Polymerase synthesis of DNA labelled with benzylidene cyanoacetamide-based fluorescent molecular rotors: Fluorescent light-up probes for DNA-binding proteins

Article abstract of DOI:10.1039/c5cc00530b

Viscosity-sensitive fluorophores, fluorescent molecular rotors based on aminobenzylidene-cyanoacetamide moiety, were tethered to 2′-deoxycytidine triphosphate via a propargylamine linker and incorporated into DNA by polymerases in primer extension, nickin

Full text of DOI:10.1039/c5cc00530b

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
Polymerase synthesis of DNA labelled with
benzylidene cyanoacetamide-based fluorescent
molecular rotors: fluorescent light-up probes for
DNA-binding proteins†
Cite this: Chem. Commun., 2015,
1, 4880
5
Received 19th January 2015,
Accepted 10th February 2015
a
a
ab
Dmytro Dziuba, Radek Pohl and Michal Hocek*
DOI: 10.1039/c5cc00530b
www.rsc.org/chemcomm
Viscosity-sensitive fluorophores, fluorescent molecular rotors based would be advantageous. In our proof-of-the-principle studies we
on aminobenzylidene–cyanoacetamide moiety, were tethered to showed that dNTPs bearing suitable environmentally sensitive
0
7
2
-deoxycytidine triphosphate via a propargylamine linker and incor- fluorescent reporter (either solvatochromic aminophthalimide or
8
porated into DNA by polymerases in primer extension, nicking GFP-fluorophore as a molecular rotor ) can be incorporated into
enzyme amplification or PCR. DNA probes incorporating modified DNA by polymerases. The resulting probes showed a light-up response
nucleosides show a light-up response upon binding to a protein.
upon binding to proteins, although the increase of fluorescence was
7,8
rather low (max. 2.5-fold). Therefore, development of more sensitive
Fluorescent probes targeting DNA-binding proteins are of great value fluorophores is highly desirable.
for cell biology to study signaling pathways, for medicinal chemistry to
Here we report improved fluorescent dNTPs based on molecular
develop drug screening assays, and for clinical chemistry to detect rotors, their enzymatic incorporation into DNA and use for sensing of
1
biomarkers. Although fluorescence spectroscopy is a powerful DNA–protein interactions. Fluorescent molecular rotors (FMRs) are a
method for studying biomolecular interactions, the existing methods class of fluorescent dyes sensitive to local viscosity. The most widely
of detection of DNA–protein binding in solutions using fluorescent used are p-(N,N-dialkylamino)benzylidene-malononitriles DCVJ and
9
,10,14
reporting groups suffer several limitations. Most of the current DNA- CCVJ (1a, 2, Fig. 1a).
based fluorescent probes are designed for the detection of proteins intensity in media of high viscosity (Fig. 1b). They found applications
These FMRs show increased fluorescence
1a
2
exhibiting enzymatic activity. Examples are nuclease and uracil in life sciences as fluorescent viscosity probes for microheterogeneous
DNA glycosylase probes based on artificial emissive nucleoside systems, such as cytoplasm and cell membranes, but the possibility
analogues quenched by stacking interactions with neighbouring to probe biomolecular interactions by emission of FMRs is only
nucleobases within the double helix. In contrast to enzymes which poorly explored. In the field of DNA studies, FMRs were used to
modify the chemical structure of DNA, many biologically important study pre-melting of DNA and probing of G-quadruplexes. Several
3
9
10
1
1
12
DNA-binding proteins, i.e. transcription factors or histones, do not emissive nucleoside analogues have been shown to be sensitive to the
13
alter the chemical composition of DNA and thus some more sophis- viscosity of the media, but none of them was used for the probing of
ticated approaches are required for their detection. Several methods interactions with proteins. In this study we hypothesized that a
1b,4
based on molecular beacons and environmentally sensitive fluor-
5
escent nucleoside analogues have been developed for this purpose.
These methods usually require de novo synthesis of the probe for each
particular target via solid phase phosphoramidite synthesis, which
make them cost-consuming and can limit high-throughput applica-
6
tions. In this respect, polymerase construction of DNA-based probes
using chemically modified deoxynucleoside triphosphates (dNTPs)
a
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech
Republic, Gilead & IOCB Research Center, Flemingovo nam. 2, CZ-16610 Prague
6
, Czech Republic. E-mail: hocek@uochb.cas.cz
b
Department of Organic Chemistry, Faculty of Science, Charles University in
Prague, Hlavova 8, CZ-12843 Prague 2, Czech Republic
Fig. 1 (a) Structure of fluorescent molecular rotors DCVJ and CCVJ. (b)
Uncorrected fluorescence spectra (lex = 460 nm) of DCVJ in ethylene glycol
and glycerol; inset – photography of the solutions of DCVJ in ethylene glycol
(left) and glycerol (right) under a UV lamp. (c) Structures of FMR-labelled
nucleosides (3a–b) and triphosphates (4a–b) described in this work.
†
Electronic supplementary information (ESI) available: Additional figures and
tables, experimental details, copies of NMR and MALDI spectra. See DOI: 10.1039/
c5cc00530b
4
880 | Chem. Commun., 2015, 51, 4880--4882
This journal is ©The Royal Society of Chemistry 2015

View Article Online
Communication
ChemComm
nucleoside analogue bearing a CCVJ-type FMR attached to a nucleo-
base via a short linker will be a useful probe for protein binding. CCVJ
is known to increase fluorescence upon binding with tight hydro-
14
phobic sites of proteins, such as albumins or antibodies. Although
DNA binding proteins usually do not have hydrophobic pockets, the
layer of water molecules next to the protein interface (the hydration
shell) exhibits retarded molecular dynamics comparing to water in
15
the bulk. This might provide a rational basis for protein-induced
16
fluorescence enhancement (PIFE).
Our design of FMR-labelled nucleosides is shown in Fig. 1c. We
use a short propargylic linker to connect the p-(N,N-dialkylamino)-
benzylidene-malononitrile to position 5 of cytosine. Two distinct
X
Fig. 3 (a) The scheme of primer extension (PEX) with FMR-modified dC TPs;
(
b) PAGE analysis of PEX with template T1X and primer P1X performed by KOD
XL (lanes 1–4) and Vent(exo-) (lanes 5–8) polymerases; and (c) preparation of
N,N-dialkylamino-aryl groups were used to give a julolidine labelled ssDNA by PEX followed by magnetic separation.
VJ
derivative (dC ) and its less bulky N,N-dimethylaniline analogue
VDP
(
dC ). The FMR-nucleosides were synthesized using a modular
3
a and 3b (0.630 and 0.627, respectively) are pretty close to the
strategy based on the Sonogashira coupling of 5-iodo-dC with the
corresponding fluorophore-linked acetylene (Scheme S1 in the ESI†).
Having in hands the nucleosides we compared their photo-
physical and viscosity-sensitive properties with reference com-
theoretical limit (0.66) indicating that title compounds exhibit
highly favourable sensing properties.
Then we proceeded to the synthesis of DNA. For the enzymatic
0
1
7
incorporation, nucleosides 3a and 3b were phosphorylated at 5
pounds 1a,b (Fig. 2, Table 1). The introduction of a nucleoside
moiety blue-shifted the absorption spectra, whereas the positions
of emission maxima were not affected. The sensitivity to viscosity
18
using the procedure of Ludwig to give corresponding triphosphates
a and 4b (Fig. 1c; Scheme S1, ESI†). We examined the possibility of
4
9a,17
incorporating the modified dNTPs into DNA by enzymatic methods,
i.e. primer extension (PEX), nicking enzyme amplification reaction
was measured using the Forster–Hoffmann theory,
stating
that the logarithm of fluorescence intensity (F) depends on the
logarithm of the viscosity of the media (Z) as follows:
6c,19
(NEAR) and polymerase chain reaction (PCR).
X
At first we tested the modified dC TPs in combination with
three natural dNTPs in PEX with KOD XL and Vent(exo-) DNA
log F = x log Z + C
polymerases (Fig. 3a). PAGE analysis of the PEX reaction
here x is the viscosity sensitivity which is an intrinsic character-
istic of a molecular rotor. According to the theoretical predictions,
the maximum value of x is 0.66, whereas, for the vast majority of
FMRs, the x value ranges from 0.4 to 0.6. The sensitivity to
viscosity was slightly higher for the nucleosides compared to the
reference, whereas this improvement was more significant in the
cases of 1b and 3b (Fig. 2). Notably, the values of x for nucleosides
VDP
(Fig. 3b) shows that dC TP was a good substrate for both
enzymes tested (lanes 4 and 8) and gave full-length extended
VJ
products, whereas the bulky dC TP was less efficient, giving a
17
VDP
number of shorter products (lanes 3 and 7). Nucleotide dC
was nicely incorporated into DNA even at a high density
(Fig. S1, ESI†). Single-stranded oligonucleotides (ssONs) were
prepared by PEX using a biotinylated template and isolated by
magnetoseparation with streptavidine-coated magnetic beads
(
Fig. 3c). MALDI-TOF analysis of the ssON containing modified
x
nucleotides dC confirmed the correct full-length products
(
Fig. S1 in the ESI†) and UV-vis spectroscopy confirmed the absence
VDP
of non-specific binding of dC TP to DNA (Fig. S2, ESI†).
Then, we tested the modified triphosphates in the nicking enzyme
19b,c
amplification reaction (NEAR),
a two-step linear isothermal ampli-
fication process for synthesis of short ssONs (Fig. 4a). The results of
x
Fig. 2 The improvement of viscosity-sensitive properties of FMR-nucleoside NEAR with different templates (12-mer ON, containing one or three dC
3
b compared to 1b. (a) Absorption (EG) and uncorrected fluorescence (EG–
modifications) are shown in Fig. 4b. The NEAR works well with both
modified dC TPs, but the yield was higher in the case of less dense
glycerol) spectra of 1b and 3b; the viscosity of samples was (from bottom to
X
top): 74.0, 112.2, 170.2, 258.1 and 391.4 mPa s. (b) The Forster–Hoffmann plot
Table 1 Photophysical properties of fluorescent molecular rotors
a
abs
b
c
Compound
l
l
em
x
1
1
3
3
a
b
466
440
452
423
503
486
506
488
0.594 ꢀ 0.028
0.554 ꢀ 0.020
0.630 ꢀ 0.021
0.627 ꢀ 0.022
VJ
a (dC )
b (dC
VDP
)
a
b
Fig. 4 Enzymatic synthesis of FMR-modified DNA by NEAR and PCR.
Position in nm of the maximum of the absorption in EG. Position in
c
nm of the maximum of the emission in EG–glycerol. Viscosity sensitivity (a) Schematic representation of NEAR; (b) agarose gel analysis of NEAR
obtained from the Forster–Hoffmann equation.
products; and (c) agarose gel analysis of PCR products.
for 1b and 3b; error bars show SD of the mean for n = 3.
This journal is ©The Royal Society of Chemistry 2015
Chem. Commun., 2015, 51, 4880--4882 | 4881

View Article Online
ChemComm
Communication
VDP
labelling. Correct incorporation of modified dNTPs was also con- The probe containing modified dC
nucleotides exhibits a light-up
firmed by MALDI-TOF spectrometry. The NEAR on a semi- response upon binding to protein, which makes it prospective for
VDP
preparative scale was also performed using dC TP followed probing DNA–protein interactions under homogenous conditions.
by HPLC purification, yielding the corresponding 12-mer mod-
This work was supported by the Academy of Sciences of the
ified oligonucleotide in 1.9 nmol yield (0.5 mL scale). To further Czech Republic (RVO: 61388963), by the Czech Science Founda-
explore the applicability of the FMR-modified dNTPs, we tested tion (P206-12-G151) and by Gilead Sciences Inc. D.D. thanks the
them in PCR. A series of optimization experiments have shown IOCB for postdoctoral fellowship.
VDP
VJ
that dC
(but not dC ) can be sufficiently incorporated by
VDP
Notes and references
KOD XL DNA polymerase using dC TP as the substrate
Fig. 4c), although it requires higher concentration of dC TP,
VDP
(
1
(a) N. Dai and E. T. Kool, Chem. Soc. Rev., 2011, 40, 5756–5770; (b)
C.-H. Leung, D. S.-H. Chan, H.-Z. He, Z. Cheng, H. Yang and D.-L.
Ma, Nucleic Acids Res., 2012, 40, 941–955.
(a) M. Hawkins, Cell Biochem. Biophys., 2001, 34, 257–281; (b) A. S.
Wahba, A. Esmaeili, M. J. Damha and R. H. E. Hudson, Nucleic Acids
Res., 2010, 38, 1048–1056; (c) J.-W. Jung, S. K. Edwards and E. T. Kool,
ChemBioChem, 2013, 14, 440–444.
increased number of cycles and longer elongation time. Alto-
gether, these results indicate that dC TP is a good substrate
VDP
2
for enzymatic synthesis of DNA using PEX, NEAR and PCR.
Finally, we examined the usability of fluorescent molecular rotors
attached to DNA as reporting groups for protein–DNA interactions.
Binding studies in solution were performed using a 30-mer ssDNA
probe ON1 (Table S2 in the ESI†) obtained by PEX with magneto-
separation (Fig. 3c) and single-strand binding protein from E. coli
3
T. Ono, S. Wang, C.-K. Koo, L. Engstrom, S. S. David and E. T. Kool,
Angew. Chem., Int. Ed., 2012, 51, 1689–1692.
4 J. J. Li, X. Fang, S. M. Schuster and W. Tan, Angew. Chem., Int. Ed.,
000, 39, 1049–1052.
(a) S. Park, H. Otomo, L. Zheng and H. Sugiyama, Chem. Commun.,
014, 50, 1573–1575; (b) P. A. Hopkins, R. W. Sinkeldam and Y. Tor,
2
5
20
(
SSB), exhibiting a non-sequence specific binding to ssDNA. We
2
observed a significant 4-fold increase of fluorescence upon titration
of DNA by SSB with a stoichiometry of interaction 2 : 1 (Fig. 5). Since
at least 56 nt ssDNA is needed to wrap around the SSB tetramer
Org. Lett., 2014, 16, 5290–5293; (c) Y. Saito, A. Suzuki, Y. Okada,
Y. Yamasaka, N. Nemoto and I. Saito, Chem. Commun., 2013, 49,
5
684–5686; (d) D. Dziuba, V. Y. Postupalenko, M. Spadafora, A. S.
Klymchenko, V. Gu ´e rineau, Y. M ´e ly, R. Benhida and A. Burger, J. Am.
Chem. Soc., 2012, 134, 10209–10213; (e) M. Weinberger, F. Berndt,
R. Mahrwald, N. P. Ernsting and H.-A. Wagenknecht, J. Org. Chem.,
21
under the conditions used, the short ONs used in our study
apparently bind in a higher ratio. Notably, the maximal increase of
2013, 78, 2589–2599; ( f ) T. Kanamori, H. Ohzeki, Y. Masaki,
7,8
fluorescence was higher than with our previous probes.
A. Ohkubo, M. Takahashi, K. Tsuda, T. Ito, M. Shirouzu, K. Kuwasako,
Y. Muto, M. Sekine and K. Seio, ChemBioChem, 2015, 16, 167–176.
(a) M. Hocek and M. Fojta, Chem. Soc. Rev., 2011, 40, 5802–5814;
To rule out possible non-specific interaction between the DNA-
tethered fluorophores and protein, we also studied the influence of
bovine serum albumin (BSA), which does not bind to DNA but
6
(
b) M. Hollenstein, Molecules, 2012, 17, 13569–13591; (c) M. Hocek,
J. Org. Chem., 2014, 79, 9914–9921.
7 J. Riedl, R. Pohl, N. P. Ernsting, P. Orsag, M. Fojta and M. Hocek,
Chem. Sci., 2012, 3, 2797–2806.
J. Riedl, P. Menova, R. Pohl, P. Orsag, M. Fojta and M. Hocek, J. Org.
Chem., 2012, 77, 8287–8293.
14b
possess hydrophobic pockets where FMR can bind. No fluores-
cence enhancement was observed, indicating the absence of non-
specific binding. Finally, to exclude the possible effect of components
of SSB buffer, we titrated the ssDNA probe with PBS–glycerol, which
also did not change the fluorescence (Fig. 5b). These control experi-
ments clearly show that the increase in FMR-DNA fluorescence was
induced by its binding to the protein.
8
9 (a) M. A. Haidekker and E. A. Theodorakis, Org. Biomol. Chem., 2007,
5
, 1669–1678; (b) M. K. Kuimova, Phys. Chem. Chem. Phys., 2012, 14,
12671–12686.
1
0 W. L. Goh, M. Y. Lee, T. L. Joseph, S. T. Quah, C. J. Brown, C. Verma,
S. Brenner, F. J. Ghadessy and Y. N. Teo, J. Am. Chem. Soc., 2014, 136,
6
159–6162.
1 S. Murudkar, A. K. Mora, P. K. Singh and S. Nath, Chem. Commun.,
012, 48, 5301–5303.
To conclude, we have designed, synthesized and characterized
1
X
new fluorescent dC TPs bearing viscosity-sensitive fluorophores,
2
VJ
fluorescent molecular rotors. While the julolidine derivative (dC TP) 12 L. Liu, Y. Shao, J. Peng, C. Huang, H. Liu and L. Zhang, Anal. Chem.,
VDP
2014, 86, 1622–1631.
3 (a) R. W. Sinkeldam, A. J. Wheat, H. Boyaci and Y. Tor, ChemPhysChem,
was too bulky for enzymatic incorporation, the smaller dC TP (4b)
1
was a very good substrate for polymerases in PEX, NEAR and PCR
and can be efficiently used in enzymatic construction of DNA probes.
2011, 12, 567–570; (b) M. G. Pawar, A. Nuthanakanti and S. G. Srivatsan,
Bioconjugate Chem., 2013, 24, 1367–1377; (c) Y. Zhang, X. Yue, B. Kim,
S. Yao and K. D. Belfield, Chem. – Eur. J., 2014, 20, 7249–7253; (d) A. A.
Tanpure and S. G. Srivatsan, ChemBioChem, 2012, 13, 2392–2399.
4 (a) T. Iwaki, C. Torigoe, M. Noji and M. Nakanishi, Biochemistry, 1993, 32,
1
7589–7592; (b) Y.-Y. Wu, W.-T. Yu, T.-C. Hou, T.-K. Liu, C.-L. Huang,
I. C. Chen and K.-T. Tan, Chem. Commun., 2014, 50, 11507–11510.
5 A. C. Fogarty, E. Duboue-Dijon, F. Sterpone, J. T. Hynes and D. Laage,
Chem. Soc. Rev., 2013, 42, 5672–5683.
6 H. Hwang and S. Myong, Chem. Soc. Rev., 2014, 43, 1221–1229.
7 J. Sutharsan, D. Lichlyter, N. E. Wright, M. Dakanali, M. A. Haidekker
and E. A. Theodorakis, Tetrahedron, 2010, 66, 2582–2588.
1
1
1
1
1
8 J. Ludwig, Acta Biochim. Biophys. Acad. Sci. Hung., 1981, 16, 131–133.
9 (a) V. Raindlov ´a , R. Pohl, M. ˇS anda and M. Hocek, Angew. Chem.,
Int. Ed., 2010, 49, 1064–1066; (b) P. Menova and M. Hocek, Chem.
Commun., 2012, 48, 6921–6923; (c) P. M ´e nov ´a , V. Raindlov ´a and
M. Hocek, Bioconjugate Chem., 2013, 24, 1081–1093; (d) D. Dziuba,
R. Pohl and M. Hocek, Bioconjugate Chem., 2014, 25, 1984–1995.
0 R. D. Shereda, A. G. Kozlov, T. M. Lohman, M. M. Cox and J. L. Keck,
Critical Rev. Biochem. Mol. Biol., 2008, 43, 289–318.
Fig. 5 (a) Fluorescence spectra of the ssDNA probe ON1 (black) in the
presence of 0.5 eq. of the SSB tetramer (blue) and BSA (red); (b) relative
change in fluorescence of ON1 (1 mM) upon addition of the SSB tetramer
2
(blue), BSA (red) and PBS–glycerol (green); titration performed at pH 7.4, 21 (a) T. M. Lohman and M. E. Ferrari, Ann. Rev. Biochem., 1994, 63, 527–570;
1
00 mM NaCl; error bars show SD of the mean for n = 3.
(b) W. Bujalowski and T. M. Lohman, J. Mol. Biol., 1989, 207, 249–268.
4
882 | Chem. Commun., 2015, 51, 4880--4882
This journal is ©The Royal Society of Chemistry 2015