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A triphenylamine-based colorimetric and fluorescent probe with donor–bridge–acceptor structure for detection of G-quadruplex DNA
Accepted Manuscript A triphenylamine-based colorimetric and fluorescent probe with donor–bridgeacceptor structure for detection of G-quadruplex DNA Ming-Qi Wang, Wen-Xiang Zhu, Zhuan-Zhuan Song, Shuo Li, Yong-Zhao Zhang PII: DOI: Reference:
S0960-894X(15)30216-X http://dx.doi.org/10.1016/j.bmcl.2015.11.007 BMCL 23263
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
23 September 2015 16 October 2015 4 November 2015
Please cite this article as: Wang, M-Q., Zhu, W-X., Song, Z-Z., Li, S., Zhang, Y-Z., A triphenylamine-based colorimetric and fluorescent probe with donor–bridge-acceptor structure for detection of G-quadruplex DNA, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.11.007
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A triphenylamine-based colorimetric and fluorescent probe with donor–bridge-acceptor structure for detection of G-quadruplex DNA Ming-Qi Wanga,*, Wen-Xiang Zhua, Zhuan-Zhuan Songa, Shuo Lib,*, Yong-Zhao Zhanga a
School of Pharmacy, Jiangsu University, Zhenjiang, 212013, P. R. China. Fax: +86 511 85032801; E-mail: [email protected]
b
School of Chemical Engineering, Chongqing University of Technology, Chongqing 400054, P. R. China. E-mail: [email protected]
Abstract: In this paper, three triphenylamine-based dyes (TPA-1, TPA-2a and TPA-2b) with donor–bridge-acceptor (D–π–A) structure were designed and synthesized for the purpose of G-quadruplexes recognition. In aqueous conditions, the interactions of the dyes with G-quadruplexes were studied with the aim to establish the influence of the geometry of the dyes on their binding and probing properties. Results indicate that TPA-2b displays significant selective colorimetric and fluorescent changes upon binding of G-quadruplex DNA. More importantly, its distinct color change enables visual detection and differentiation of G-quadruplexes from single and duplex DNA structures. CD titration date reveals that TPA-2b could induce and stabilize the formation of G-quadruplex structure. All these remarkable properties of TPA-2b suggest that it should have promising application in the field of G-quadruplexes research.
Keywords: G-quadruplex, Triphenylamine, Donor–bridge-acceptor, Visual detection Single-stranded nucleic acids containing repetitive guanine-rich bases are known to adopt non-canonical four-stranded secondary structures called G-quadruplexes (G4s) which are composed of several planar stacks of guanine quartets stabilized by hydrogen bonds and metal cations (usually K+ or Na+).1 During the past two decades, G4 structures have been well characterized by nuclear magnetic resonance and X-ray crystallography and they can exhibit diversity topological conformations, depending on the primary sequence, strand orientation and numbers of strands involved.2 In spite of being well studied in vitro, the evidence for the
presence of G4 structures in vivo remains mainly controversial, but is currently supported by a growing wealth of biochemical and molecular genetics data.3-5 Because of their abundance in functional genomic regions, such as telomere, promoter regions of important oncogenes and the untranslated regions of mRNAs, they have possible implication in a range of biological functions, including telomere maintenance, transcription and translation regulation, the DNA damage response, genome rearrangements and anti-tumor chemotherapy.6-9 In this context, the recognition of G4s with small-molecule probes has emerged as a strategy to design: chemical tools to detect these biological processes and potential anticancer drugs.10, 11 In recently years, fluorescence-based detection method has received great attention due to its simple operation, high selectivity and sensitivity. Therefore, great efforts have been made to develop small-molecule-based fluorescent probes for G4s.12-19 One of the common limitations is that most of them have only displayed one type of output, which are prone to be disturbed in quantitative detection by many factors, thus it would highly desire to develop probes containing different outputs. Such probes will take advantage of different methods for more reliable measurements and versatile applications. Recently, colorimetric detection with visual readouts has been attracted much interest because it is rapid, facile and even allow instrument-free.20,
21
To this end, design a probe for detection G4s with colorimetric and
fluorescent properties is a promising strategy. Triphenylamine-based dyes have been widely used in opto- and electro-active materials, fluorescence probes and cell imaging for their good electron donating and transporting capability.22-24 Moreover, the triphenylamine moiety usually plays the role of donor in various systems and can form a donor–acceptor (D-A) system to increase the fluorescence. Some triphenylamine derivatives have demonstrated the ability of interaction with AT regions, but rarely reported to have the optical response to G4s.22a, 25 Inspired by those studies, we attempted to screen and discover new dual probes for G4 structures based on triphenylamine dyes with push-pull architectures (Scheme 1). TPA-1 and TPA-2a were designed by linking an indolium hemicyanine and a N-methyl-quinoline unit to the triphenylamine skeleton via a methine chain respectively, whereas TPA-2b possesses an additional functional amine side chain compared to TPA-2a. The N-methylated heterocycles were chosen because of their electron-accepting as well as DNA binding properties.26 The
rotation of the methine-bridge would impact the push–pull effects between the donor and acceptor moieties, and also may be restricted after binding to the specific DNA structure, and give rise to a different spectroscopy response, which could be utilized to distinguish the DNA structures. The amino-side group could be protonated at physiological pH, which may further increases solubility in aqueous solution.
Scheme 1. Chemical structures of triarylamine-based dyes.
The desired dyes were synthesized in our laboratories. TPA-1 and TPA-2a were prepared from commercially available materials using simple methylation and condensation procedures (Scheme S1). TPA-2b, an amine-side chain derivative of TPA-2a, was obtained through a facile three-component one-pot reaction (Scheme S1). All of the new compounds were characterized by 1H NMR, 13C NMR and MS (see the Supplementary Information). With the dyes in hand, their photophysical properties were first elucidated in different solvents. In buffered solution of relatively high ionic strength (10 mM Tris-HCl, 60 mM KCl, pH 7.4), all the dyes displayed broad absorption bands and blue shifted with respected to the bands observed in organic solvents (Figures S1-S3, Table S2 for the detailed date). The spectral shifts may be attributed to specific solute-solute and solute-solvent interactions or charge-transfer interactions. It also has been found that the three dyes were almost non-fluorescent in buffered solutions, with very weak and broad fluorescence bands in the long wavelength region between 400 and 580 nm and thus display remarkable Stockes shifts of about 100 nm for dyes TPA-1 and TPA-2a and about 90 nm for TPA-2b. Taken together, these spectral features (i.e., broad, long-wavelength absorption bands, large Stokes shifts) are characteristic of donor–acceptor systems exhibiting internal charge transfer upon excitation. In this regard, the absorption maxima of the dyes TPA-1 and TPA-2a are redshifted with respect
to the TPA-2b, which gives evidence for more pronounced charge-transfer interactions in TPA-1 and TPA-2a. The interactions of the dyes with G4 DNA were studied using absorption spectra titrations. Electronic absorbance spectroscopy is one of the most useful ways to investigate the interactions of compounds with DNA.27 Compound binding to DNA usually results in hypochromism and red shift. The extent of the hypochromism and red shift parallels the binding affinity.28 Titrations of dyes TPA-1, TPA-2a and TPA-2b in the absence and presence of Ckit1 as well as representative G4 DNA were obtained at room temperature. The changes in the spectral profiles were shown in Figure 1 and Supplementary Figures S4 and S5. With the increase in concentration of Ckit1, the intensity of TPA-2b at 450 nm gradually decreased, and a notable redshift of the maximum was observed with an isosbestic point at 485 nm, which led to a new peak at appromximately 495 nm. The hypochromism (H%), as defined by H% = 100% (Afree-Abound)/Afree, of band at 480 nm of TPA-2b were 41.3%. As compared to TPA-2b, addition of Ckit1 led to a 20 nm red shift and 31.2% hypochromism for TPA-2a and only 11.9% hypochromism for TPA-1. The exact date was shown in Table S3. In order to further compare quantitatively the binding affinities of the three dyes, the intrinsic binding constants Kb of TPA-1, TPA-2a and TPA-2b to Ckit1 were measured to be 0.12 × 106, 5.47 × 106 and 14.15 × 106 M-1, respectively, using the equation from Eq.(1) (see supplementary information). The larger binding constant Kb of TPA-2b, indicating TPA-2b binds to Ckit1 more tightly than the other two dyes. As the three dyes containing the same triphenylamine skeleton, this may be caused the quinolinium moiety may also interact with G-quartet plane by π-π stacking or with the phosphate backbone via electrostatic interaction.29 In addition, introduction of amine side chain would also increase the stabilizing ability of TPA-2b to Ckit1. This might be related to that introduction of the amine side chain into the quinolinium ring led to binding mode of ligands with G4 DNA change to a certain extent. Interesting, the specific red-shift of TPA-2b gave rise to a marked and vivid color change from pale yellow to pink under daylight, which was valuable for the naked-eye detection of Ckit1 (Figure 1C-D).
Figure 1. (A) Absorbance spectra of TPA-2b in buffer (10 mM Tris-HCl, pH 7.4, 60 mM KCl) in the presence of increasing amounts of Ckit1. TPA-2b = 8 μM, [Ckit1] = 0-2.5 μM from top to bottom. Arrows indicate the change in absorbance upon increasing the Ckit1 concentrations. (B). Corresponding half-reciprocal plot of TPA-2b (R = 0.9978). (C) Photograph of 8 μM TPA-2b (a) and 8 μM TPA-2b with 3 μM Ckit1 mixture (b) in buffer taken under daylight. (D) Color changes of TPA-2b (8 μM) upon addition of Ckit1 (0-4 μM) under daylight.
The detailed fluorescence properties of the dyes with G4 DNA were explored by using fluorescence titration assays. As shown in Figure 2A, TPA-2b alone in buffer displayed weak fluorescence emission. With the gradual addition of the G4 DNA Ckit1, an emission peak at approximately 627 nm was significantly enhanced and accompanied by a 75 nm red shift, indicating the interaction occurred with G4 DNA. The change of (F/F0-1) versus Ckit1 concentration can be satisfactorily fitted to the Boltzmann function (Figure 2B). This fluorescence enhancement might be caused by conformational changes in the excited state of TPA-2b, most likely by the rotation restriction around the vinyl-bridge that separates the triphenylamine and N-methylated quinolinium motieties, as clearly shown by the emission enhancement in a viscous medium (Figure S7).20b It can be seen that there was a good linear relationship between the concentration of Ckit1 (0.4-1.6 μM). and the fluorescence intensity. The limit of detection was estimated to be 0.158 μM according to the standard method (Supplementary information, Figure S8). However, TPA-1 and TPA-2a did not show any significant fluorescence enhancement after adding Ckit1 (Supplementary Figure S6). Taken
together with the results of absorption and fluorescence spectral experiments, it can be concluded that TPA-2b has great potential as a colorimetric and fluorescent G4 DNA probe. Therefore, TPA-2b was chosen as the most promising probe for further detailed investigation.
Figure 2. (A) Fluorescence spectra of TPA-2b (2 μM) upon titration with prefolded Ckit1 (0-2 μM) in 10 mM Tris-HCl buffer (pH 7.4) and 60 mM KCl. (B) Plots of F/F0-1 at 627 nm versus Ckit1 concentration. λex = 462 nm.
The interactions with different DNA oligomers, including G4, single- and double-stranded DNAs under identical salt conditions were conducted to examine the selectivity. Changes in absorption spectra were shown in Figure 3 and Figure S9, the interactions with G4s, including Ckit1, CM22, HTG-21, were characterized by the clearly enhanced the absorbance at 495nm and formation of new bands redshifted about 45 nm. In the case of single-stranded DNA (ss26) and double-stranded DNAs (ds26, polyd(A-T)9 and polyd(G-C)9), the new bands showed slight decrease and much smaller redshift (by 6-26 nm) (Figures 3A-B and S9). Of note, the red-shift of the absorption bands point out to differences in the TPA-2b binding affinities in single-, duplex and G4 DNA. Moreover, TPA-2b also exhibited excellent selectivity for G4s over single- and double-stranded DNAs (Figures 3C-D and S10) by fluorescence measurements, which was consistent with the absorption titration studies. The fluorescence quantum yield values of TPA-2b with different nucleic were summarized in Table S4. Meanwhile, as shown in Figure 3E, the incubation of G4s with TPA-2b caused significant color change under daylight. And other DNAs did not cause noticeable color change, suggesting that G4s could be
easily detected and differentiated from other forms of DNA with just the naked eyes.
Figure 3. (A)The absorbance enhancement of TPA-2b (8 μM) at 495 nm with different amounts of DNAs. (B) Absorbance response of TPA-2b to DNAs, the y-axis corresponds to the shift in the absorbance maximum (λmax). (C) The fluorescence intensity enhancement of TPA-2b (2 μM) with different amounts of DNAs. (D) Distribution of the values of the F/F0 for TPA-2b with DNAs. λex = 462 nm. (E) Photograph of the aqueous solutions of TPA-2b/DNA mixtures taken under daylight. Conditions: 10 mM Tris-HCl buffer (pH 7.4, 60 mM KCl).
It is known that cationic species play an important role in determining the structure of G4s.1 Therefore, the fluorescence measurements were also performed with different cationic species (K+ or Na+), or without any metals ions (Figure 4). The binding affinity of TPA-2b to the K+ was found to be stronger than that of Na+, indicating the higher fluorescence enhancement in the buffer solution with KCl (14-fold) than that with NaCl (9-fold). In other words, G4 structure formed in the presence of K+ is stable than that in the presence of Na+. Interestingly,
in the absence of any metal ions, the TPA-2b-G4 system shows a fluorescence turn-on up to 32-fold, which is larger in the case of K+ or Na+. Besides, after adding Ckit1 without prefolding, the absorption spectra showed 50 nm red-shift and approximately 43% hypochromism (Figure S11, Table S5), which is similar to the result of absorption spectra in the presence of K+. These might indicate that the positively charged quinolinium group and amine side chain in TPA-2b increased the solubility of the dye in the aqueous buffer and strengthened its interaction with the negatively charged Ckit1 via electrostatic interaction. When the positively charged K+ or Na+ ions added, the TPA-2b was partially detached and driven into the aqueous buffer, probably due to its unfavorable docking on the G-quadruplex surface.
Figure 4. Fluorescence intensity enhancement (F/F 0) of TPA-2b (2 μM) plotted against the Ckit1 in 10 mM Tris-HCl buffer (pH 7.4) in the presence of 60 mM KCl, 60 mM NaCl and absence of any metal ions. λex = 462 nm.
To validate the assumption, the structural changes in Ckit1 caused by TPA-2b were studied using Circular dichroism (CD) measurements. CD spectroscopy is an extremely useful technique to study nucleic acids conformation, so it is widely used to study the G4 conversion induced by probes.14 In the presence of 60 mM K+ ions and absence of any molecules, the CD spectrum of Ckit1 was of the typical parallel G-quadruplex, with the positive peak at 264 nm and a negative peak at 240 nm.30 After addition of TPA-2b to the solution, the characteristic peaks of the parallel G4 had no obvious change, which the positive peak at 264 nm enhanced
slightly (Figure 5). In addition, we tested the effect of Na+ ions on conformation of G-quadruplex. As shown in Figure S12, it was found that the spectrum was almost unchanged on addition of different concentrations of TPA-2b. These results indicated the TPA-2b had no influence on the conformation of G4 structure in monovalent cations. However, the slight enhancement of the positive peak attribute to the stabilization of the G4 structure. On the other hand, as shown in 5B, Ckit1 sequence adopted a parallel structure in the absence of monovalent cations. Upon addition of TPA-2b, significant changes in ellipticity were observed. And the positive band at 260 nm increased significantly and the negative band at 238 nm also increased. The results indicated that TPA-2b could induce and stabilize the Ckit1 sequence to form parallel structure of quadruplex in the absence of salt.
Figure 5. CD titrations spectra of TPA-2b for Ckit1 G4 DNA (4 μM) in 10 mM Tris-HCl buffer (pH 7.4) in the presence(A) and absence (B) of 60 mM KCl. [TPA-2b]/μM: (1) 0; (2) 2; (3) 4; (4) 8.
Competition titrations were performed to further confirm the selective of TPA-2b binding to G4 DNA, in which the ability of TPA-2b to retain an enhanced fluorescence intensity with the addition of G4 DNA challenged by single-strand or duplex DNA. As shown in Figures 6 and S9, in the presence of an excess of duplex DNA (ds26, 50 μM) or single-strand DNA (ss26, 50 μM), very slightly increased the intensity of TPA-2b (about 2 folds) was observed, however, upon addition of an increasing amount of Ckit1 led to a significant enhancement of the
fluorescence intensity (about 10 folds). Similar results were found by using absorption spectra. Although the absorbance of the dye was affected by the presence of dsDNA and ssDNA, the addition of the Ckit1 led to much larger red-shifts of the absorption bands.(Figure S13) The finding shows that detection of G4 DNA is indeed possible under these conditions. These results demonstrate the high potential of TPA-2b to serve as quadruplex-sensitive probe in the competitive biological environment.
Figure 6. Spectrofluorimetric titrations of Ckit1 (0-4 μM) to the dye TPA-2b in the presence of an excess of dsDNA (50 μM) or ssDNA (50 μM). The concentration of TPA-2b was 2 μM in buffer. λex = 462 nm.
In conclusion, three new dyes with D-π-A structure were rationally designed and synthesized for G4 recognition. Results show the TPA-2b can be used as a colorimetric and fluorescent dual probe for specific detection of G4s, based on its significant absorbance red-shift and fluorescence enhancement in the presence of G4s. Furthermore, the distinct color changes of TPA-2b offered a convenient visualization method for discriminating G4s from other DNA forms (including single- and double-strand DNAs). The CD studies showed that TPA-2b could induce and stabilize G4 structure. Further studies on the exploration of biomedical applications of this probe are ongoing in our laboratories.
Acknowledgements This work was financially supported by the Senior Talent Cultivation Program of Jiangsu University (Nos. 15JDG068). This work was also supported by Natural Science Foundation Project of CQ CSTC (cstc2013jcyjA50012), Scientific and Technological Research Program of Chongqing Municipal Education Commission (Grant No. KJ130820), China Postdoctoral Science Foundation Funded Project (No. 2014M562326). We thank Analytical & Testing Center of Jiangsu University for NMR, MS and CD analysis.
Supplementary date Experimental procedures, fluorescence and absorption spectra of the probe; Copies of 1H and 13C NMR, MS spectra for new compounds.
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[16] Vummidi, B. R.; Alzeer, J.; Luedtke, N. W. ChemBioChem 2013, 14, 540. [17] Zhang, W.; Ma, Z.; Du, L.; Li, M. Analyst 2014, 139, 2641. [18] Ma, D. L.; Zhang, Z.; Wang, M.; Lu, L.; Zhong, H. J.; Leung, C. H. Chem. Biol. 2015, 22, 812. [19] Laguerre, A.; Stefan, L.; Larrouy, M.; Genest, D.; Novotna, J.; Pirrotta, M.; Monchaud, D. J. Am. Chem. Soc. 2014, 136, 12406. [20] (a) Yan, J. W.; Ye, W. J.; Chen, S. B., Wu, W. B.; Hou, J. Q.; Ou, T. M.; Tan, J. H.; Li, D.; Gu, L. Q.; Huang, Z. S. Anal. Chem. 2012, 84, 6288. (b) Yan, J. W.; Chen, S. B.; Liu, H. Y.; Ye, W. J.; Ou, T. M., Tan. J. H.; Li, D.; Gu, L. Q.; Huang, Z. S. Chem. Commun. 2014, 50, 6927. (c) Yan, J. W.; Tian, Y. G.; Tan, J. H.; Huang, Z. S. Analyst 2015, In print. (d) Chen, S. B.; Wu, W. B.; Hu, M. H.; Ou, T. M.; Gu, L. Q.; Tan. J. H.; Huang, Z. S. Chem. Commun. 2014, 50, 12173. (e) Hu, M. H.; Chen, S. B.; Guo, R. J.; Ou, T. M.; Huang, Z. S.; Tan. J. H. Analyst 2015, 140, 4616. [21] Jin, B.; Zhang, X.; Zheng, W.; Liu, X.; Zhou, J.; Zhang, N.; Wang, F.; Shangguan, D. Anal. Chem. 2014, 86, 7063. [22] (a) Lai, H.; Xiao, Y.; Yan, S.; Tian, F.; Zhong, C.; Liu, Y.; Weng, X.; Zhou, X. Analyst, 2014, 139, 1834. (b) Wang, C. C.; Yan, S. Y.; Chen, Y. Q.; Zhou, Y. M.; Zhong, C.; Guo, P.; Huang, R.; Weng, X. C.; Zhou, X. Chinese Chem. Lett. 2015, 26, 323. [23] Dumat, B.; Bordeau, G.; Aranda, A. I.; Mahuteau-Betzer, F.; Harfouch, Y. E.; Metge, G.; Charra, F.; Fiorini-Debuisschert, C.; Teulade-Fichou, M. P. Org. Biomol. Chem. 2012, 10, 6054. [24] Sakong, C.; Kim, H. J.; Kim, S. H.; Namgoong, J. W.; Park, J. H.; Ryu, J. H.; Kim, B.; Ko, M. J.; Kim, J. P. New J. Chem. 2012, 36, 2025. [25] Blaise, D.; Guillaume, B.; Elodi, F. P.; Florence, M. B.; Nicolas, S.; Germain, M.; Celine, F. D.; Fabrice, C.; Marie-Paule, T. F. J. Am. Chem. Soc. 2013, 135, 12697. [26] Bunkenborg, J.; Gadjev, N. I.; Deligeorgiew, T.; Jacobsen, J. P. Bioconjugate Chem. 2000, 11, 861. [27] Liu, Z. C.; Wang, B. D.; Yang, Z. Y.; Li, Y.; Qin, D. D.; Li, T. R. Eur. J. Med. Chem. 2009, 44, 4477. [28] Tarushi, A.; Kljun, J.; Turel, I.; Pantazaki, A. A.; Psomas G.; Kessissoglou, D. P. New J. Chem. 2013, 37, 342. [29] Liu, Z. Q.; Zhuo, S. T.; Tan, J. H.; Ou, T. M.; Li, D.; Gu, L. Q. Huang, Z. S. Tetrahedron 2013, 69, 4922. [30] Hong, Y.; Xiong, H.; Wing, J.; Lam, Y.; Häubler, M.; Liu, J.; Yu, Y.; Zhong, Y.; Sung, H. H. Y.; Williams, I. D.; Wong, K. S.; Tang, B. Z. Chem. Eur. J. 2010, 16, 1232.
Graphical abstract
A colorimetric and fluorescent dual probe with D–π–A structure for G-quadruplexes was successfully developed. Its significant and distinct changes in color enable the label-free and visual detection of and differentiation of G-quadruplexes from single and duplex DNA structures.
S0960-894X(15)30216-X http://dx.doi.org/10.1016/j.bmcl.2015.11.007 BMCL 23263
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
23 September 2015 16 October 2015 4 November 2015
Please cite this article as: Wang, M-Q., Zhu, W-X., Song, Z-Z., Li, S., Zhang, Y-Z., A triphenylamine-based colorimetric and fluorescent probe with donor–bridge-acceptor structure for detection of G-quadruplex DNA, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.11.007
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A triphenylamine-based colorimetric and fluorescent probe with donor–bridge-acceptor structure for detection of G-quadruplex DNA Ming-Qi Wanga,*, Wen-Xiang Zhua, Zhuan-Zhuan Songa, Shuo Lib,*, Yong-Zhao Zhanga a
School of Pharmacy, Jiangsu University, Zhenjiang, 212013, P. R. China. Fax: +86 511 85032801; E-mail: [email protected]
b
School of Chemical Engineering, Chongqing University of Technology, Chongqing 400054, P. R. China. E-mail: [email protected]
Abstract: In this paper, three triphenylamine-based dyes (TPA-1, TPA-2a and TPA-2b) with donor–bridge-acceptor (D–π–A) structure were designed and synthesized for the purpose of G-quadruplexes recognition. In aqueous conditions, the interactions of the dyes with G-quadruplexes were studied with the aim to establish the influence of the geometry of the dyes on their binding and probing properties. Results indicate that TPA-2b displays significant selective colorimetric and fluorescent changes upon binding of G-quadruplex DNA. More importantly, its distinct color change enables visual detection and differentiation of G-quadruplexes from single and duplex DNA structures. CD titration date reveals that TPA-2b could induce and stabilize the formation of G-quadruplex structure. All these remarkable properties of TPA-2b suggest that it should have promising application in the field of G-quadruplexes research.
Keywords: G-quadruplex, Triphenylamine, Donor–bridge-acceptor, Visual detection Single-stranded nucleic acids containing repetitive guanine-rich bases are known to adopt non-canonical four-stranded secondary structures called G-quadruplexes (G4s) which are composed of several planar stacks of guanine quartets stabilized by hydrogen bonds and metal cations (usually K+ or Na+).1 During the past two decades, G4 structures have been well characterized by nuclear magnetic resonance and X-ray crystallography and they can exhibit diversity topological conformations, depending on the primary sequence, strand orientation and numbers of strands involved.2 In spite of being well studied in vitro, the evidence for the
presence of G4 structures in vivo remains mainly controversial, but is currently supported by a growing wealth of biochemical and molecular genetics data.3-5 Because of their abundance in functional genomic regions, such as telomere, promoter regions of important oncogenes and the untranslated regions of mRNAs, they have possible implication in a range of biological functions, including telomere maintenance, transcription and translation regulation, the DNA damage response, genome rearrangements and anti-tumor chemotherapy.6-9 In this context, the recognition of G4s with small-molecule probes has emerged as a strategy to design: chemical tools to detect these biological processes and potential anticancer drugs.10, 11 In recently years, fluorescence-based detection method has received great attention due to its simple operation, high selectivity and sensitivity. Therefore, great efforts have been made to develop small-molecule-based fluorescent probes for G4s.12-19 One of the common limitations is that most of them have only displayed one type of output, which are prone to be disturbed in quantitative detection by many factors, thus it would highly desire to develop probes containing different outputs. Such probes will take advantage of different methods for more reliable measurements and versatile applications. Recently, colorimetric detection with visual readouts has been attracted much interest because it is rapid, facile and even allow instrument-free.20,
21
To this end, design a probe for detection G4s with colorimetric and
fluorescent properties is a promising strategy. Triphenylamine-based dyes have been widely used in opto- and electro-active materials, fluorescence probes and cell imaging for their good electron donating and transporting capability.22-24 Moreover, the triphenylamine moiety usually plays the role of donor in various systems and can form a donor–acceptor (D-A) system to increase the fluorescence. Some triphenylamine derivatives have demonstrated the ability of interaction with AT regions, but rarely reported to have the optical response to G4s.22a, 25 Inspired by those studies, we attempted to screen and discover new dual probes for G4 structures based on triphenylamine dyes with push-pull architectures (Scheme 1). TPA-1 and TPA-2a were designed by linking an indolium hemicyanine and a N-methyl-quinoline unit to the triphenylamine skeleton via a methine chain respectively, whereas TPA-2b possesses an additional functional amine side chain compared to TPA-2a. The N-methylated heterocycles were chosen because of their electron-accepting as well as DNA binding properties.26 The
rotation of the methine-bridge would impact the push–pull effects between the donor and acceptor moieties, and also may be restricted after binding to the specific DNA structure, and give rise to a different spectroscopy response, which could be utilized to distinguish the DNA structures. The amino-side group could be protonated at physiological pH, which may further increases solubility in aqueous solution.
Scheme 1. Chemical structures of triarylamine-based dyes.
The desired dyes were synthesized in our laboratories. TPA-1 and TPA-2a were prepared from commercially available materials using simple methylation and condensation procedures (Scheme S1). TPA-2b, an amine-side chain derivative of TPA-2a, was obtained through a facile three-component one-pot reaction (Scheme S1). All of the new compounds were characterized by 1H NMR, 13C NMR and MS (see the Supplementary Information). With the dyes in hand, their photophysical properties were first elucidated in different solvents. In buffered solution of relatively high ionic strength (10 mM Tris-HCl, 60 mM KCl, pH 7.4), all the dyes displayed broad absorption bands and blue shifted with respected to the bands observed in organic solvents (Figures S1-S3, Table S2 for the detailed date). The spectral shifts may be attributed to specific solute-solute and solute-solvent interactions or charge-transfer interactions. It also has been found that the three dyes were almost non-fluorescent in buffered solutions, with very weak and broad fluorescence bands in the long wavelength region between 400 and 580 nm and thus display remarkable Stockes shifts of about 100 nm for dyes TPA-1 and TPA-2a and about 90 nm for TPA-2b. Taken together, these spectral features (i.e., broad, long-wavelength absorption bands, large Stokes shifts) are characteristic of donor–acceptor systems exhibiting internal charge transfer upon excitation. In this regard, the absorption maxima of the dyes TPA-1 and TPA-2a are redshifted with respect
to the TPA-2b, which gives evidence for more pronounced charge-transfer interactions in TPA-1 and TPA-2a. The interactions of the dyes with G4 DNA were studied using absorption spectra titrations. Electronic absorbance spectroscopy is one of the most useful ways to investigate the interactions of compounds with DNA.27 Compound binding to DNA usually results in hypochromism and red shift. The extent of the hypochromism and red shift parallels the binding affinity.28 Titrations of dyes TPA-1, TPA-2a and TPA-2b in the absence and presence of Ckit1 as well as representative G4 DNA were obtained at room temperature. The changes in the spectral profiles were shown in Figure 1 and Supplementary Figures S4 and S5. With the increase in concentration of Ckit1, the intensity of TPA-2b at 450 nm gradually decreased, and a notable redshift of the maximum was observed with an isosbestic point at 485 nm, which led to a new peak at appromximately 495 nm. The hypochromism (H%), as defined by H% = 100% (Afree-Abound)/Afree, of band at 480 nm of TPA-2b were 41.3%. As compared to TPA-2b, addition of Ckit1 led to a 20 nm red shift and 31.2% hypochromism for TPA-2a and only 11.9% hypochromism for TPA-1. The exact date was shown in Table S3. In order to further compare quantitatively the binding affinities of the three dyes, the intrinsic binding constants Kb of TPA-1, TPA-2a and TPA-2b to Ckit1 were measured to be 0.12 × 106, 5.47 × 106 and 14.15 × 106 M-1, respectively, using the equation from Eq.(1) (see supplementary information). The larger binding constant Kb of TPA-2b, indicating TPA-2b binds to Ckit1 more tightly than the other two dyes. As the three dyes containing the same triphenylamine skeleton, this may be caused the quinolinium moiety may also interact with G-quartet plane by π-π stacking or with the phosphate backbone via electrostatic interaction.29 In addition, introduction of amine side chain would also increase the stabilizing ability of TPA-2b to Ckit1. This might be related to that introduction of the amine side chain into the quinolinium ring led to binding mode of ligands with G4 DNA change to a certain extent. Interesting, the specific red-shift of TPA-2b gave rise to a marked and vivid color change from pale yellow to pink under daylight, which was valuable for the naked-eye detection of Ckit1 (Figure 1C-D).
Figure 1. (A) Absorbance spectra of TPA-2b in buffer (10 mM Tris-HCl, pH 7.4, 60 mM KCl) in the presence of increasing amounts of Ckit1. TPA-2b = 8 μM, [Ckit1] = 0-2.5 μM from top to bottom. Arrows indicate the change in absorbance upon increasing the Ckit1 concentrations. (B). Corresponding half-reciprocal plot of TPA-2b (R = 0.9978). (C) Photograph of 8 μM TPA-2b (a) and 8 μM TPA-2b with 3 μM Ckit1 mixture (b) in buffer taken under daylight. (D) Color changes of TPA-2b (8 μM) upon addition of Ckit1 (0-4 μM) under daylight.
The detailed fluorescence properties of the dyes with G4 DNA were explored by using fluorescence titration assays. As shown in Figure 2A, TPA-2b alone in buffer displayed weak fluorescence emission. With the gradual addition of the G4 DNA Ckit1, an emission peak at approximately 627 nm was significantly enhanced and accompanied by a 75 nm red shift, indicating the interaction occurred with G4 DNA. The change of (F/F0-1) versus Ckit1 concentration can be satisfactorily fitted to the Boltzmann function (Figure 2B). This fluorescence enhancement might be caused by conformational changes in the excited state of TPA-2b, most likely by the rotation restriction around the vinyl-bridge that separates the triphenylamine and N-methylated quinolinium motieties, as clearly shown by the emission enhancement in a viscous medium (Figure S7).20b It can be seen that there was a good linear relationship between the concentration of Ckit1 (0.4-1.6 μM). and the fluorescence intensity. The limit of detection was estimated to be 0.158 μM according to the standard method (Supplementary information, Figure S8). However, TPA-1 and TPA-2a did not show any significant fluorescence enhancement after adding Ckit1 (Supplementary Figure S6). Taken
together with the results of absorption and fluorescence spectral experiments, it can be concluded that TPA-2b has great potential as a colorimetric and fluorescent G4 DNA probe. Therefore, TPA-2b was chosen as the most promising probe for further detailed investigation.
Figure 2. (A) Fluorescence spectra of TPA-2b (2 μM) upon titration with prefolded Ckit1 (0-2 μM) in 10 mM Tris-HCl buffer (pH 7.4) and 60 mM KCl. (B) Plots of F/F0-1 at 627 nm versus Ckit1 concentration. λex = 462 nm.
The interactions with different DNA oligomers, including G4, single- and double-stranded DNAs under identical salt conditions were conducted to examine the selectivity. Changes in absorption spectra were shown in Figure 3 and Figure S9, the interactions with G4s, including Ckit1, CM22, HTG-21, were characterized by the clearly enhanced the absorbance at 495nm and formation of new bands redshifted about 45 nm. In the case of single-stranded DNA (ss26) and double-stranded DNAs (ds26, polyd(A-T)9 and polyd(G-C)9), the new bands showed slight decrease and much smaller redshift (by 6-26 nm) (Figures 3A-B and S9). Of note, the red-shift of the absorption bands point out to differences in the TPA-2b binding affinities in single-, duplex and G4 DNA. Moreover, TPA-2b also exhibited excellent selectivity for G4s over single- and double-stranded DNAs (Figures 3C-D and S10) by fluorescence measurements, which was consistent with the absorption titration studies. The fluorescence quantum yield values of TPA-2b with different nucleic were summarized in Table S4. Meanwhile, as shown in Figure 3E, the incubation of G4s with TPA-2b caused significant color change under daylight. And other DNAs did not cause noticeable color change, suggesting that G4s could be
easily detected and differentiated from other forms of DNA with just the naked eyes.
Figure 3. (A)The absorbance enhancement of TPA-2b (8 μM) at 495 nm with different amounts of DNAs. (B) Absorbance response of TPA-2b to DNAs, the y-axis corresponds to the shift in the absorbance maximum (λmax). (C) The fluorescence intensity enhancement of TPA-2b (2 μM) with different amounts of DNAs. (D) Distribution of the values of the F/F0 for TPA-2b with DNAs. λex = 462 nm. (E) Photograph of the aqueous solutions of TPA-2b/DNA mixtures taken under daylight. Conditions: 10 mM Tris-HCl buffer (pH 7.4, 60 mM KCl).
It is known that cationic species play an important role in determining the structure of G4s.1 Therefore, the fluorescence measurements were also performed with different cationic species (K+ or Na+), or without any metals ions (Figure 4). The binding affinity of TPA-2b to the K+ was found to be stronger than that of Na+, indicating the higher fluorescence enhancement in the buffer solution with KCl (14-fold) than that with NaCl (9-fold). In other words, G4 structure formed in the presence of K+ is stable than that in the presence of Na+. Interestingly,
in the absence of any metal ions, the TPA-2b-G4 system shows a fluorescence turn-on up to 32-fold, which is larger in the case of K+ or Na+. Besides, after adding Ckit1 without prefolding, the absorption spectra showed 50 nm red-shift and approximately 43% hypochromism (Figure S11, Table S5), which is similar to the result of absorption spectra in the presence of K+. These might indicate that the positively charged quinolinium group and amine side chain in TPA-2b increased the solubility of the dye in the aqueous buffer and strengthened its interaction with the negatively charged Ckit1 via electrostatic interaction. When the positively charged K+ or Na+ ions added, the TPA-2b was partially detached and driven into the aqueous buffer, probably due to its unfavorable docking on the G-quadruplex surface.
Figure 4. Fluorescence intensity enhancement (F/F 0) of TPA-2b (2 μM) plotted against the Ckit1 in 10 mM Tris-HCl buffer (pH 7.4) in the presence of 60 mM KCl, 60 mM NaCl and absence of any metal ions. λex = 462 nm.
To validate the assumption, the structural changes in Ckit1 caused by TPA-2b were studied using Circular dichroism (CD) measurements. CD spectroscopy is an extremely useful technique to study nucleic acids conformation, so it is widely used to study the G4 conversion induced by probes.14 In the presence of 60 mM K+ ions and absence of any molecules, the CD spectrum of Ckit1 was of the typical parallel G-quadruplex, with the positive peak at 264 nm and a negative peak at 240 nm.30 After addition of TPA-2b to the solution, the characteristic peaks of the parallel G4 had no obvious change, which the positive peak at 264 nm enhanced
slightly (Figure 5). In addition, we tested the effect of Na+ ions on conformation of G-quadruplex. As shown in Figure S12, it was found that the spectrum was almost unchanged on addition of different concentrations of TPA-2b. These results indicated the TPA-2b had no influence on the conformation of G4 structure in monovalent cations. However, the slight enhancement of the positive peak attribute to the stabilization of the G4 structure. On the other hand, as shown in 5B, Ckit1 sequence adopted a parallel structure in the absence of monovalent cations. Upon addition of TPA-2b, significant changes in ellipticity were observed. And the positive band at 260 nm increased significantly and the negative band at 238 nm also increased. The results indicated that TPA-2b could induce and stabilize the Ckit1 sequence to form parallel structure of quadruplex in the absence of salt.
Figure 5. CD titrations spectra of TPA-2b for Ckit1 G4 DNA (4 μM) in 10 mM Tris-HCl buffer (pH 7.4) in the presence(A) and absence (B) of 60 mM KCl. [TPA-2b]/μM: (1) 0; (2) 2; (3) 4; (4) 8.
Competition titrations were performed to further confirm the selective of TPA-2b binding to G4 DNA, in which the ability of TPA-2b to retain an enhanced fluorescence intensity with the addition of G4 DNA challenged by single-strand or duplex DNA. As shown in Figures 6 and S9, in the presence of an excess of duplex DNA (ds26, 50 μM) or single-strand DNA (ss26, 50 μM), very slightly increased the intensity of TPA-2b (about 2 folds) was observed, however, upon addition of an increasing amount of Ckit1 led to a significant enhancement of the
fluorescence intensity (about 10 folds). Similar results were found by using absorption spectra. Although the absorbance of the dye was affected by the presence of dsDNA and ssDNA, the addition of the Ckit1 led to much larger red-shifts of the absorption bands.(Figure S13) The finding shows that detection of G4 DNA is indeed possible under these conditions. These results demonstrate the high potential of TPA-2b to serve as quadruplex-sensitive probe in the competitive biological environment.
Figure 6. Spectrofluorimetric titrations of Ckit1 (0-4 μM) to the dye TPA-2b in the presence of an excess of dsDNA (50 μM) or ssDNA (50 μM). The concentration of TPA-2b was 2 μM in buffer. λex = 462 nm.
In conclusion, three new dyes with D-π-A structure were rationally designed and synthesized for G4 recognition. Results show the TPA-2b can be used as a colorimetric and fluorescent dual probe for specific detection of G4s, based on its significant absorbance red-shift and fluorescence enhancement in the presence of G4s. Furthermore, the distinct color changes of TPA-2b offered a convenient visualization method for discriminating G4s from other DNA forms (including single- and double-strand DNAs). The CD studies showed that TPA-2b could induce and stabilize G4 structure. Further studies on the exploration of biomedical applications of this probe are ongoing in our laboratories.
Acknowledgements This work was financially supported by the Senior Talent Cultivation Program of Jiangsu University (Nos. 15JDG068). This work was also supported by Natural Science Foundation Project of CQ CSTC (cstc2013jcyjA50012), Scientific and Technological Research Program of Chongqing Municipal Education Commission (Grant No. KJ130820), China Postdoctoral Science Foundation Funded Project (No. 2014M562326). We thank Analytical & Testing Center of Jiangsu University for NMR, MS and CD analysis.
Supplementary date Experimental procedures, fluorescence and absorption spectra of the probe; Copies of 1H and 13C NMR, MS spectra for new compounds.
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Graphical abstract
A colorimetric and fluorescent dual probe with D–π–A structure for G-quadruplexes was successfully developed. Its significant and distinct changes in color enable the label-free and visual detection of and differentiation of G-quadruplexes from single and duplex DNA structures.