Dendrimer-Based Signal Amplification of Click-Labelled DNA in Situ

Article abstract of DOI:10.1002/cbic.201700209

The in vivo incorporation of alkyne-modified bases into the genome of cells is today the basis for the efficient detection of cell proliferation. Cells are grown in the presence of ethinyl-dU (EdU), fixed and permeabilised. The incorporated alkynes are then efficiently detected by using azide-containing fluorophores and the Cu^I-catalysed alkyne–azide click reaction. For a world in which constant improvement in the sensitivity of a given method is driving diagnostic advancement, we developed azide- and alkyne-modified dendrimers that allow the establishment of sandwich-type detection assays that show significantly improved signal intensities and signal-to-noise ratios far beyond that which is currently possible.

Full text of DOI:10.1002/cbic.201700209

Accepted Article
Title: Dendrimer-based signal amplification of click-labelled DNA in situ
Authors: Nada Raddaoui, Samuele Stazzoni, Leonhard Möckl, Bastien
Viverge, Florian Geiger, Hanna Engelke, Christoph Bräuchle,
and Thomas Carell
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To be cited as: ChemBioChem 10.1002/cbic.201700209
Link to VoR: http://dx.doi.org/10.1002/cbic.201700209
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ChemBioChem
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COMMUNICATION
Dendrimer-based signal amplification of click-labelled DNA in situ
#
#
Nada Raddaoui , Samuele Stazzoni , Leonhard Möckl, Bastien Viverge, Florian Geiger, Hanna Engelke,
Christoph Bräuchle and Thomas Carell*[a]
The in vivo incorporation of alkyne modified bases into the genome of
cells is today the basis for efficient detection of cell proliferation. Cells
are grown in the presence of ethinyl-dU (EdU), fixed and
permeabilized. The incorporated alkynes are then efficiently detected
using azide-containing fluorophores and the Cu(I) catalyzed alkyne-
azide click reaction. In a world where constant improvement of the
sensitivity of a given method is driving diagnostic advancement, we
developed azide and alkyne modified dendrimers that allow to
establish sandwich-type detection assays that show significantly
improved signal intensities and signal to noise ratios far beyond of
what is currently possible.
sandwich-type approach with alkyne and azide containing
dendrimers 1 and 2 (Scheme 1) that allows significant chemical
signal amplification. The method was shown to provide
unprecedented detection sensitivities of proliferating cells. The
synthesis of the needed amplifying tetraazide/alkyne molecules 1
and 2 is depicted in Scheme 1 (and S1, S2). In both cases, the
principle design idea was to stay as close as possible to
polyethyleneglycol based structures because of the needed high
solubility in water. Starting point towards 1 is the ethylene glycol
derivative 3, which we converted first into the azide 4. The
hydroxyl group was subsequently tosylated to 5 to enable the
fourfold substitution reaction with ethylenediamine to give the
desired tetraazide compound 1.
The proliferation rate of cells is a key parameter that requires in
many contexts precise determination.[1] Cell proliferation assays
help for example routinely to evaluate the toxicity of compounds
in the framework of the development of new pharmaceuticals.[2]
Also in cancer diagnostics, it is required to measure the
proliferation of cells with high precision.[3] Particularly in this field
highest sensitivity is desired in order to detect at best even single
cancer cells in a patient sample. Today the most precise way to
measure cell proliferation is to culture the cells in the presence of
C5-ethinyl-dU (EdU), which is incorporated into the genome of
proliferating cells as a typical anti-metabolite.[4] The amount of
incorporated EdU is subsequently measured by reacting the
alkynes within the DNA with azido-modified fluorescent dyes
using the Cu(I) catalysed alkyne-azide click reaction[5] and
detected using fluorescent microscopy.[6] This reactions proceed
on DNA with extreme efficiency likely because the Cu(I) is loosely
pre-coordinated to the electron rich centers at the nucleobases.[6]
This technology is used in established commercially available kits
(
EdU-Click kit from baseclick, Click-iT from Thermo Fisher).
However in all available methods, the sensitivity is limited by the
number of alkynes, which are incorporated during the culturing
phase of the experiment in the presence of EdU (one alkyne). This
creates the problem that slowly proliferating, but still
Scheme 1. Synthesis of the ethylene glycol based tetraazide 1 and of the
tetraalkyne 2 needed for the study. Reagents and conditions: a) NaN , DMF,
3
9
0°C, o/n, 96%. b) TsCl, NEt
LiBr, DMF, 60°C, o/n, 63%. d) TsCl, NEt
bromide, NaH, THF, 0°C to rt, 82%. f) 8, K CO
3
, CH
2
Cl
2
, o/n, 90%. c) ethylenediamine, 5, KOH,
, THF, rt, o/n, 92%. e) propargyl
, acetone, 80°C, o/n, 31%.
3
2
3
[
7]
cancerogenous cells often escape detection. We report here a
The synthesis of the tetraalkyne 2 started with the ethyleneglycol
derivative 6, which was monotosylated in excellent yield to
provide 7. Reaction with propargyl bromide furnished compound
8, which was used for a fourfold substitution reaction with
pentaerythritol 9 to give the dendrimer 2. Both compound 1 and 2
were subsequently purified by flash column chromatography.
[
a]
M.Sc. N. Raddaoui, M.Sc. S. Stazzoni, M.Sc. B. Viverge, Dr. L.
Möckl, M.Sc. F. Geiger, Dr. H. Engelke, Prof. Dr. C. Bräuchle and
Prof. Dr. T. Carell
M
In a first approach to amplify the cell proliferation signal, we used
the polyethyleneglycol based tetraazide molecule 1 as shown in
Figure 1A and S3. For the experiments, we grew HeLa cells in µ-
slides in the presence of 10 M EdU for 2 h. The medium was
removed and 3.7% formaldehyde in phosphate buffered saline
supplemented with 0,02% Tween (1x PBS-T) buffer was added to
fix the cells. After two time washing with 1x PBS-T, the cells were
permeabilized with 0.5% Triton X-100 in 1x PBS-T for 20 min at
Center for Integrated Protein Science (CiPS ) at the Department of
Chemistry, LMU München
Butenandtstr. 5-13, 81377 München
Fax: (+) 49 2180 77756
E-mail: Thomas.Carell@lmu.de
Homepage: www.carellgroup.de
These authors contributed equally to this work.
#
Supporting information for this article is given via a link at the end of the
document.
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ChemBioChem
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COMMUNICATION
room temperature (RT). We then added the click-cocktails
successively. In one experiment, we just added the Tamra-azide
possibly because alkynes are known to react to some extent with
nucleophiles. Thiol-containing nucleophiles are abundantly
present in cells. Control experiments performed without the
addition of the tetraazide 1 confirmed this hypothesis and showed
a still high background signal even without using our dendrimers
(Fig. S4).
10 (20 M) as a positive control. In the other experiment, we
performed a first reaction with the dendrimer 1 (20 M) for 1 h
followed by a second click reaction with the Tamra-alkyne 11 (5

M) for 30 minutes (For more detailed characterization of the
reaction products, determined with a symplified model using
synthetic oligonucleotides see SI). In both experiments, we
washed the fixed cells twice with 3% BSA in PBS buffer. In the
dendrimer amplified experiment with the Tamra-alkyne 11 we
noted an unusually high background even after these intensive
washing steps. Screening for appropriate washing conditions
showed that best results were obtained when we washed with a
solution of guanidinium isocyanate (Fig. S4). We then determined
the Tamra fluorescence using a fluorescence microscope. The
data are depicted in Fig. 1B. Clearly visible is that the prior click
with the dendrimer-azide 1 furnished a six-fold increase of the
fluorescence signal. This was particularly visible in the
fluorescence microscopic evaluation of the click-modified cells
In order to solve the background problem and to further increase
the sensitivity (signal to noise ratio) of detection, we experimented
next with a double-dendrimer approach (Fig. 2) where we first
reacted the fixed DNA with the tetraazide 1, followed by an
additional click reaction with the tetraalkyne-dendrimer 2. This
was then followed by a final click reaction with the Tamra-azide
10 (Fig. S5). In this way, we planned to circumvent the use of the
problematic dye-alkynes.
(
Fig. 1C).
Figure 2. A) Depiction of the double dendrimer (triple click) amplified cell
proliferation assay. Cells were grown in the presence of 5-ethinyl-dU. After
fixation and permeabilization, the present alkynes are first reacted with the
tetraazide-dendrimer 1 in the presence Cu(I). The multiple azide containing
DNA is then reacted with the tetraalkyne 2. The so double modified DNA (triple
click) is finally detected with an azide-modified dye 10 using again the Cu(I)
catalyzed click reaction. B) The control experiment is performed using the
dendrimer-free standard proliferation assay. Triple click shows data after double
dendrimer amplification. C) Fluorescence microscopy pictures of cells detected
with the standard EdU assay as control (top) and after double dendrimer
amplification with triple click (bottom). Red arrows show cells in the early S-
phase. Green arrows show cells in late S-phase. Scale bars, 20 µm.
Figure 1. A) Depiction of the single dendrimer (double click) amplified cell
proliferation assay. Cells were grown in the presence of 5-ethinyl-dU. The cells
were fixed and the present alkynes reacted with a tetraazide-dendrimer 1 in the
presence of Cu(I) (double click). The multiple azide containing DNA is then
detected in situ with an alkyne-modified dye 11 using again the Cu(I) catalyzed
click reaction. B) The control experiment is performed using the dendrimer-free
standard proliferation assay with 10. Double click shows data after dendrimer
amplification. C) Fluorescence microscopy pictures of cells detected with the
standard assay as control (top) and after dendrimer amplification (bottom). Red
arrays show cells in the early S-phase with partial EdU incorporation. Green
arrows show cells in late S-phase, where the DNA synthesis is almost finished
and EdU is incorporated into the whole genome. Scale bars, 20 µm.
For this experiment, we again cultured HeLa cells in µ-slides in
the presence of 10 M EdU for 2 h. The medium was removed
and 3.7% formaldehyde in 1x PBS-T was again added to fix the
cells. We washed the cells two times with 1x PBS-T and
permeabilized the cells with 0.5% Triton in 1x PBS-T for 20 min at
RT. We then added the click-cocktails successively: First we
We noted that after extensive washing with guanidinium
isocyanate, the background was reduced but steadily higher than
in the non-dendrimer experiments. We speculated that this
background problem may be caused by the dye-alkyne 11
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ChemBioChem
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COMMUNICATION
added Cu(I) and tetraazide 1. We washed the cells twice with a
In summary, click-based detection of cell proliferation is today
state-of-art technology. We show here that by using dendrimer-
type tetraazide (1) and dendrimer-type tetraalkyne (2) compounds
sandwich type detection assays can be established that yield
strongly improved signal intensities with low background giving
higher signal to noise ratios for imaging and high throughput
content assays. We expect that the so improved cell proliferation
assay will be able to detect either slowly or even single
proliferating cancer cells with unprecedented sensitivity.
0
1
.2 M acetate buffer pH 4.7 followed by two washing steps with
x PBS-T and performed subsequently the second click reaction
with tetraalkyne 2 and Cu(I) for 1 h. The cells were again washed
twice with 1x PBS-T. Finally we added Cu(I) and the Tamra-azide
10 to the cells and allowed the final click cocktail to penetrate the
cells for 30 minutes. After again two time washing with
guanidinium isocyanate we studied the cells by fluorescence
microscopy. This time the experiment was a full success. We
detected a strongly reduced background, not higher than in the
control experiment with just EdU (Fig. 2B,C). The obtained
fluorescence signal was highly improved by a factor of 2.5. Most
importantly, the direct inspection of the cells by fluorescence
microscopy shows a strongly improved signal to noise ratio (Fig.
Acknowledgements
We thank the Deutsche Forschungsgemeinschaft for financial
support via SFB1032 (TP-A5), SFB749 (TP-A4), SPP1784,
2C, S5).
M
CA275 and the Excellence Cluster CiPS . Further support from
Next, the new single and double dendrimer based methods were
applied for high throughput screening (HTS). This method is the
most widely used tool not only for the development of new
pharmaceuticals compounds but also needed for the
measurement of the response of cells to different nutrients,
mitogens, cytokines, growth factors and toxic agents . With the
signal amplification provided by our dendrimers, we were able to
detect a strong, specific signal even when only a very small
number of cells like just 100 cells were present per well (Fig. 3).
This is a significant improvement over contemporary methods that
need 500 to 1000 cells per well, which allows now the reliable
detection of small number of proliferating cells that otherwise
escape staining and detection. What we noted, however, is a
reduction of the signal intensity in the double-dendrimer approach,
which is likely due to self-quenching of the then densly packed
fluorophores. To solve this, optimization of the dendrimers is now
required.
the European Union via the Marie Curie International Training and
Mobility Network "Clickgene" (grant No. 642023) is acknowledged.
Keywords: click chemistry • cell proliferation • dendrimer •
fluorescence microscopy • high throughput screening
[
8]
[1] a) M. G. Daidone, R. Silvestrini, J. Natl. Cancer Inst. Monogr. 2001, 27-
35; b) S. H. Torp, C. F. Lindboe, U. S. Granli, T. M. Moen, T. Nordtomme,
Clin. Neuropathol. 2001, 20, 190-195; c) M. A. Aleskandarany, A. R.
Green, A. A. Benhasouna, F. F. Barros, K. Neal, J. S. Reis-Filho, I. O.
Ellis, E. A. Rakha, Breast Cancer Res. 2012, 14, R3; dM. Wang, J. Wu, Y.
Guo, X. Chang, T. Cheng, Mol Med Rep 2017, 15, 1607-1612; e) T.
Wieder, E. Brenner, H. Braumuller, O. Bischof, M. Rocken, Cancer
Metastasis Rev. 2017; f) A. S. Thorat, N. A. Sonone, V. V. Choudhari, R.
M. Devarumath, K. H. Babu, 3 Biotech. 2017, 7, 16; g) H. Chen, X. Gu, Y.
Liu, J. Wang, S. E. Wirt, R. Bottino, H. Schorle, J. Sage, S. K. Kim, Nature
2011, 478, 349-355; h) K. D. Poss, L. G. Wilson, M. T. Keating, Science
2002, 298, 2188-2190.
[
2] a) X. Lu, J. W. Horner, E. Paul, X. Shang, P. Troncoso, P. Deng, S. Jiang,
Q. Chang, D. J. Spring, P. Sharma, J. A. Zebala, D. Y. Maeda, Y. A.
Wang, R. A. DePinho, Nature 2017, 543, 728-732; b) N. V. Jordan, A.
Bardia, B. S. Wittner, C. Benes, M. Ligorio, Y. Zheng, M. Yu, T. K.
Sundaresan, J. A. Licausi, R. Desai, R. M. O'Keefe, R. Y. Ebright, M.
Boukhali, S. Sil, M. L. Onozato, A. J. Iafrate, R. Kapur, D. Sgroi, D. T.
Ting, M. Toner, S. Ramaswamy, W. Haas, S. Maheswaran, D. A. Haber,
Nature 2016, 537, 102-106; c) M. A. Erb, T. G. Scott, B. E. Li, H. Xie, J.
Paulk, H. S. Seo, A. Souza, J. M. Roberts, S. Dastjerdi, D. L. Buckley, N.
E. Sanjana, O. Shalem, B. Nabet, R. Zeid, N. K. Offei-Addo, S. Dhe-
Paganon, F. Zhang, S. H. Orkin, G. E. Winter, J. E. Bradner, Nature 2017,
543, 270-274.
2
2
1
1
500
000
500
000
1
1
20
00
8
6
4
0
0
0
20
0
[
[
3] a) G. I. Evan, K. H. Vousden, Nature 2001, 411, 342-348; b) G. Evan, T.
Littlewood, Science 1998, 281, 1317-1322.
4] a) A. Salic, T. J. Mitchison, Proc. Natl. Acad. Sci. U. S. A. 2008, 105,
2415-2420; b) M. Ababou, V. Dumaire, Y. Lecluse, M. Amor-Gueret,
5
00
0
Oncogene 2002, 21, 2079-2088; c) B. L. Cavanagh, T. Walker, A. Norazit,
A. C. Meedeniya, Molecules 2011, 16, 7980-7993.
5] a) H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. Int .Ed. 2001,
[
40, 2004-2021; b) P. M. Gramlich, C. T. Wirges, A. Manetto, T. Carell,
1
00
500
1000
2000
Angew. Chem. Int. Ed. 2008, 47, 8350-8358.
[
[
6] J. Gierlich, G. A. Burley, P. M. Gramlich, D. M. Hammond, T. Carell, Org.
Lett. 2006, 8, 3639-3642.
Number of cells
7] S. Nik-Zainal, P. Van Loo, D. C. Wedge, L. B. Alexandrov, C. D.
Greenman, K. W. Lau, K. Raine, D. Jones, J. Marshall, M. Ramakrishna,
A. Shlien, S. L. Cooke, J. Hinton, A. Menzies, L. A. Stebbings, C. Leroy,
M. Jia, R. Rance, L. J. Mudie, S. J. Gamble, P. J. Stephens, S. McLaren,
P. S. Tarpey, E. Papaemmanuil, H. R. Davies, I. Varela, D. J. McBride, G.
R. Bignell, K. Leung, A. P. Butler, J. W. Teague, S. Martin, G. Jonsson, O.
Mariani, S. Boyault, P. Miron, A. Fatima, A. Langerod, S. A. Aparicio, A.
Tutt, A. M. Sieuwerts, A. Borg, G. Thomas, A. V. Salomon, A. L.
Richardson, A. L. Borresen-Dale, P. A. Futreal, M. R. Stratton, P. J.
Campbell, C. Breast Cancer Working Group of the International Cancer
Genome, Cell 2012, 149, 994-1007.
Figure 3: Application of the single and double dendrimer amplified assay in high
throughput screening. Cells were grown on microplate with different densities
100, 500, 1000 and 2000 cells/well) and incubated for 2 h with 5-ethinyl-dU at
7°C. Negative control cells were grown without EdU labeling. The cells were
fixed and permeablized and the present alkynes reacted with a tetraazide
dendrimer 1 and Tamra-alkyne (double click, gray bars) or with tetraazide 1,
tetraalkyne 2 and Tamra-azide (triple click, orange bars) in the presence of Cu(I)
in situ. Positive control cells were reacted with Tamra-azide in presence of Cu(I)
(
3
(
control, blue bars). The cellular signal of duplicate samples was measured with
a Tecan microplate reader. After subtraction of the background fluorescence, it
was possible to detect a stronger signal even with only 100 cells. Blue: Standard
click protocol. Grey: Single dendrimer approach with dendrimer 1. Orange:
Double dendrimer approach with the dendrimers 1 and 2.
[
8] F. Zanella, J. B. Lorens, W. Link, Trends in Biotechnology, 28, 237-245.
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ChemBioChem
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COMMUNICATION
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201700209
COMMUNICATION
COMMUNICATION
#
#
Finding a needle in a haystack: A
new method to detect proliferating
cells in situ using multiple
consecutive click reactions with
N. Raddaoui , S. Stazzoni , L.
Möckl, B. Viverge, F. Geiger, H.
Engelke, C. Bräuchle and T.
Carell*[a]
dendrimeric
molecules
and
clickable dyes is presented, that
allows to reach outstanding
sensitivities
Page No. – Page No.
Dendrimer-based signal
amplification of click-labelled
DNA in situ **
.
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