Selective parallel G-quadruplex recognition by a NIR-to-NIR two-photon squaraine

Article abstract of DOI:10.1039/C8SC02882F

Fluorescence imaging probes for specific G-quadruplex (G4) conformations are of considerable interest in biomedical research. Herein, we present the synthesis and the binding properties of a new water-soluble near-infrared (NIR) amphiphilic squaraine dye (CAS-C1) which is capable of selective detection of parallel over non-parallel and non G4 topologies. The striking changes in its linear optical response upon binding to parallel G4s give rise to high fluorescence quantum yields (Φ_f ≈ 0.7) and one-photon molecular brightness in the far-red-NIR region. The outstanding recognition process of CAS-C1 for parallel G4s via end-stacking provides binding constants in the nanomolar regime (K_b = 10⁷ to 10⁸ M^?1) awarding it as one of the most potent parallel G4 binders currently available. Moreover, the CAS-C1-parallel G4 system exhibits large two-photon absorption (TPA) cross-sections and molecular brightness in the second NIR biological transparency window (λ ≈ 1275 nm), making it an ideal candidate for NIR-to-NIR ultrasensitive two-photon procedures.

Full text of DOI:10.1039/C8SC02882F

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Selective parallel G-quadruplex recognition by
a NIR-to-NIR two-photon squaraine†
Cite this: DOI: 10.1039/c8sc02882f
Vincenzo Grande,‡ab Chia-An Shen,‡a Marco Deiana,c Marta Dudek,c
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
c
c
ab
*
and Frank Wurthner
¨
*
Joanna Olesiak-Banska,
Katarzyna Matczyszyn
Fluorescence imaging probes for specific G-quadruplex (G4) conformations are of considerable interest in
biomedical research. Herein, we present the synthesis and the binding properties of a new water-soluble
near-infrared (NIR) amphiphilic squaraine dye (CAS-C1) which is capable of selective detection of parallel
over non-parallel and non G4 topologies. The striking changes in its linear optical response upon binding
to parallel G4s give rise to high fluorescence quantum yields (Ff z 0.7) and one-photon molecular
brightness in the far-red-NIR region. The outstanding recognition process of CAS-C1 for parallel G4s via
end-stacking provides binding constants in the nanomolar regime (Kb ¼ 107 to 108
M
ꢀ1) awarding it as
Received 29th June 2018
Accepted 26th August 2018
one of the most potent parallel G4 binders currently available. Moreover, the CAS-C1-parallel G4 system
exhibits large two-photon absorption (TPA) cross-sections and molecular brightness in the second NIR
biological transparency window (l z 1275 nm), making it an ideal candidate for NIR-to-NIR ultrasensitive
two-photon procedures.
DOI: 10.1039/c8sc02882f
rsc.li/chemical-science
majority of the small optical imaging probes for G4 detection are
commonly photoexcited by relatively high-to-moderate energy
light (330–600 nm)14,15 in order to generate the emission between
Introduction
Single-stranded guanine-rich oligonucleotides can fold into
noncanonical compact, highly stable four-stranded helices
known as G-quadruplexes (from here denoted as G4s).1 Recent
studies have demonstrated the occurrence of G4s in human cells
and their implication in several key biological events especially
those that are disease- and aging-related.2,3 As a consequence,
they are recognized as potential drug targets. To date, over
700 000 G4s have been identied across the human genome,4
especially located in the telomeres at the ends of chromosomes
and in the promoter regions of oncogenes. Although indisputable
progresses have been made over the past years in the design of
G4-binders,5–10 the development of more complex multitasking
compounds that combine the optimization of both optical
performances (excitation and emission wavelengths > 600 nm
and high brightness) and G4 structural selectivity/affinity (G4
over duplex binding and among the G4 topologies, Fig. 1)
remains highly challenging.11–13 Indeed, the overwhelming
the visible and far-red region of the electromagnetic spectrum.
Such energies, in particular UV or blue, are in themselves dele-
terious to cells as they can irreversibly damage DNA and/or
induce formation of cytotoxic reactive oxygen species.16 Further-
more, their excitation and emission wavelengths are conned
outside the rst and second near-infrared (NIR) biological
transparency windows (BTWs, NIR-I: 700–950 nm and NIR-II:
1000–1350 nm)17 where absorption and scattering from tissues
and oxy/deoxygenated blood is signicant, limiting their rele-
vance to 2-D cell cultures and precluding their use in more real-
istic diagnosis environments, such as in thick tissues or in vivo
models. The lack of biocompatible uorescent organic probes in
the BTWs has prevented the use of these highly sensitive spectral
ranges for in-depth imaging.17 In addition, the dynamic nature of
the G4 structure enable a range of different topologies (e.g.
parallel, hybrid and antiparallel) to potentially form under
different physiological conditions, making molecular recognition
particularly arduous. In our research on G4-probes, we have
recently shown that a dicyanovinyl squaraine dye (SQgI, Fig. 1a)18
could behave as G4-specic NIR light-up probe. In the complexed
state, SQgl is emissive at 700 nm upon excitation at 661 nm
through one-photon absorption (OPA) with an impressive
quantum yield of FF z 0.6. The probe exhibited good selectivity
of G4 over duplex, with the dicyanovinyl squaraine dye capable to
impart outstanding selectivity over non-G4 structures and
remarkable specicity toward parallel G4 topologies. However,
this probe presented inherent optical, practical and affinity
a
¨
¨
¨
¨
Universitat Wurzburg, Institut fur Organische Chemie, Am Hubland, 97074 Wurzburg,
Germany. E-mail: wuerthner@uni-wuerzburg.de
b
¨
Center for Nanosystems Chemistry & Bavarian Polymer Institute (BPI), Universitat
¨
¨
Wurzburg, Theodor-Boveri-Weg, 97074 Wurzburg, Germany
cAdvanced Materials Engineering and Modelling Group, Faculty of Chemistry, Wroclaw
University of Science and Technology, Wybrzeze Wyspianskiego 27, 50-370 Wroclaw,
Poland. E-mail: katarzyna.matczyszyn@pwr.edu.pl
† Electronic supplementary information (ESI) available: Synthesis, titration
experiments, optical spectra, NMR and HRMS spectra. See DOI:
10.1039/c8sc02882f
‡ These authors contributed equally.
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squaraine core scaffold with brush substituents carrying six
triethylene glycol chains (synthesis and characterization in ESI).
The massive presence of oligoethylene glycol chains is not only
benecial for the solubility of the ligand, but it is also known to
strengthen the affinity of the ligand toward the parallel G4s
upon noncovalent interactions within the grooves. This
approach is reported in the literature and has been exploited in
the design of selective G4-ligands.21–23 The molecular design of
dicyanovinyl squaraine revealed a suitable choice in terms of
affinity and selectivity with G4s.18 Additionally, the donor–
acceptor–donor (D–A–D) nature of the chromophores24
appeared very promising for multi-photon absorption processes
at long-infrared wavelengths. The synthesis of the newly
designed squaraine according to the route shown in Fig. 1a
starts from the reported brush precursor 1 that was brominated
to give the corresponding bromide 2, which underwent
a nucleophilic substitution with methyl-benzothiazole to give 3.
Following the reported synthetic procedure for dicyanovinyl
squaraines,24 CAS-C1 was obtained aer condensation of the
squaric ester 4 with two equivalents of methyl-benzothiazolium
3. Squaraine CAS-C1 revealed fairly good solubility in various
organic solvents. The absorption and emission spectra in these
Fig. 1 (a) Synthetic route to CAS-C1. (b) Schematic illustration of the
multiphotonic selective detection mechanism: the squaraine shows
amphiphilic assembly in water into micelles. The formation of a stable
complex with parallel G-quadruplex (left) is able to displace the
squaraine from the micellar aggregate and trigger the fluorescent
emission in the NIR-I biological window upon both one-photon
excitation in the NIR-I and two-photon excitation in the NIR-II. CAS-
C1 bestows high selectivity of interactions over non-quadruplex and
non-parallel G4 structures (right).
solvents revealed
a moderate negative solvatochromism
(Fig. S2†) and can be assigned to monomeric species (Fig. S1†).
Aggregation in water
In aqueous solution, CAS-C1 absorption spectra reveal
a different band shape and a strongly blue-shied absorption
maximum at 632 nm with an apparent extinction coefficient
ꢀ3max ¼ 6.2 ꢁ 104 Mꢀ1 (Fig. 2a, red line) which are rst evidences
for an H-type exciton coupling between aggregated dyes.25
Atomic force microscopy (AFM) revealed the presence of
small particles with an irregular diameter of 10–30 nm and
a height of 0.6–3.0 nm (Fig. 2b) which suggests the formation of
limitations including: (i) moderate solubility, (ii) moderate
binding constants (Kb values comprised between 105 to 106
Mꢀ1 18–20 and (iii) excitation wavelengths outside the BTWs. Thus,
)
we speculated that exploring the chemical space around the
squaraine core should enable optimization of both optical and
DNA binding performances and we decided to decorate the
dicyanovinylene squaraine moiety with bulkier water-soluble
brush-type groups.
Herein, we present the rst example of a water-soluble
amphiphilic squaraine dye (hereaer CAS-C1, Fig. 1a) exhibit-
ing G4-selective uorescence light-up upon photoexcitation
both within the NIR-I (lmax z 720 nm and FF z 0.7) through
OPA, and within the NIR-II (two-photon absorption cross-
section, s2, of z 300 GM and two-photon molecular bright-
ness, s2 ꢁ FF, of z 200 GM at 1275 nm) through TPA. More-
over, the unprecedented nonlinear optical properties of CAS-C1
are combined with the outstanding selectively for parallel over
non-parallel G4 architectures and duplex morphologies of any
length and composition awarding it as the best two-photon
parallel G4-specic probe currently available.
Fig. 2 (a) UV/Vis absorption spectra of an aqueous CAS-C1 solution (c
¼ 3.0 ꢁ 10ꢀ6 M, 25 ꢂC, red line) and upon addition of increasing
amounts of SDS. The inset shows the incorporation of the squaraine
dye in the SDS micelle upon addition of the surfactant with a critical
concentration of 2.1 ꢁ 10ꢀ3 M. (b) AFM height images on mica after
spin coating the CAS-C1 aqueous solution (3.0 ꢁ 10ꢀ6 M). Z scale is
Results and discussion
Design and synthesis
With the aim to increase the binding selectivity and the solu-
bility of ligand SQgl, we decorated the dicyanovinylene 1.2 nm. (c) Cross-section from yellow dashed lines 1–10.
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micellar structures. The blue-shied absorption band indicates KRAS 22, bcl2 VAV1), in the vascular endothelial growth factor
a co-facial stacking of CAS-C1 molecules, most likely in an (VEGF) gene, in the telomeric transcripts (TERRA) and the
antiparallel fashion in order to cancel the dyes' large dipole thrombin-binding DNA aptamer (TBA) (see ESI† for details
moment.26 Such arrangement explains well the strong H-type concerning the G4 topologies and their folding process).
excitonic coupling and the quenched uorescence emission. Indeed, upon addition of parallel G4s (e.g. VAV-1, c-myc and
Due to the massive presence of ethylene glycol chains, CAS-C1 is VEGF) the disappearance of the absorption band of CAS-C1
particularly suited to form such micellar structure where the aggregate along with the formation of a new bathochromically
hydrophilic parts are predominantly directed to the outside shied band centered at z 700 nm indicate the formation of G4
(Fig. 1b).
(parallel):CAS-C1 complexes (Fig. 3, S6 and S7†). Concomitant
ꢂ
Upon heating to 95 C, CAS-C1 micellar aggregates become with absorption changes, the steady-state emission of the
disfavored for entropic reasons leading to a new band at 676 nm squaraine uorophore upon excitation at lexc ¼ 660 nm, almost
with isosbestic point at 653 nm. These changes are reversible fully quenched in its aggregated unbound state (lem ¼ 698 nm),
and are ascribed to the formation of the monomeric species in lights-up in the NIR-I at lem z 720 nm (Fig. 3a, S8 and S9†).
water, with the typical uorescence emission peaked at 699 nm
Notably, both the absorption and the emission bands are z
(Fig. S3A†). Global tting of the temperature-dependent 20 nm redshied compared to the spectrum of the monomeric
measurement in the range 550–750 nm is in good agreement squaraine in water (Fig. S3†). This is a clear indication of
with an isodesmic aggregation mechanism (Fig. S3B†). Despite a direct interaction of the cyanine p-surface of CAS-C1 with the
the fact that the pure monomeric state is not fully reached in guanines of the parallel quadruplexes. The excitation spectra
water, the extrapolation of the tting at 676 nm predicts and the lifetime decays recorded for CAS-C1 in the presence of
a reasonable value for monomeric CAS-C1 of ꢀ3max ¼ 9.0 ꢁ G4s indicate that the strong photoluminescence observed arises
104 Mꢀ1 cmꢀ1. In accordance with the disaggregation model, mainly from the CAS-C1 : G4 complex through geometrical and
a uorescence emission in water lights-up at 699 nm (Fig. S3A†, structural restrictions which inhibit the nonradiative relaxation
dotted line). In contrast, the emission prole of CAS-C1 in its pathways, hence the increased lifetime and FF (Fig. S10–S12†).
aggregated form is fully quenched due to nonradiative relaxa- TCSPC decay trace of the CAS-C1 : G4 excited states (Fig. S1 and
tion pathways in its H-aggregate state. In order to investigate the S12†) showed the presence of only one species, ascribed to the
photophysical properties of the excited states, time-correlated complexed squaraine, in accordance with the monoexponential
single photon counting (TCSPC) measurements of CAS-C1 in function (eqn S1, ESI†). The singlet state lifetimes obtained by
water at 80 ꢂC were performed. The time-dependent decay tted tting of time-resolved uorescence decays of the CAS-C1 : G4
better with the biexponential function (eqn S2, ESI†) suggesting complexes are generally longer than the ones in organic
the coexistence of two different species: one with longer lifetime solvents and comprised in the range of 5–6 ns (Fig. S2B†). This
(s1 ¼ 4.76 ns), consistently ascribed to the excited state of the relatively long lifetimes with outstanding FF in the 700–800 nm
free monomer in solution; the other one with shorter lifetime range makes CAS-C1 particularly intriguing candidate as
(s2 ¼ 1.35 ns), ascribed to the species aggregated in the micellar a uorescence lifetime-based probe for G4 live-cell visualization
structure (Fig. S4†). The dramatic decrease of the lifetime and by uorescence lifetime imaging microscopy (FLIM).
emission intensity is, therefore, in accordance with the forma-
tion of additional radiationless pathways in the aggregated between parallel and antiparallel G4s as well as single- and
form. double-stranded DNA of any length and composition indicates
Comparison of absorption and uorescence responses
A more efficient approach to disassemble CAS-C1 into a clear-cut binding preference of CAS-C1 for G4 parallel topol-
monomeric dyes is provided by the addition of surfactant ogies (Fig. 3b). This was further conrmed by duplex DNA
molecules. Thus, we performed titration with sodium dodecyl competition titration assays (Fig. S19 and S20†) which revealed
sulfate (SDS) (Fig. 2a). The surfactant affects moderately the very modest interference of interaction with c-myc and VAV-1,
formation of the micelles at lower SDS concentration. Above the even in the presence of 50 eq. of the ds-DNA.
critical concentration of 2.1 ꢁ 10ꢀ3 M, SDS induces a marked
It turned out that probe accommodation within the hydro-
breakup of CAS-C1 micelles by dissolving the CAS-C1 monomer phobic G4 scaffolds exert changes in the polarity of the
N
within the SDS micelles (Fig. 2a).
surrounding that well-match a ET value of 0.6 (Fig. S2†). The
optical changes arising in the absorption and emission proles
of the CAS-C1-parallel G4 systems result in excellent one-photon
brightness (dened as the product 3 ꢁ FF) in the NIR region
G4 binding
In a similar fashion to the disaggregation induced by SDS, with values comprised between 56 and 84 ꢁ 103 Mꢀ1 cmꢀ1
molecular recognition of CAS-C1 to suitable p-surfaces should (Table 1).
also cause the disassembly of the micelle. This idea prompted
As suggested by recent publications,27–29 the issue of the
us to study the recognition process of the squaraine toward binding stoichiometry of the complexes in solution was
a large panel of biologically relevant G4 topologies such as those assessed by Job's plot titration assays (Fig. S13–S15†), the
found in the human telomeres (Tel-22) and minisatellite tangent method and a global tting based on both the 2 : 1, 1 : 1
(Ceb25), in the proto-oncogene involved in the tumorigenesis of and 1 : 2 binding models (Fig. S16–S18†). If compared with the
urinary bladder cancer (hRAS1), in the promoter region of myc, previously reported SQgl, CAS-C1 also showed multiple binding
kit, KRAS, bcl2, and vav genes (c-myc, c-kit2, c-kit87up, KRAS 32, with the G4s, except for sG4, VAV-1 and c-myc.
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Fig. 3 (a) Visible/NIR absorption (solid lines) and fluorescence spectra (dotted lines, excitation on the isosbestic point at 660 nm) of a CAS-C1
buffered solution in water (1.66 ꢁ 10ꢀ6 M, TRIS buffer c ¼ 10 mM, pH ¼ 7.2, KCl c ¼ 100 mM) upon addition of c-myc G4 at 25 ꢂC (red and blue
lines correspond to the spectra at 0 eq. and 3.4 eq. respectively. Superimposed black dotted line results from a global fitting with a 1 : 1 binding
model (Kb ¼ 9.7 ꢁ 106 Mꢀ1). (b) Fluorescence titrations of the CAS-C1 buffered solution in water upon addition of various G4s sequences at 25 ꢂC:
the plots show a clear-cut preference of the squaraine for the parallel G4s (in red). (c and d) Docking lowest energy binding models for CAS-C1
with c-myc (score function DE ¼ꢀ5.9 kcal molꢀ1).
A detailed quantitative binding data analysis that takes into for the previously reported squaraine SQgI (see ESI† for details
account 1 : 1 and 1 : 2 CAS-C1 : G4 stoichiometries with asso- concerning the quantitative binding data analysis).18 These
ciated (anti-)cooperative effects indicates that CAS-C1 exhibits results are ascribed to the highly accessible parallel G-quartets
higher binding constants for parallel topologies such as c-myc which, being devoid of adjacent lateral or diagonal loops,
(Fig. 3c and d) with respect to antiparallel and non-G4 topolo- provide better p-stacking platforms for the accommodation of
gies with values that exceed by two-orders of magnitude those the squaraine core, thereby resulting in a better planarity and
Table 1 Summary of the spectroscopic properties and the binding constants of the complexes of CAS-C1 with various G4sa
lmax
3max
(103 Mꢀ1 cmꢀ1
lem
3max ꢁ FF
G-quadruplex
(nm)
)
(nm)
K11 (Mꢀ1
)
K12 (Mꢀ1
)
s (ns)
FF
(103 Mꢀ1 cmꢀ1
)
sG4
699
699
699
698
699
699
699
699
699
699
114
118
108
108
113
116
103
113
103
92
722
722
721
719
721
721
721
721
721
720
8.1 ꢁ 106
1.2 ꢁ 107
1.0 ꢁ 107
9.0 ꢁ 105
1.6 ꢁ 102
1.5 ꢁ 107
5.9 ꢁ 106
36
—
—
—
—
5.8
5.6
5.5
6.0
5.7
5.4
5.6
—
0.74 ꢃ 0.07
84
79
56
71
84
—
—
—
—
—
VAV-1
c-myc
25Ceb
VEGF
Kras 22
Kras 32
bcl-2
0.67 ꢃ 0.02
0.52 ꢃ 0.03
0.66 ꢃ 0.01
3.7 ꢁ 109
1.7 ꢁ 106
4.1 ꢁ 105
6.0 ꢁ 108
4.3 ꢁ 108
4.0 ꢁ 108
0.74 ꢃ 0.08
—
—
—
—
—
ckit-87up
ckit-2
3.1 ꢁ 102
5.5
—
5.0 ꢁ 102
a
Data tting with 1 : 1 and 1 : 2 models was performed according to ref. 28 and the soware provided at the website http://supramolecular.org. We
like to note that the credibility of the derived values for the 1 : 2 case is lower than for the 1 : 1 case. This is due to the presence of three species and
unknown uorescence quantum yields of the 1 : 1 and the 1 : 2 complexes, and changing amounts of these complexes during the titration
experiment. To give the same weight to UV/Vis and uorescence experiments we evaluated both titrations independently and took the average
values.
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thus in a more efficient rigidication as desired for a uores- properties in the NIR, a chromophore should also possess high
cence increase. On the other hand, the negative effect of the s2 values to be considered as a suitable probe for two-photon
external quartets surrounded by loops in antiparallel G4s such microscopy procedures in NIR-to-NIR congurations. The TPA
as 22AG and c-kit hampers to some extent the end-stacking spectra of CAS-C1 complexed with parallel G4s including VAV1,
ability of CAS-C1 inducing a lower overall optical response. VEGF and c-myc were measured by two-photon excited uo-
The aforementioned rationale is conrmed by using the closely rescence (TPEF) method in the range 1000–1550 nm and are
related c-myc G4 analogue sG4, featuring no anking overhang reported in Fig. 4 as s2 ꢁ FF.
residues at both 50 and 30 ends that limit the steric hindrance, to
In general, all the complexes show similar TPA spectra
enhance the light-up ability of CAS-C1. Indeed, complexation of composed of an intense high energy TPA band blue shied
the squaraine with sG4 provides a FF of 0.74 which is among the compared to the one-photon transition energy and a weak low
highest values reported for nucleic acid complexes within energy TPA band that matches well the wavelength-doubled 1PA
comparable spectral range (Table 1).30,31 Structural insights into spectra. These spectral features are usually associated with
the binding mode of CAS-C1 with G4s were provided by docking centrosymmetric quadrupolar molecules, in which selection
binding analysis on the G4s whose structures in solution had rules for 1PA and 2PA electronic transitions are mutually
been derived by NMR studies under similar experimental exclusive. CAS-C1 presents a non-centrosymmetric character
conditions. The elucidated structures of G4s are referenced in with a quasi-C2v symmetry and can be treated as bent quad-
Table S1†. In the case of c-myc, the squaraine chromophore is rupolar chromophore, of which the quadrupolar conformation
the recognition unit leading to a perfect complementary match gives rise to an intense hypsochromically shied TPA while the
with the 30-quartet (Fig. 3c). However, the substitution of side residual dipolar character results in a weak TPA transition
groups at the squaraine chromophore has surely an impact in overlapped with the 1PA maximum. Similar conclusions were
the binding. On one side, the massive number of glycol chains recently reported for extended Michler's ketone, heptamethine,
are responsible for the enhanced interaction compared to SQgl diarylboryl and anthracenyl derivatives.35–38 The TPA cross-
by establishing strong electrostatic interactions with the polar section (s2) values for the CAS-C1 : G4 systems follow the
backbone of the G4. Presumably, the glycol chains displace order: CAS-C1 : VAV1 s2 ¼ 312 GM, CAS-C1 : VEGF s2 ¼ 278 GM
water molecules from the grooves and thereby provide an and CAS-C1 : c-myc s2 ¼ 273 GM at 1275 nm (Fig. S25†).
entropic contribution to the stability of the G4-CAS-C1 complex, Interestingly, the CAS-C1 aggregate also shows TPA absorption
especially for the parallel ones,32 which explains the strength- close to half energy of the corresponding OPA, in agreement
ening of the binding affinity compared with SQgl. On the other with a centrosymmetric aggregate (Fig. S26†).
hand, it is reasonable to assume that the steric hindrance of the
The high values of s2 combined with the associated 2PA
benzyloxy unit, which is accommodated in the hydrophobic maximum are an optimal combination for NIR-to-NIR cong-
pockets in the side of the quartet (Fig. 3c and d), largely urations since excitation of CAS-C1 : G4 systems can be per-
contribute to the selectivity of the interaction.
formed within the NIR-II window by TPA (lex(2PA) ¼ 1275 nm)
Two-photon absorption
A fundamental optical limitation is encountered at the nano-
scale level owing to Abbe's law of diffraction, which limits the
maximum spatial resolution to approximately half the wave-
length of light used to image the specimen.33 One way to over-
come this limitation involves the use of nonlinear optical
procedures based on multi-photon absorption processes. In
this approach, the excitation of a chromophore is performed by
the simultaneous absorption of two-photons with half the
energy required for the corresponding one-photon absorption
process. As a result, the excitation wavelength is conned in the
rst and second NIR biological transparency windows where the
diffusion and absorption of light by inherently biological
matrices is minimized, and thus allowing deep tissue penetra-
tion. Moreover, the low-energy excitation prevents photo-
bleaching and cellular damages in biological samples.
Importantly, the TPA is limited to a small femtoliter volume and
occurs only at the focal point of a focused laser beam resulting Fig. 4 Two-photon molecular brightness (s2 ꢁ FF) plot of CAS-C1 in
the presence of VEGF, VAV-1 and c-myc (10 mM Tris, 100 mM K+, pH
in 3D image resolution. Despite the lower energy of the IR light
compared to visible and UV light, this excitation mode has been
proven to induce damage to the samples when using classical
uorophore that possess low TPA cross-section (s2 < 50 GM).34 It
7.2) at molar ratio (r) ¼ 7. The values of the FF used are tabulated in
Table 1. As a comparison, the OPA corresponding to the double of the
energy of the TPA is shown (top and right axis): the absorption (dotted
lines) and emission (dashed line) spectra corresponding to the CAS-C1
turns out that, besides having signicant photoluminescence complexes with the G4s are shown.
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with emissions centered within the NIR-I spectral region hand, to increase the selectivity of the interaction by exploiting
(lem z 720 nm). In line with the trend depicted for s2 also the the more easily accessible G-quartet surface of parallel G4s and,
two-photon molecular brightness (s2 ꢁ FF), as a result of the on the other hand, the massive presence of glycol chains in the
strong uorescence enhancement of CAS-C1 bound to parallel hydrophobic pocket of the G4 imparted strong affinity. The
G4s, follow the same order (i.e. CAS-C1 : VAV1 s2 ꢁ FF ¼ 209 complexes of CAS-C1 with parallel G4s also possess strong two-
GM, CAS-C1 : VEGF s2 ꢁ FF ¼ 206 GM and CAS-C1 : c-myc photon absorption with maxima centered at 1275 nm, which
s2 ꢁ FF ¼ 142 GM at 1275 nm) indicating the suitability of make them ideally suited for NIR-to-NIR imaging procedures.
CAS-C1 to be used as a nonlinear biomarker for parallel G4 Ultimately, the enhanced uorescence quantum yields and the
detection by using ultrasensitive uorescence microscopy two-photon absorption cross-section enable one to obtain
techniques. These values are nevertheless lower than exceptionally high two-photon molecular brightness values
those observed for monomeric CAS-C1 in organic solvent within the NIR-II window that are desired for virtually all
(s2 ꢁ FF ¼ 386 GM in isopropanol, Fig. S24†). This result is in ultrasensitive uorescence spectroscopy and microscopy tech-
agreement with recent results from Goodson and co-workers39 niques. These overall ndings award CAS-C1 as the best two-
and some of us40,41 that relate the changes in the TPA cross- photon parallel G4-specic probe currently available.
section to the different DNA binding modes. In particular,
groove binders exhibit an enhanced TPA cross-section due to
the DNA electric eld induced enhancement of the transition Conflicts of interest
dipole moment, while intercalators or end-stackers show
a decrease in the TPA cross-section when complexed with DNA.
There are no conicts to declare.
Accordingly, this nding also supports our proposal that CAS-
C1 binds to parallel G4s mainly via end-stacking mode in
agreement with the docking results. The end-stacking ability of
Acknowledgements
CAS-C1 was further conrmed through electronic circular
We thank Prof. Pall Thordarson (Sydney, Australia) for his
helpful advice for the analysis of the titration experiments. The
dichroism (ECD) measurements (Fig S27†).42 Indeed, the native
conformation of the parallel G4s was almost not affected by the
¨
research at the University of Wurzburg was nancially sup-
presence of the squaraine dye indicating no or very weak
perturbation on the quadruplex morphologies. By analogy with
previously reported data for a G4 intercalator,41 the absence of
conformational changes observed for CAS-C1-parallel G4
systems constitutes a clear evidence of the ability of CAS-C1
to coordinate the external G-tetrads, ruling out the possible
intercalative binding behavior of our probe known to give
rise to pronounced structural changes associated with
a partially disruption of the stacking interactions that govern
G4 formation.
ported by the State of Bavaria with the establishment of the
KeyLab for Supramolecular Polymers of the Bavarian Polymer
Institute (BPI). At the Wroclaw University of Science and Tech-
nology, nancial support from a statutory activity subsidy from
the Polish Ministry of Science and Higher Education for the
Faculty of Chemistry of WUST, KNOW – center of excellence in
biotechnology project, and Harmonia grant DEC-2016/22/M/
ST4/00275 are highly acknowledged.
Notes and references
Conclusions
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towards parallel G4 motif open up a new research line for this
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