The development of a light-up red-emitting fluorescent probe based on a G-quadruplex specific cyanine dye

Abstract

A cyanine dye-dimethylindole red containing an extending polymethine chain, a sterically bulky dimethylindole heterocycle and an anionic propylsulfonate substituent on the quinoline ring was found to behave as a high specific light-up G-quadruplex probe in the red-emitting region above 650 nm, especially for parallel G-quadruplex c-myc. The 10 to 70-fold enhancement in the fluorescence quantum yield of dimethylindole red when incubating with G-quadruplexes may benefit more accurate definition of the distribution of G-quadruplexes across the genome.

---

Introduction

Guanine-rich DNA can fold into a G-quadruplex structure owing to a multilayered stack of planar G-quartets via Hoogsteen hydrogen bonds.¹ In the human genome, guanine-rich sequences with the potential to form G-quadruplexes exist in the telomere (e.g. HT 22) as well as in promoter regions of HIV-1 ² and certain oncogenes, such as c-myc,^3,4 bcl-2,⁵ c-kit,^6,7 Kras,⁸ Hras.^9,10 These G-quadruplexes are believed to be involved in the regulation of several key biological processes, including cellular aging and cancer physiology.¹¹ Hence, G-quadruplexes have been the object of intensive study to determine their function and as emerging therapeutic targets in oncology.^11–14 Since G-quadruplex structures were verified to be stable and detectable in human genomic DNA in vivo,¹⁵ the identification and sensing of these sequences by fluorescent probes in cell has sparked great interests.

A considerable number of probes have been evaluated for their ability to selectively bind the quadruplex DNA structures, for instance, cyanine dyes, triphenylmethane dyes, carbazoles, porphyrins and metal complexes (Pt, Ru, Ir).^16–20 Notably, red to near infrared fluorescent probes offer certain advantages for photon penetration and eliminative background in bioimaging.²¹ Therefore, intense efforts with the aim to design red to near infrared fluorescent G-quadruplex probes have been made.^22–36 Cyanine dyes possess high fluorescent quantum yield, facile synthesis and controllable properties, have received great attention on the design of DNA staining probes.³⁷ Variation of the length of the polymethine bridge, and the identity of the heterocycles and bridge allow the tuning of absorption and emission spectra throughout the visible and near-infrared regions.³⁷ However, among the reported cyanine probes that have selectivity to G-quadruplex over duplex DNA,^{31–35,38–41} only a very few of probes exhibited strong emission bands above 650 nm, such as squaraines (CSTS³⁴ and TSQ1 ³²), ISCH-1 ³¹ and CyC-M716.⁴⁰ Most G-quadruplex probes are with positively charged substituents, and have inevitably enhanced the interaction with the negative charged phosphate backbone of DNA. Ligands with negative charged substituents, such as CSTS,³⁴ have been proven to dampen the non-specific interaction with DNA.

Herein, we presented an anionic substituted red-emitting dye, dimethylindole red (Dir, Scheme 1), initially synthesized by Armitage group as fluorescent dye for RNA aptamers or K7 single chain variable fragment (scFv) antibodies.^42,43 Addition of one extra methine bridge induced Dir a ca. 87 nm vinyl shift of absorbance band, by comparing with thiazole orange (TO), a well-known dye for duplex DNA. To introduce the bulky dimethylindole heterocycle and anionic propylsulfonate substituent, Dir exhibited weak fluorescence in the presence of excess amount of calf thymus DNA and synthetic dsDNA, thereby making it great potential for high specific light-up probe of G-quadruplex with long wavelength emitting.

Scheme 1 A high specific red-emitting G-quadruplex probe, dimethylindole red (Dir).

Experimental

Materials and instruments

All reagents and solvents were obtained from commercial sources and used without further purification. The ¹H NMR and ¹³C NMR spectra were recorded on an Agilent Technologies 800/54 premium compact using DMSO-d₆ as solvent. The mass spectrometry (ESI-MS) experiments were run on a Waters UPLC-Premier XE. The purity was determined by Waters UPLC. The melting point was recorded on Shenguang SGW X-4A melting point apparatus. Absorbance spectra were recorded by Perkin Elmer Lambda 25 UV/Vis spectrometer. Fluorescent spectra were performed on Hitachi F-4600 fluorescence spectrophotometer (Japan). CD spectra were recorded on the Chirascan plus CD Spectrometer (Applied Photophysics).

Oligonucleotides

Oligonucleotides were purchased from Sangon Biotech (Shanghai, China). The concentration of oligonucleotide was determined on the basis of UV absorption at 260 nm on Eppendorf Biophotometer (Germany) and the corresponding extinction coefficients were obtained from the OligoAnalyzer 3.1. Calf thymus DNA was dissolved overnight in 20 mM Tris–HCl with 100 mM KCl (pH 7.0) buffer. The calf thymus DNA concentration per nucleotide was determined by absorption spectroscopy using the molar absorption coefficient (6600 M⁻¹ cm⁻¹) at 260 nm. G-Quadruplex sequences were dissolved by 20 mM Tris–HCl with 100 mM KCl or 10 mM Tris–HCl buffer. G-Quadruplex was heated to 90 °C for 5 min and then cooled to room temperature overnight in order to form the G-quadruplex. All solutions were stored at −20 °C and diluted immediately before use.

Synthesis of Dir

The dye was synthesized according to the literature.⁴² Dir was obtained as yellow-reddish powder, yield 20%. Melting point: 237.4 to 238.4 °C. ¹H NMR (DMSO-d₆): 8.61 (1H, d, J = 7.2 Hz, H-1); 8.56 (1H, d, J = 8.0 Hz, H-5); 8.30–8.28 (2H, m, H-β and H-8); 8.07 (1H, d, J = 6.4 Hz, H-2); 7.99 (1H, t, J = 7.2 Hz, H-7); 7.75 (1H, t, J = 7.2 Hz, H-6); 7.46 (1H, d, J = 7.2 Hz, H-10); 7.28 (1H, t, J = 7.2 Hz, H-12); 7.24 (1H, d, J = 13.6 Hz, H-α); 7.15 (1H, d, J = 7.2 Hz, H-13); 7.06 (1H, t, J = 7.2 Hz, H-11); 6.18 (1H, d, J = 12.0 Hz, H-α′); 4.81 (2H, t, J = 7.2 Hz, H-a); 3.41 (3H, s, H-18); 2.51 (2H, t, J = 7.2 Hz, H-c); 2.16 (2H, quint, J = 7.2 Hz, H-b); 1.65 (6H, s, H-19 and H-19′). ¹³C NMR in ppm: 26.0 (C-b); 28.6 (C-19 and C-19′); 30.4 (C-18); 47.9 (C–c and C-16); 53.9 (C-a); 100.7 (C-α′); 109.6 (C-13); 111.9 (C-α); 112.9 (C-2); 118.8 (C-8); 122.5 (C-10); 123.1 (C-11); 125.3 (C-4); 125.9 (C-5); 127.7 (C-6); 128.5 (C-12); 134.3 (C-7); 138.3 (C-9); 140.5 (C-15); 143.4 (C-14); 143.8 (C-1); 144.4 (C-β); 152.1 (C-3); 168.7 (C-17). ESI-MS m/z: 449.6 (M + H⁺), calculated 448.58. The purity was identified 98.69% by UPLC. It is suitable for the further research.

Photostability experiments

The photostability of Dir was investigated under continuously irradiated by a 500 W xenon lamp for different times. Taking the commercial organic dye Cy5 as reference, Dir and Cy5 were diluted with in Tris–HCl buffer (10 mM Tris–HCl and 100 mM KCl, pH 7.4). The samples were irradiated with a 500 W xenon lamp for different time intervals, and then the fluorescence spectra of Dir and Cy5 were recorded, respectively.

Absorbance experiments

The DNA sequences used in the absorbance experiments were listed in Table S1 (ESI†). The absorbance of Dir (5 μM) was performed in Tris–HCl buffer (10 mM Tris–HCl, pH 7.4) with 100 mM KCl. Dir solution (5 μM) was treated with DNA stock solution. The absorbance of Dir–DNA solution was recorded.

Excitation spectra experiments

The excitation spectra of Dir in the presence of DNA were performed in Tris–HCl buffer (10 mM Tris–HCl, pH 7.4) with 100 mM KCl. The DNA sequences used in the fluorescence assay were listed in Table S1 (ESI†). Dir solution (0.5 μM) was treated with DNA stock solution (15 μM). The emission wavelength was set at 651 nm, then the excitation spectra of Dir–DNA solution were recorded.

Fluorescence titration experiments

The DNA sequences used in the fluorescence assay were listed in Table S1 (ESI†). The fluorescence titration of Dir was performed in Tris–HCl buffer (20 mM Tris–HCl, pH 7.4) with 100 mM KCl. Excitation wavelength was set at 600 nm, and emission spectrum between 620 nm and 850 nm was recorded. Dir solution (0.5 μM) was treated with increments of DNA stock solution. Dir–DNA solution was allowed to incubate for 2 min before the fluorescence spectrum was recorded. To identify the selectivity of Dir to G-quadruplex DNA, we also carried out the fluorescence titration of Dir subjected to the calf thymus DNA, dsDNA 27 and random single stranded DNA. The binding affinities of Dir to quadruplexes and duplex DNA were determined by monitoring the changes in fluorescence intensity upon increasing DNA concentration.

The apparent binding equilibrium constant K_a was obtained based on the fluorescence titration assay. The data from the spectral titrations were analyzed according to the independent site model by nonlinear fitting to eqn (1).^22,34,44

whereas, F₀ represents the fluorescence intensity of free Dir, F_max represents the fluorescence intensity of totally bound Dir in the presence of G-quadruplex, and n represents the putative number of binding sites on a given DNA matrix.

Fluorescence quantum yield determination

The quantum yield of Dir in Tris–HCl buffer (10 mM Tris–HCl, pH 7.4) with 100 mM KCl was determined relative to a standard with a known quantum yield (Cy5 with a quantum yield of 0.27 in PBS). A series of Dir solutions in Tris–HCl buffer solution and Cy5 solution in PBS were prepared. To get the quantum yield of Dir in the presence of DNA, a series of Dir–DNA mixtures (the ratio of DNA to Dir at 30 : 1) were prepared. Make sure all of the absorbance values were smaller than 0.1. The integrated emission of dye solutions was plotted against the absorbance at the excitation wavelength. The quantum yield of Dir (Φ_x) was calculated by eqn (2):whereas, Φ_st represents the quantum yield of Cy5, and S_x represents the slope of Dir. S_st represents the slope of Cy5, and n_x represents the refractive index of the solvent for Dir. n_st represents the refractive index of PBS, the solution for Cy5.

Fluorescence differentiation under irradiation

Dir (10 μM) was incubated with various DNA solution (30 μM) in Tris–HCl buffer (10 mM Tris–HCl, pH 7.4) with 100 mM KCl. The samples were irradiated by UV light (302 nm). Photos of the samples under irradiation were taken by a camera.

CD experiments

c-myc, HT 22, Hras were used in the CD studies, which represented three different kinds of conformation of G-quadruplexes (parallel, hybrid, antiparallel). CD experiments were performed at a fixed concentration of DNA (4 μM) with the increased amount of Dir. The solution was allowed to equilibrate for 5 min after each addition of Dir. The CD experiments were carried out at the condition of Tris–HCl buffer (10 mM Tris–HCl, pH 7.4) without KCl and Tris–HCl buffer with 100 mM KCl.

Results and discussion

The desired probe Dir was synthesized and prepared by the condensation of reactive hemidye and indolium under basic condition (Scheme 1, see Fig. S1–S5 in ESI†).⁴² The maximum absorbance of Dir in the red region of the visible spectrum was 599 nm in Tris buffer solution (Fig. 1). Addition of one extra methine bridge induced Dir a ca. 87 nm vinyl shift of absorbance band, by comparing with TO, a well-known dye for duplex DNA. To introduce the bulky dimethylindole heterocycle and anionic substituent, Dir exhibited weak fluorescence in buffer solution, and even in the presence of excess amount of calf thymus DNA and synthetic dsDNA (Fig. 2), thereby making it great potential for high specific light-up probe of G-quadruplex with long wavelength emitting. The photostability of Dir was also performed under continuously irradiated by a 500 W xenon lamp for different times.^45,46 Taking the organic dye Cy5 as reference, similar changes in the fluorescence intensity could be observed by comparison on the normalized fluorescence intensity of Cy5 and Dir (Fig. S6, ESI†) under irradiation up to 180 min, which suggested the photostability of Dir was comparable with the commercial dye Cy5.

Fig. 1 UV-Vis spectra of 5 μM Dir in the absence and presence of DNA (20 μM) with different comformations: (A) parallel G-quadruplexes (c-myc, bcl-2, kit-1, Kras); (B) non-parallel G-quadruplexes (HT 22, Hras, TBA); (C) dsDNA (dsDNA 27, ct-DNA) and single stranded DNA (ssDNA 1, ssDNA 2) in 20 mM Tris–HCl buffer, 100 mM KCl, pH 7.4.

Fig. 2 (A) Fluorescence titration of Dir (0.5 μM) with c-myc (0.5–15 μM), λ_ex = 600 nm; (B) the fluorescence intensity enhancement of Dir (0.5 μM) at 651 nm against the ratio of [DNA]/[Dir]; (C) distribution of the values of F/F₀ at 651 nm for Dir with all the tested samples.

In this work, we systematically investigated the quadruplex binding nature (affinity and selectivity) and unique fluorescent properties of Dir. The selective binding of Dir was validated by G-quadruplexes and duplex DNA with different structural elements through absorbance assays and fluorescence experiments. G-Quadruplex sequences presenting in the promoter region (c-myc, bcl-2, kit-1, Kras, Hras), human telomere region (HT 22) and thrombin binding aptamer (TBA) were employed (Table S1, ESI†).

The absorption spectra of Dir in the presence of G-quadruplexes were carried out first. When incubating with 4-fold excess of G-quadruplex DNA, the absorption spectra of Dir showed moderate hypochromism effect and apparent bathochromic shift to long wavelength. The absorbance maximum band at 599 nm gave obvious decrease accompanying with red shifts (10–14 nm) in the existence of parallel G-quadruplex sequences (Fig. 1A, Table 1). c-myc, especially induced the most tense of red shift, up to 14 nm. These great red shifts indicated that there was a strong interaction between Dir and the parallel G-quadruplexes. For a hybrid G-quadruplex, HT 22 caused 7 nm red shift, smaller than the parallel G-quadruplexes (Fig. 1B). But for the antiparallel G-quadruplexes, Hras caused 4 nm red shift, and TBA only induced 1 nm red shift (Fig. 1B). In general, the bathochromic shift of Dir was most pronounced with parallel than non-parallel G-quadruplexes. To compare the binding selectivity of Dir to G-quadruplexes over dsDNA and ssDNA, both natural duplexes (calf-thymus DNA, or ct-DNA), synthetic duplexes (dsDNA 27), random single stranded DNA (ss-DNA 1 and its complementary sequence, ss-DNA 2) were utilized (Table S1, ESI†). In the presence of duplex DNA and single-stranded DNA, the absorbance of Dir was slightly decreased, but a negligible red-shift of absorbance was observed (Fig. 1C). The absorbance band of Dir exhibited no change in the existence of ct-DNA. dsDNA 27 and single stranded DNA (ssDNA 1 and ssDNA 2) caused a small red-shift of 1 nm. The binding of Dir and G-quadruplexes with different structural elements exhibited different spectral features. It seemed that G-quadruplexes made great effects on the absorbance spectra of Dir than dsDNA and ssDNA, especially parallel G-quadruplexes.

Table 1 The oligonucleotides mainly used in this work, the character, the apparent binding equilibrium constants (K_a), the fluorescent quantum yield (QY), the relative fluorescence enhancement value (F/F₀) and the absorbance band red shifts of Dir when bound with DNA in Tris–HCl buffer with KCl

Name	Character/in KCl	Ka/10^7 M^−1	QY^a/%	F/F0	Red shift^b/nm
a The QY of Dir in 20 mM Tris–HCl buffer with 100 mM KCl is 0.7%.b The red shifts were determined by the UV-Vis spectroscopy.
c-myc	Parallel	1.12 ± 0.29	49	36	14
bcl-2	Parallel	1.81 ± 0.45	21	20	10
kit-1	Parallel	1.78 ± 0.42	20	19	10
Kras	Parallel	2.59 ± 0.58	15	18	11
HT 22	Hybrid	3.41 ± 1.57	12	16	7
Hras	Anti-para	3.97 ± 1.27	8.2	7.0	4
TBA	Anti-para	—	6.3	3.2	2
ssDNA1	Single	—	—	1.0	1
ssDNA2	Single	—	—	1.6	1
dsDNA 27	Duplex	—	—	1.2	1
ct-DNA	Duplex	—	—	1.9	0

---

To demonstrate that Dir may act as a quadruplex-selective fluorescent probe, its unique fluorescent properties were assessed. The excitation spectra of Dir against oligonucleotides with different structural elements were recorded (Fig. S7, ESI†). The excitation intensity of Dir alone was quite low in solution. In contrast, DNA made Dir a very interesting structure-dependent enhancement in the excitation intensity. A series of fluorescent titrations were thus preformed with Dir against oligonucleotides with different structural elements. The fluorescence of Dir was totally quenched in solution. When incubated with four parallel sequences (c-myc, bcl-2, kit-1, Kras), Dir displayed 18 to 36-fold fluorescence enhancement (Fig. 2B). Specifically, the relative fluorescent enhancement elicited up to 36-fold at 651 nm with a 12 nm bathochromic shift in the presence of c-myc, a parallel-stranded G-quadruplex (Fig. 2A). For the hybrid type G-quadruplex, HT 22 caused 16-fold fluorescence intensity increase (Fig. 2B). On the contrary, Hras and TBA adopted antiparallel-stranded conformation, and the fluorescence caused 7.0-fold increase for Hras, 3.2-fold enhancement for TBA. More importantly, in the existence of dsDNA (ct-DNA and dsDNA 27) or the random single-stranded DNA (ssDNA1, ssDNA2), Dir remained low fluorescent (Fig. 2B). The profound fluorescence enhancement of Dir in the existence of specific quadruplex, is likely due to torsional restriction of the excited state when confining by a tightly interaction.

TO is well known to be a light-up dye for almost all forms of nucleic acids, and widely used in DNA and RNA staining.^47,48 Therefore, TO does not exhibit the marked selectivity for duplex DNA structures or quadruplex–DNA.^49,50 By comparing the fluorescence response of Dir against various DNA, the results inferred the specific binding of quadruplexes over dsDNA and single-stranded DNA (Fig. 2C). Comparing with TO, the distinct features of Dir were the existence of the extra methine bridge, dimethylindole heterocycle and the anionic propylsulfonate substituent on the quinoline ring. Addition of the extra methine typically induced Dir a ca. 87 nm vinyl shift of absorbance band, and further decreased the background fluorescence of Dir. Meanwhile, dyes based on dimethylindole heterocycle have been proved to inhibit π–π stacking to base pairs in DNA or RNA due to the obvious steric effect caused by the bulky dimethylindole, while the benzothiazole heterocycle of TO would benefit to the intercalation of TO with the nucleobases.⁵¹ The anionic propylsulfonate substituent on the quinoline ring allows further suppress the non-specific electrostatic interaction of Dir to negative phosphate backbone of DNA. Therefore, such great improvement in quadruplex binding selectivity should be attributed to the existence of dimethylindole heterocycle and the anionic propylsulfonate substituent on the quinoline ring.

We extended fluorescence titration experiments to determine apparent binding equilibrium constants, K_a (Table 1, see Fig. S8, ESI†). The values of quadruplex binding constant (K_a) for Dir were in the order of 10⁷. These values were 10-fold higher than those of V-shaped bisbenzimidazole derivatives with parallel G-quadruplex,⁴⁴ and 100-fold higher than those of asymmetric distyrylpyridinium dyes.²² The nonlinear curve fitting results revealed that the putative number of binding sites (n) was about 0.1. The much smaller binding site (n < 1) indicated that the interaction between Dir and G-quadruplex may be mainly surface aggregation on G-quadruplex or groove binding rather than intercalation.^52,53

Dir exhibited weak fluorescence in buffer, and the quantum yield was only 0.7%, based on the standard dye Cy5. The quantum yield of Dir was significantly improved after binding with G-quadruplexes. The parallel G-quadruplex, c-myc induced 70-fold enhancement of the quantum yield (Table 1, see Fig. S9, ESI†). The quantum yield of Dir in the presence of c-myc increased to 49%. This value was approximately 4 times higher than the value for hybrid-type telomeric G-quadruplex HT 22, 6 times higher for antiparallel G-quadruplex Hras, and 8 times higher for the antiparallel G-quadruplex TBA. Notably, the quantum yield and the relative fluorescence enhancement were in agreement with the results of absorbance assays and fluorescence titration experiments. These results indicated that the probe has proven to be a high specific G-quadruplex light-up probe, especially for c-myc.

Interestingly, such remarkable G-quadruplex binding can even be achieved by its ability to enlighten quadruplex samples under UV light irradiation (302 nm). Photos of Dir with various DNAs under UV light irradiation were undertaken (Fig. 3). Dir against G-quadruplexes can be easily differentiated and displayed a conspicuous red light, except for the antiparallel G-quadruplex TBA. By incubated with duplex DNA or single-stranded DNA, Dir remained quenched. Thus Dir is a very promising probe for easy and selective visualization of quadruplexes.

Fig. 3 Fluorescence differentiation of Dir (10 μM) with various DNA structures (30 μM) under UV light (302 nm).

Folding, unfolding and refolding of G-rich DNA have direct effect on G-quadruplex structure and its function. What roles Dir may play in the formation of DNA G-quadruplex is meaningful for understanding the probe-quadruplex interaction. The study of the conformational switch of G-quadruplex structure in the absence and presence of Dir was investigated by circular dichroism (CD) spectroscopy. The CD spectra of the parallel G-quadruplex c-myc exhibited a characteristic positive peak at 265 nm and a negative peak at 240 nm. As shown in Fig. 4A, Dir had a negligible impact on the characteristic peaks of c-myc. Similar results were observed when antiparallel G-quadruplex Hras or hybrid-type G-quadruplex HT 22 were titrated by Dir in Tris–HCl buffer in the absence (see Fig. S10, ESI†) and presence of K⁺ (Fig. 4). And no induced CD signal peaks in the wavelength longer than 350 nm could be detected (Fig. 4 and S10, ESI†). The probe Dir had a negligible effect on the formation of G-quadruplex structures. The results were also observed by other cyanine dyes.^34,54 Such a G-quadruplex probe without influencing the topology of G-quadruplex may reveal the distribution of intracellular formed G-quadruplexes, and may also avoid the debate whether the detectable G-quadruplexes present or are induced by the probes.

Fig. 4 CD spectra of 4 μM G-quadruplex-forming oligonucleotides c-myc (A), HT 22 (B) and Hras (C) in the absence and presence of Dir (8 μM) in 20 mM Tris–HCl buffer with 100 mM KCl, pH 7.4.

Most cyanine dyes were believed to bind with parallel G-quadruplexes via end-stacking, whereas bind with hybrid G-quadruplex via loop-mediated interaction.^34,54 Thus, it was understandable that the better fluorescence response of Dir to parallel quadruplex over non parallel quadruplex may be due to more restricted torsional motion of the dye excited state when end-stacking into parallel G-quadruplexes. Furthermore, the fluorescence enhancement of c-myc quadruplex was much higher than the other parallel quadruplex. The variability in G-quadruplexes is not only the direction of the G-tracts base composition, which is commonly classified as parallel, antiparallel or hybrid, but also the type and the length of the loop. There is great difference in the structural polymorphism of the parallel G-quadruplexes in this work. c-myc is a chair-type parallel structure with two lateral two-base loops, and an orthogonal three-base bridging loop.³ bcl-2 exhibits basket-type parallel structure with three chain-reversal loops of one, thirteen, and one-base.⁵ kit-1 folds into basket-type parallel structure with two reversal one-base loops, a two-base loop, and a five-base stem-loop.⁶ Kras adopts a basket-type parallel topology with three chain-reversal loops of three, eleven, and two-base.⁸ It is suggested that each parallel G-quadruplex adopts specific topology with variations in connecting loop types, numbers of G-quartets and glycosyl torsion angles, which will greatly influence the specific binding sites of dyes. With this in mind, chair-type c-myc quadruplex with two lateral two-base loops and an orthogonal three-base bridging loop would be more accessible for ligand stacking on the external guanine tetrad, which may be the reason of the higher fluorescence enhancement of Dir to c-myc quadruplex over the other parallel quadruplexes.

Conclusions

In summary, we have successfully developed a high specific red-emitting G-quadruplex probe, especially for parallel G-quadruplex, c-myc. This probe with the sterically bulky dimethylindole heterocycle and an anionic propylsulfonate substituent on the quinoline ring greatly mitigated nonspecific DNA binding. The extended polymethine chain of Dir endowed the light-up function in red-emitting region above 650 nm. Notably, the fluorescence quantum yield of Dir was greatly enhanced when incubating with G-quadruplexes. The probe Dir had a negligible effect on the topology of the G-quadruplexes. Therefore, these unique abilities could make Dir potentially usable for selective visualization of intracellular quadruplexes, and for detecting folding, unfolding and refolding of G-quadruplexes.

Acknowledgements

This work is supported by the National Natural Science Foundation of China (No. 21575154, 21275156).

Notes and references

1. M. Gellert, M. N. Lipsett and D. R. Davies, Proc. Natl. Acad. Sci. U. S. A., 1962, 48, 2013–2018.
2. S. Amrane, A. Kerkour, A. Bedrat, B. Vialet, M.-L. Andreola and J.-L. Mergny, J. Am. Chem. Soc., 2014, 136, 5249–5252.
3. A. Siddiqui-Jain, C. L. Grand, D. J. Bearss and L. H. Hurley, Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 11593–11598.
4. H. You, J. Wu, F. Shao and J. Yan, J. Am. Chem. Soc., 2015, 137, 2424–2427.
5. P. Agrawal, C. Lin, R. I. Mathad, M. Carver and D. Yang, J. Am. Chem. Soc., 2014, 136, 1750–1753.
6. A. T. Phan, V. Kuryavyi, S. Burge, S. Neidle and D. J. Patel, J. Am. Chem. Soc., 2007, 129, 4386–4392.
7. S. Rankin, A. P. Reszka, J. Huppert, M. Zloh, G. N. Parkinson, A. K. Todd, S. Ladame, S. Balasubramanian and S. Neidle, J. Am. Chem. Soc., 2005, 127, 10584–10589.
8. S. Cogoi and L. E. Xodo, Nucleic Acids Res., 2006, 34, 2536–2549.
9. A. Membrino, S. Cogoi, E. B. Pedersen and L. E. Xodo, PLoS One, 2011, 6, e24421.
10. S. Cogoi, A. E. Shchekotikhin and L. E. Xodo, Nucleic Acids Res., 2014, 42, 8379–8388.
11. M. L. Bochman, K. Paeschke and V. A. Zakian, Nat. Rev. Genet., 2012, 13, 770–780.
12. D. Monchaud and M.-P. Teulade-Fichou, Org. Biomol. Chem., 2008, 6, 627–636.
13. S. Neidle, Curr. Opin. Struct. Biol., 2009, 19, 239–250.
14. S. Balasubramanian, L. H. Hurley and S. Neidle, Nat. Rev. Drug Discovery, 2011, 10, 261–275.
15. E. Y. N. Lam, D. Beraldi, D. Tannahill and S. Balasubramanian, Nat. Commun., 2013, 4, 1796.
16. A. C. Bhasikuttan and J. Mohanty, Chem. Commun., 2015, 51, 7581–7597.
17. B. R. Vummidi, J. Alzeer and N. W. Luedtke, ChemBioChem, 2013, 14, 540–558.
18. D.-L. Ma, Z. Zhang, M. Wang, L. Lu, H.-J. Zhong and C.-H. Leung, Chem. Biol., 2015, 22, 812–828.
19. H.-Z. He, D. S.-H. Chan, C.-H. Leung and D.-L. Ma, Nucleic Acids Res., 2013, 41, 4345–4359.
20. D.-L. Ma, D. S.-H. Chan and C.-H. Leung, Acc. Chem. Res., 2014, 47, 3614–3631.
21. S. A. Hilderbrand and R. Weissleder, Curr. Opin. Chem. Biol., 2010, 14, 71–79.
22. X. Xie, B. Choi, E. Largy, R. Guillot, A. Granzhan and M.-P. Teulade-Fichou, Chem.–Eur. J., 2013, 19, 1214–1226.
23. L. Zhang, J. C. Er, K. K. Ghosh, W. J. Chung, J. Yoo, W. Xu, W. Zhao, A. T. Phan and Y.-T. Chang, Sci. Rep., 2014, 4, 3776.
24. J. Alzeer, B. R. Vummidi, P. J. Roth and N. W. Luedtke, Angew. Chem., Int. Ed., 2009, 48, 9362–9365.
25. H. Lai, Y. Xiao, S. Yan, F. Tian, C. Zhong, Y. Liu, X. Weng and X. Zhou, Analyst, 2014, 139, 1834–1838.
26. L. Xu, D. Zhang, J. Huang, M. Deng, M. Zhang and X. Zhou, Chem. Commun., 2010, 46, 743–745.
27. F. Doria, A. Oppi, F. Manoli, S. Botti, N. Kandoth, V. Grande, I. Manet and M. Freccero, Chem. Commun., 2015, 51, 9105–9108.
28. M. Zuffo, F. Doria, V. Spalluto, S. Ladame and M. Freccero, Chem.–Eur. J., 2015, 21, 17596–17600.
29. J.-w. Yan, Y.-g. Tian, J.-h. Tan and Z.-s. Huang, Analyst, 2015, 140, 7146–7149.
30. X. Xie, A. Renvoise, A. Granzhan and M.-P. Teulade-Fichou, New J. Chem., 2015, 39, 5931–5935.
31. J.-W. Yan, S.-B. Chen, H.-Y. Liu, W.-J. Ye, T.-M. Ou, J.-H. Tan, D. Li, L.-Q. Gu and Z.-S. Huang, Chem. Commun., 2014, 50, 6927–6930.
32. Y. Chen, S. Yan, L. Yuan, Y. Zhou, Y. Song, H. Xiao, X. Weng and X. Zhou, Org. Chem. Front., 2014, 1, 267–270.
33. Y.-J. Lu, Z.-Y. Wang, D.-P. Hu, Q. Deng, B.-H. Huang, Y.-X. Fang, K. Zhang, W.-L. Wong and C.-F. Chow, Dyes Pigm., 2015, 122, 94–102.
34. B. Jin, X. Zhang, W. Zheng, X. Liu, J. Zhou, N. Zhang, F. Wang and D. Shangguan, Anal. Chem., 2014, 86, 7063–7070.
35. Y.-J. Lu, S.-C. Yan, F.-Y. Chan, L. Zou, W.-H. Chung, W.-L. Wong, B. Qiu, N. Sun, P.-H. Chan and Z.-S. Huang, Chem. Commun., 2011, 47, 4971–4973.
36. M. Wang, Z. Mao, T.-S. Kang, C.-Y. Wong, J.-L. Mergny, C.-H. Leung and D.-L. Ma, Chem. Sci., 2016, 2016(7), 2516–2523.
37. B. Le Guennic and D. Jacquemin, Acc. Chem. Res., 2015, 48, 530–537.
38. M. Nikan, M. Di Antonio, K. Abecassis, K. McLuckie and S. Balasubramanian, Angew. Chem., Int. Ed., 2013, 52, 1428–1431.
39. D. Lin, X. Fei, Y. Gu, C. Wang, Y. Tang, R. Li and J. Zhou, Analyst, 2015, 140, 5772–5780.
40. L. Zhang, J. C. Er, X. Li, J. J. Heng, A. Samanta, Y.-T. Chang and C.-L. K. Lee, Chem. Commun., 2015, 51, 7386–7389.
41. P. Yang, A. De Cian, M.-P. Teulade-Fichou, J.-L. Mergny and D. Monchaud, Angew. Chem., Int. Ed., 2009, 48, 2188–2191.
42. T. P. Constantin, G. L. Silva, K. L. Robertson, T. P. Hamilton, K. Fague, A. S. Waggoner and B. A. Armitage, Org. Lett., 2008, 10, 1561–1564.
43. N. I. Shank, K. J. Zanotti, F. Lanni, P. B. Berget and B. A. Armitage, J. Am. Chem. Soc., 2009, 131, 12960–12969.
44. B. Jin, X. Zhang, W. Zheng, X. Liu, C. Qi, F. Wang and D. Shangguan, Anal. Chem., 2014, 86, 943–952.
45. S. Zhao, M. Lan, X. Zhu, H. Xue, T.-W. Ng, X. Meng, C.-S. Lee, P. Wang and W. Zhang, ACS Appl. Mater. Interfaces, 2015, 7, 17054–17060.
46. Y. He, H.-T. Lu, L.-M. Sai, Y.-Y. Su, M. Hu, C.-H. Fan, W. Huang and L.-H. Wang, Adv. Mater., 2008, 20, 3416–3421.
47. J. Nygren, N. Svanvik and M. Kubista, Biopolymers, 1998, 46, 39–51.
48. D. L. Boger, B. E. Fink, S. R. Brunette, W. C. Tse and M. P. Hedrick, J. Am. Chem. Soc., 2001, 123, 5878–5891.
49. D. Monchaud, C. Allain, H. Bertrand, N. Smargiasso, F. Rosu, V. Gabelica, A. De Cian, J. L. Mergny and M. P. Teulade-Fichou, Biochimie, 2008, 90, 1207–1223.
50. D. Monchaud, C. Allain and M.-P. Teulade-Fichou, Bioorg. Med. Chem. Lett., 2006, 16, 4842–4845.
51. B. A. Armitage, in Heterocyclic Polymethine Dyes: Synthesis, Properties and Applications, ed. L. Strekowski, Springer Berlin Heidelberg, Berlin, Heidelberg, 2008, pp. 11–29.
52. P. K. Sasmal, A. K. Patra, M. Nethaji and A. R. Chakravarty, Inorg. Chem., 2007, 46, 11112–11121.
53. S. Anbu, A. Paul, A. P. C. Ribeiro, M. F. C. Guedes da Silva, M. L. Kuznetsov and A. J. L. Pombeiro, Inorg. Chim. Acta, 2016, 450, 426–436.
54. S.-B. Chen, W.-B. Wu, M.-H. Hu, T.-M. Ou, L.-Q. Gu, J.-H. Tan and Z.-S. Huang, Chem. Commun., 2014, 50, 12173–12176.

---

Footnote

† Electronic supplementary information (ESI) available: Experimental details and other data. See DOI: 10.1039/c6ra11152a

---