaddition between cyclooctynes and azides represents an important alternative since it
allows a catalystꢀfree means of labeling. For this purpose, we recently established the
carboxymethylmonobenzocyclooctyne (COMBO)29 group as a structurally simple, nonꢀ
fluorinated copperꢀfree “click” reagent that offers easy access to bioconjugates through its
carboxy function and exhibits lower lipophilicity in comparison to its doubly benzofused
alternatives.29 Moreover, COMBO exhibits good chemical stability, thus enable the
preparation and use of a new DNA building block with the COMBO group attached to the 5ꢀ
position of a 2’ꢀdeoxyuridine in DNA allowing rapid and efficient copperꢀfree postsynthetic
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modification with azidoꢀdyes.26 The fast and efficient labeling potential of COMBO was also
recently demonstrated by us in combination with tetrazineꢀtagged nucleic acids.30 We applied
this building block for representative labelling with fluorescent dye 3b in order to track down
any effects that might affect fluorescence properties that are associated with the kind of
linkage to the DNA. Accordingly, oligonucleotide DNA10b and its corresponding double
stranded DNA12 were synthesized. The fluorescence quantum yield of this DNA double
strand is unspectedly low (9%) which was attributed to the rather rigid and inflexible ethynyl
linker between the 2'ꢀdeoxyuridine part and the fluorophore which interferes with best
possible interactions of the dye with the DNA. Hence, a new DNA building block 13 that also
carries the COMBO group attached to the 5ꢀposition of 2'ꢀdeoxyuridine but linked via a
saturated alkyl and thereby a flexible linker (Figure 6) was synthesized.31,32,33,34
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Figure 6. Synthesis of the phosphoramidite 13 as new COMBOꢀmodified DNA building block, and
sequences of DNA8 and DNA9. a) 20% Pd(OH)2/C, HSiEt3, MeOH, rt, 24 h; b) DMTCl, Et3N,
pyridine, 40 °C, 18 h; c) NH4OH; d) COMBO, HBtU, HOBt·H2O, DMF, DIPEA, rt, 2 h; e)
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Cl(iPr)2NPO(CH2)2CN, DIPEA, CH2Cl2, rt, 1 h. The corresponding single strands are named DNA10b
and DNA11b for double stranded DNA12 and DNA13, respectively.
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The synthesis applies the trifluoroacetylꢀprotected 5ꢀ(3’’ꢀaminopropynyl)ꢀ2’ꢀdeoxyuridine 14
as starting material that can be obtained following literature procedures.34 The key step is the
hydrogenation of the ethynyl linker that can be achieved by treatment with HSiEt3 using 20%
Pd(OH)2/C as catalyst.35 The new and saturated nucleoside 15 is obtained in 70% yield. The
subsequent steps are very similar to the previously published procedure for the COMBOꢀ
modified DNA building block with the ethynyl linker.26 First, the DMT group is introduced to
the 5'ꢀposition, then the TFA protecting group is removed from the amine, which allows
subsequently to couple COMBO using a peptide coupling procedure (HBtU/HOBt). Finally,
the introduction of the phosphoramidite group to the 3'ꢀposition completes the synthesis of 13.
The oligonucleotide DNA11b was synthesized on solid phase using automated standard
conditions, including workup, purification by HPLC and identification by MALDIꢀTOF mass
spectrometry. The correspondingly modified double strand DNA13 shows, as expected, a
significantly increased fluorescence quantum yield. In fact, the quantum yield of DNA13 is
nearly identical to that of DNA9.
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3b
3b
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DNA13
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λ/nm
Figure 7. Comparison of the absorption (left) and fluorescence (right) of 3b, DNA10b, DNA11b,
DNA12 and DNA13.
Finally, we performed live cell experiments in order to demonstrate the potential of the newly
synthesized dyes in fluorescent imaging. Prior to treatment with the corresponding azidoꢀ
dyes, 5x 104 HeLa cells were transiently transfected with a 15 pmol single stranded DNA that
was modified with COMBO through the flexible linker (DNA11a), and Screenfect®, for 24
hours. The DNA11a transfected cells were then treated with 30 pmol of dyes 1aꢀ3b for 24
hours, then imaged by confocal fluorescent microscopy (Figure 8). In comparison to nonꢀ
transfected control cells specific fluorescent staining could be observed, indicating that all
dyes tested are taken up by the cells irrespective of having +1 or zero net charge. This is in
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accordance with literature examples (e.g. Rhodamineꢀdyes or ATTOꢀdyes).36 Contrary to
these previous examples, however, the dyes tested in this work show no measurable nonꢀ
specific background fluorescence that could originate from adsorption to hydrophobic, and/or
negatively charged surfaces (e.g. DNA). The fluorescence images reflect the quantum yield
differences between the dyes. The dyes show different accumulation preferences depending
on their cellular uptake mechanism. Remarkably, coumarin derivatives (2a, 3a) showed
specific DNA staining in the nuclei, while benzofuraneꢀ tagged DNA molecules (2b, 3b) were
accumulated preferably in endosomal vesicles. MTT assays revealed that none of the dyes are
toxic at the concentration levels used for this labeling experiments (0.15 µM) (SI Figure 17ꢀ
22). Therefore, all dyes are well suited for in vivo click reactions.
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We also compared these new dyes with our previously reported lepidiniumꢀdyes, (1a,b). Dye
1b showed fluorescence intensity that so low, that the results are hard to interpret. On the
other hand, 1a exhibited similar features to the newly synthesized series. We also noticed that
dyes 2ꢀ3 are much more soluble in aqueous media than lepidinium dyes 1. Overall, these
results show clearly, that the newly prepared dyes have a significant potential for lowꢀ
background live cell imaging schemes, especially those that bear higher quantum yields.
CONCLUSIONS
In conclusion, we have synthesized a set of new benzothiazolium containing dimethine dyes
harboring an azide function enabling bioorthogonal conjugation of the dyes to alkyne
functionalized targets. Photophysical characterization of the respective triazolic products
revealed that they are all excitable in the visible regime of the spectrum with emission
maxima in the farꢀred, near infrared region. Water solubility of the dyes improved remarkably
with respect to their lepidiniumꢀbased predecessors and large Stokes’ shifts were observed in
each case. As expected the fluorescent quantum yields were low in aqueous solutions,
however, in each case, marked fluorescence enhancements were observed when the dyes were
conjugated to DNA targets. This effect was much more pronounced for the a series of the
dyes, suggesting that the coumarin moiety interacts with the DNA bases in a different manner
than the benzofurane (series b) at the expense of stabilization of the double strand.
Furthermore, we have investigated the possibility of cyclooctyne motif incorporation into the
oligonucleotide strands in order to enable copperꢀfree labeling protocols. Of the linkers
through which the cyclooctyne is attached to the DNA base we found that the more flexible
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linker allows similar interactions between the dyes and DNA, which realized in nearly
identical enhancement of fluorescence upon copperꢀfree conjugation of dye 3b to DNA13 as
observed in the case of copperꢀcatalyzed construction of DNA9 conjugate. We also tested the
in vivo applicability of these new azidoꢀdyes on HeLa cells, transfected previously with
cyclooctynylatedꢀDNA (DNA11a). All dyes were taken up by the cells with no observed
toxicity and could specifically tag the target DNA. Nonꢀtransfected cells on the other hand
showed no appreciable fluorescence indicating that these dyes are suitable for backgroundꢀ
fluorescence free imaging of alkyne modified biomolecular targets using farꢀred light.
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Figure 8. In vivo click reaction of DNA11a and dyes 1aꢀ3b; 1x 104 Hela cells are transfected with
DNA11a and subsequently incubated with dyes 1aꢀ3b. Left column fluorescence confocal image, right
column: Merge of fluorescence image with bright field. Scale bar 10 µm.
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ACKNOWLEDGEMENTS
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Present work was supported by the Hungarian Scientific Research Fund (OTKA, NNꢀ110214)
and the “Lendület” Program of the Hungarian Academy of Sciences (LP2013ꢀ55/2013) and
by the Deutsche Forschungsgemeinschaft (grant Wa 1386/16ꢀ1) and by the GRK 2039
(funded by DFG) and by the Helmholtz Program “Biointerfaces in Technology and Medicine”
(BIFꢀTM).
Supporting Information Available:
Details of synthetic procedures, characterization data and spectra are available. Details
of DNA labeling experiments and toxicity tests are also provided. This material is available
free of charge via the Internet at http://pubs.acs.org.
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Graphical abstract
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