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Journal Name ARTICLE Development of a highly sensitive fluorescent light-up probe for G-quadruplexes Received 00th January 20xx, Accepted 00th January 20xx DOI: 10.1039/x0xx00000x www.rsc.org/
Ming-Hao Hu, Shuo-Bin Chen, Rui-Jun Guo, Tian-Miao Ou, Zhi-Shu Huang, and Jia-Heng Tan * G-quadruplexes are higher-order nucleic acid structures that have attracted extensive attention because of their biological significance and potential applications in supramolecular chemistry. An ever-increasing interest in G-quadruplexes has promoted the development of selective and sensitive fluorescent probes as research tools for these structures. However, most current studies primarily focus on the improved selectivity of probes for G-quadruplexes. Their detection limits or ways to improve their detection limits are rarely described. In this study, a new set of di-substituted triarylimidazole fluorescent probes were designed and synthesized, with the aim of upgrading the detection limit of a lead triarylimidazole IZCM-1 for G-quadruplexes. Among these compounds, IZCM-7 was the most promising candidate. The limit of detection (LOD) value of IZCM-7 for G-quadruplex was up to 3 nM in solution and up to 5 ng in a gel matrix. These values were significantly improved in comparison with those of IZCM-1. Further biophysical studies revealed that the fluorescence quantum yield and binding affinity of IZCM-7 for G-quadruplex were markedly increased, and these two factors might be responsible for the significantly improved detection limit of IZCM-7. In addition, the sensitive and selective fluorescence performance of IZCM-7 for G-quadruplex remained the same even in the presence of large amounts of non-G-quadruplex competitors, suggesting its promising application prospect.
Introduction The development of highly sensitive and selective probes to qualitatively and quantitatively detect nucleic acids is of profound importance to a wide range of investigations, such as 1-3 biochemistry and clinical diagnosis. Apart from their primarily double helical structures, DNA or RNA can adopt higher-order and functionally-useful structures, such as G4 quadruplexes. G-quadruplexes are unique four-stranded structures formed by guanine-rich nucleic acid sequences. The building blocks of G-quadruplexes are G-quartets, which are derived from the association of four guanines into a cyclic Hoogsteen hydrogen-bonding arrangement. The G-quartets stack up with one on top of another to form G-quadruplex 5,6 structures. G-quadruplexes can be basically divided into the following three primary topologies: parallel, antiparallel, and 7 hybrid-type structures. G-quadruplexes are widely dispersed in eukaryotic genomes, including telomeric DNA, rDNA and a series of gene 8 promoter regions. During the past two decades, G-quadruplex structures have attracted extensive attention because of their biological significance and potential applications in 9-11 supramolecular chemistry. An ever-increasing interest in G-
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China. E-mail: [email protected].
†Electronic
Supplementary Information (ESI) available: [Synthesis and characterization of IZCM-2 to IZCM-8, and other additional supporting figures]. See DOI: 10.1039/x0xx00000x
quadruplexes has promoted the development of rapid and simple approaches for the selective and sensitive detection of these structures. Thus, the discovery of fluorescent probes for G-quadruplexes has become an extremely active area of research.12-14 Several probes specific for G-quadruplexes have previously been reported.15-26 Nevertheless, most of these studies primarily focused on the improvement of probe selectivity for G-quadruplexes. Their detection limits are rarely described. Notably, the selectivity and detection limit are both important criteria for evaluating an ideal probe. Designing fluorescent probes for G-quadruplexes when only considering their selectivity would hamper their further application. Recently, we discovered a novel fluorescent probe called IZCM-1 for detecting G-quadruplexes by incorporating a coumarin fluorophore into the 2,4,5-triarylimidazole framework.27,28 Notably, the fluorescence quantum yield of IZCM-1 increased by approximately 100 times when binding to some G-quadruplexes. However, its detection limit is not satisfactory. The most outstanding limit of detection (LOD) value of IZCM-1 for G-quadruplexes in solution is 40 nM. When employing IZCM-1 as a staining reagent for G-quadruplexes in electrophoresis gels, the corresponding detection limit is more than 200 ng (see the ESI†). Such data are much higher than data for commercial gel EtBr stains and SYBR® dyes with detection limits of less than 10 ng for most nucleic acids. Nevertheless, EtBr and SYBR® dyes are non-selective for Gquadruplexes and other nucleic acids.29-31 It is hard to note the G-quadruplex bands when using these dyes to stain mixed nucleic acid samples that are separated in a gel matrix. Thus,
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the research gaps impelled us to synthesize new triarylimidazole fluorescent probe aiming not only to improve its detection limit but also to maintain its selectivity for Gquadruplexes. The sensitivity of commercial nucleic acid probes is caused by a high affinity for nucleic acids, in combination with a high 32 quantum yield upon binding. In studying IZCM-1, we also found significant and positive correlations between the binding affinity and fluorescence performance of IZCM-1 in the 28 presence of different G-quadruplexes. These results made us envisage the possibility of upgrading the probe´s detection limit by improving its binding affinity and fluorescence quantum yield with G-quadruplexes. In considering the structure of IZCM-1, which includes a 1-methylpiperazine group, this terminal cationic group makes an important contribution to the electrostatic interaction of IZCM-1 with Gquadruplexes. However, this group alone is likely insufficient because previous experience indicated that the presence of at least two cationic side chains to the chromophore is usually 33,34 required to gain high G-quadruplex binding potency. In addition, the nitrogen atom in the junction between the side chain and phenyl group could also be changed because the substituent connected to the chromophore sometimes 35,36 influenced the fluorescence quantum yield of a compound. Based on these considerations, we tried to modify the structure of IZCM-1, and we thereby designed a new set of disubstituted triarylimidazole fluorescent probes.
fluorescence performance on G-quadruplexes. The detailed interactions of the most outstanding probe with various Gquadruplexes, double-stranded and single-stranded nucleic acids and the corresponding photophysical changes were studied. In addition, the probe's detection limits for various nucleic acids were examined in both the solution and a gel matrix, with the aim of developing a selective and sensitive triarylimidazole fluorescent probe for G-quadruplexes.
Results The synthesis of triarylimidazole fluorescent probes
The designed probes (IZCM-2 to IZCM-8) were prepared by condensing coumarin aldehyde and the corresponding substituted diphenylethanedione moiety according to our previous studies.27,28 Their structure and purity were confirmed by 1H and 13C NMR spectrometry, HRMS spectrometry, and HPLC analysis (Scheme S1 and Figure S1-28, ESI†). Screening triarylimidazole fluorescent probes Table 1. Optical data and fluorescence quantum yields (ΦF) of disubstituted triarylimidazole fluorescent probes in buffer (10 mM Tris-HCl, pH=7.2, 100 mM KCl) and in the presence of different nucleic acids ΦF
a
λabs
λem
(nm)
(nm)
Buffer
T21
ds26
Pu22
IZCM-2
449
526
0.012
0.043
0.041
0.257
IZCM-3
459
528
0.004
0.007
0.009
0.226
IZCM-4
448
529
0.041
0.046
0.045
0.079
IZCM-5
444
529
0.050
0.084
0.084
0.454
IZCM-6
449
529
0.020
0.053
0.082
0.308
IZCM-7
448
528
0.012
0.059
0.077
0.518
IZCM-8
447
532
0.006
0.010
0.014
0.086
a
1 μM of each compound and 10 μM of each DNA were used in the determination of ΦF.
Figure 1. The structures of IZCM-1 and di-substituted triarylimidazole fluorescent probes IZCM-2 to IZCM-8. As shown in Figure 1, the designed probes (IZCM-2 to IZCM8) bear two identical substituents in comparison with IZCM-1. The substituents include an amine side chain and a carboxyl side chain, which would be helpful to understand the electrostatic impact. In addition, oxygen atoms were introduced instead of nitrogen atoms in the junctions between the side chains and phenyl groups. In this study, these designed probes were synthesized and screened for their
To identify the most promising triarylimidazole fluorescent probe, newly synthesized compounds were screened for fluorescence performance with a focus on the fluorescence quantum yield and selectivity for G-quadruplexes. Three representative oligonucleotides, including single-stranded DNA T21, double-stranded DNA ds26 and G-quadruplex DNA formed by the oligonucleotide Pu22, were employed in the assays. As shown in Table 1, these candidate probes exhibited a slight difference in their absorption and emission maximums in buffer solution. Their fluorescence emissions were weak, even in the presence of singlestranded and double-stranded DNA. By contrast, most of the candidates displayed strong fluorescence when adding Gquadruplex Pu22 except IZCM-4 and IZCM-8. It was also noteworthy that the fluorescence quantum yield values were always more pronounced for candidates bearing oxygen atoms in the junctions between side chains and phenyl groups (IZCM-5, IZCM-6 and IZCM7). Among these three candidates, IZCM-7 was the best probe in
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view of its outstanding fluorescence quantum yields upon binding to G-quadruplex Pu22. In addition, IZCM-7 displayed weaker fluorescence emissions in buffer solution or in the presence of single-stranded and double-stranded DNA relative to IZCM-5 and IZCM-6, thus showing its better selectivity towards G-quadruplexes. From among the candidates, IZCM-7 was chosen as the most promising triarylimidazole fluorescent probe for further detailed investigation. The Optical properties of IZCM-7 The detailed optical properties of IZCM-7 were then investigated by absorption and fluorescence spectroscopy in different solvents. As shown in Table 2, IZCM-7 exhibited a slight difference in the absorption maximum in different solvents and showed weak fluorescence emission in most low-viscosity liquids. Interestingly, a remarkable fluorescence enhancement of IZCM-7 arose in the highviscosity glycerol solution (60% aqueous glycerol solution), suggesting that the fluorescence intensity of IZCM-7 could be tunable. It was also noteworthy that there was a positive correlation between the fluorescence quantum yield and solvent viscosity, showing that the quantum yield of IZCM-7 increased significantly with the increasing glycerol content in the glycerolwater mixed solution. The double-logarithmic plot of the quantum 2 yield vs. the viscosity was almost linear (R = 0.9786, Figure S29, ESI†). In addition, IZCM-7 had little propensity to aggregate in buffer according to its concentration-dependent absorbance spectrum (Figure S30, ESI†). These results closely resembled those found in the IZCM-1 studies, suggesting that the variations in fluorescence emission may be caused by conformational changes in the excited state of the 2,4,5-triarylimidazole-derived chromophore, most likely based on the rotational state of the single 37 bonds around the imidazole moiety. Table 2. Optical properties of IZCM-7 in different solvents a
Solvent
η (cP )
λabs (nm)
λem (nm)
ΦF
Acetone
0.32
444
525
0.028
Dichloromethane
0.44
448
527
0.058
Chloroform
0.57
453
530
0.120
Toluene
0.59
447
520
0.033
Methanol
0.60
441
529
0.109
Ethanol
1.20
441
528
0.097
Water
1.00
448
528
0.019
60% glycerol
10.80
440
528
0.329
a
7 in high-viscosity solution, suggesting that G-quadruplexes could similarly lock IZCM-7 into an active fluorescent conformation. In addition, a significant lighting up in the fluorescence was also observed when IZCM-7 was treated with other types of Gquadruplexes, including KRAS, ILPR, THTG, bcl-2, c-kit2, c-kit3, HRAS and htg22 (Figure 2B). By contrast, negligible fluorescence enhancement was observed when titrating IZCM-7 with singlestranded DNA (Py22, T21 and A21), double-stranded DNA (ds26, 19AT, dx12 and ct-DNA), and protein BSA. This trend in fluorescence intensities could be observed by the naked eye under UV light (Figure S31, ESI†).
Figure 2. (A) The fluorescence titration of 1 µM IZCM-7 with the stepwise addition of the G-quadruplex Pu22 (arrow: 0–10 mol equiv.) in 10 mM Tris-HCl buffer, 100 mM KCl, pH 7.2. The fluorescence emissions of IZCM-7 with and without Pu22 under UV light were shown in the inner panel. (B) The fluorescence intensity enhancement of 1 µM IZCM-7 at 525 nm against the sample concentrations, λex = 450 nm.
Viscosity at 25 °C
Fluorescence spectroscopic studies of IZCM-7 interactions with nucleic acid The detailed fluorescence properties of IZCM-7 with various Gquadruplexes and other nucleic acids were explored by using a fluorescence titration assay. As shown in Figure 2A, IZCM-7 alone in buffer displayed weak fluorescence emission. With the gradual addition of G-quadruplex DNA Pu22, an emission peak at approximately 525 nm was significantly enhanced. This remarkable fluorescence enhancement closely resembled the behavior of IZCM-
Figure 3. (A) The fluorescence titration of 1 µM IZCM-7 with the stepwise addition of ct-DNA in 10 mM Tris-HCl buffer containing 1 µM G-quadruplex Pu22, 100 mM KCl, pH 7.2. (B) The fluorescence titration of 1 µM IZCM-7 with the stepwise addition of the Gquadruplex Pu22 without and with 200 µM ct-DNA in 10 mM TrisHCl buffer, 100 mM KCl, pH 7.2. Competition titrations were performed to further confirm the selective fluorescence response of IZCM-7 binding to Gquadruplexes, in which the ability of IZCM-7 to retain an enhanced
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fluorescence intensity with the addition of G-quadruplexes was 18 challenged by duplex DNA. As shown in Figure 3A, in the presence of various amounts of duplex ct-DNA, the enhanced fluorescence of IZCM-7 with G-quadruplex Pu22 was only slightly affected, even at the 200 μM duplex competitor concentration. In addition, when gradually adding the Pu22 G-quadruplex into the solution containing IZCM-7 and 200 μM ct-DNA, the enhanced fluorescence emissions were practically identical to those in the experiment without a duplex competitor (Figure 3B). Collectively, these results demonstrated the promising potential of IZCM-7 to serve as a promising G-quadruplex fluorescent probe even in a competitive environment. Table 3. The fluorescence quantum yields of IZCM-7 with different a nucleic acids and BSA protein Sample
ΦF
Relative ΦF
Tris
0.012
1
KRAS
0.595
50
ILPR
0.582
49
THTG
0.553
46
bcl-2
0.528
44
Pu22
0.518
43
c-kit2
0.479
40
HRAS
0.454
38
c-kit3
0.439
37
htg22
0.403
34
ds26
0.077
6
19AT
0.074
6
dx12
0.070
6
ct-DNA
0.022
2
py22
0.077
6
T21
0.059
5
A21
0.018
1
BSA
0.021
2
(HRAS, c-kit3, htg22). This behavior in IZCM-7 partly resembled that of IZCM-1 in its display of a selective fluorescence response towards parallel G-quadruplexes. However, fluorescence emission enhancement and quantum yield values of IZCM-7 with non-parallel G-quadruplexes were apparently upgraded. The discrepancy between parallel and non-parallel G-quadruplexes was reduced. The detection limits of IZCM-7 for G-quadruplex Encouraged by the strong fluorescence emission enhancement and improved quantum yield values of IZCM-7 with Gquadruplexes, we then investigated the detection limits associated with using IZCM-7 to identify G-quadruplexes in solution. The LOD values were calculated on the basis of the equation LOD = K × 38,39 Sb/m. The K value is generally taken to be 3 according to the International Union of Pure and Applied Chemistry (IUPAC) recommendation. The Sb value represents the standard deviation for multiple measurements (n = 20) of blank solution. The m value is the slope of the calibration curve, which is derived from the linear range of the IZCM-7 fluorescence titration curve with different nucleic acids and represents the sensitivity of this method (Figure S32, ESI†). The linear ranges of the fluorescence titration curve for IZCM-7 with most of the G-quadruplexes ranged from 0 to 400 nM. The m values were then obtained by fitting these curves and the corresponding LOD values are shown in Table 4. Clearly, the LODs of IZCM-7 for parallel G-quadruplexes (KRAS and Pu22) in solution were below 10 nM. These values were better than that of antiparallel G-quadruplex c-kit3 and hybrid-type G-quadruplex htg22, which were below 20 nM. In addition, we could not obtain the exact LOD values for double-stranded DNA ds26 because of its negligible fluorescence enhancement during titration. It was also noteworthy that the LOD values of IZCM-7 for G-quadruplex in solution were significantly improved relative to IZCM-1. The LOD values of IZCM-1 for G-quadruplex KRAS, Pu22, c-kit3 and htg22 were 13 nM, 17 nM, 118 nM and 165 nM, respectively (Figure S33, ESI†). Accordingly, the improving detection limits of IZCM-7 for Gquadruplex revealed the importance of two new amine chains and oxygen atoms in the junctions. Table 4. The detection limits and linear ranges of IZCM-7 for different nucleic acids in solution
a
1 μM of IZCM-7 and 10 μM of each sample were used in the determination of ΦF. The fluorescence quantum yield values of IZCM-7 with different nucleic acids and BSA protein are summarized in Table 3. Notably, these values were consistent with the results for fluorescence titration and competitive experiments showing that fluorescence emission enhancement was always more pronounced for Gquadruplex structures. The quantum yield values of IZCM-7 with Gquadruplexes reached 0.51 on average. Typically, the quantum yield value of IZCM-7 was up to 0.60 in the presence of KRAS Gquadruplex. These data were 50% higher than that of IZCM-1 (0.40) 28 under the same conditions. This structural modification significantly improved the quantum yield values of IZCM-7. On the other hand, the fluorescence emission enhancement and the quantum yield values of IZCM-7 with parallel G-quadruplexes (KRAS, ILPR, THTG, bcl-2, Pu22, and c-kit2) were slightly but perceptibly higher than those with non-parallel G-quadruplexes
Nucleic Acid
LOD ( nM )
Linear Range ( nM )
KRAS
3
0 - 200
Pu22
8
0 - 400
c-kit3
15
0 - 400
htg22
19
0 - 400
ds26
> 1000
nd
a
a
nd: not determined
In addition to the LOD values of IZCM-7 in solution, we were also interested in its performance in staining G-quadruplex bands after gel electrophoresis. G-quadruplex DNA (KRAS, Pu22, c-kit3 and htg22), double-stranded DNA (ds26) and single-stranded DNA (mPu22, a mutant of Pu22) were employed in the polyacrylamide gel electrophoresis (PAGE) experiment. After electrophoresis, the polyacrylamide gels were immersed in 4 µg/mL IZCM-7 staining solution for 20 minutes. As shown in Figure 4A, clear dose-response
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fluorescence bands were accompanied by an increasing concentration of G-quadruplex DNA, and the bands of doublestranded DNA and single-stranded DNA were hardly detectable. In addition, parallel G-quadruplexes (KRAS and Pu22) were much easier to detect compared with antiparallel G-quadruplex c-kit3 and hybrid-type G-quadruplex htg22. These findings were consistent with the trends observed from LOD values of IZCM-7 for nucleic acids in solution. However, the PAGE experiments were repeated by using commercial SYBR® Green I stain as a benchmark. Compared with IZCM-7, SYBR® Green I was a stain for all types of nucleic acids (Figure 4B). Most importantly, the performance of IZCM-7 in staining G-quadruplex bands was comparable to that of SYBR® Green I and was much stronger than that of IZCM-1 (Figure S34, ESI†). These results could also be observed from their detection limits as summarized in Table 5. Notably, the detection limits of IZCM-7 for G-quadruplex KRAS was identical to that of SYBR® Green I, suggesting the potential commercial value of IZCM-7 as a selective stain for G-quadruplexes.
Figure 4. The dose-response staining of G-quadruplexes (KRAS, Pu22, HRAS, and htg22), duplex DNA (ds26) and single-stranded DNA (mPu22) by (A) IZCM-7 and (B) SYBR® Green I. The conditions were as follows: 10 μL of nucleic acids was loaded on to 20% acrylamide in 1 × TBE buffer containing 100 mM KCl. The nucleic acid concentration was increased from 0.05 to 6.4 μM.
After electrophoresis, the polyacrylamide gels were immersed in a 4 µg/mL IZCM-7 staining solution for 20 minutes and SYBR® Green I was used as a benchmark. As shown in Figure 5A, clear concentration-dependent bands for G-quadruplex Pu22 were observed when applying IZCM-7 as a gel stain. The background was clean. By contrast, mixed bands were observed when applying SYBR® Green I as gel stain. It was hard to note the band of Gquadruplex Pu22 owing to the interference of double-stranded DNAs. In addition, the detection limit of IZCM-7 for G-quadruplex Pu22 under competition conditions was also 7 ng, demonstrating the practicability of using IZCM-7 as a specific stain for Gquadruplexes, even in the presence of abundant double-stranded DNA. Taking all of these results together, IZCM-7 displayed promising application prospects for the selective and sensitive Gquadruplexes in both the solution and gel matrix.
Figure 5. The dose-response staining of G-quadruplex Pu22 in the presence of mixed double-stranded DNAs (dx12, ds26, 19AT, Hairpin-4T, Hairpin-6T, Hairpin-8T and Hairpin-10T) by (A) IZCM-7 and (B) SYBR® Green I. Conditions: 10 μL of nucleic acids were loaded onto 20% acrylamide in 1 × TBE buffer containing 100 mM KCl. The concentration of G-quadruplex Pu22 was increased from 0.05 to 6.4 μM. The content of each double-stranded DNA was maintained at 200 ng. UV-Vis spectroscopic studies of IZCM-7 interactions with Gquadruplex
Table 5. The detection limits of IZCM-7 and SYBR® Green I as gel a stains for different nucleic acids LOD ( ng ) Nucleic Acid IZCM-7
SYBR® Green I
KRAS
5
5
Pu22
7
3
c-kit3
14
7
htg22
28
3
ds26
510
Analyst Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: M. Hu, S. Chen, R. Guo, T. Ou, Z. Huang and J. Tan, Analyst, 2015, DOI: 10.1039/C5AN00761E.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
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Journal Name ARTICLE Development of a highly sensitive fluorescent light-up probe for G-quadruplexes Received 00th January 20xx, Accepted 00th January 20xx DOI: 10.1039/x0xx00000x www.rsc.org/
Ming-Hao Hu, Shuo-Bin Chen, Rui-Jun Guo, Tian-Miao Ou, Zhi-Shu Huang, and Jia-Heng Tan * G-quadruplexes are higher-order nucleic acid structures that have attracted extensive attention because of their biological significance and potential applications in supramolecular chemistry. An ever-increasing interest in G-quadruplexes has promoted the development of selective and sensitive fluorescent probes as research tools for these structures. However, most current studies primarily focus on the improved selectivity of probes for G-quadruplexes. Their detection limits or ways to improve their detection limits are rarely described. In this study, a new set of di-substituted triarylimidazole fluorescent probes were designed and synthesized, with the aim of upgrading the detection limit of a lead triarylimidazole IZCM-1 for G-quadruplexes. Among these compounds, IZCM-7 was the most promising candidate. The limit of detection (LOD) value of IZCM-7 for G-quadruplex was up to 3 nM in solution and up to 5 ng in a gel matrix. These values were significantly improved in comparison with those of IZCM-1. Further biophysical studies revealed that the fluorescence quantum yield and binding affinity of IZCM-7 for G-quadruplex were markedly increased, and these two factors might be responsible for the significantly improved detection limit of IZCM-7. In addition, the sensitive and selective fluorescence performance of IZCM-7 for G-quadruplex remained the same even in the presence of large amounts of non-G-quadruplex competitors, suggesting its promising application prospect.
Introduction The development of highly sensitive and selective probes to qualitatively and quantitatively detect nucleic acids is of profound importance to a wide range of investigations, such as 1-3 biochemistry and clinical diagnosis. Apart from their primarily double helical structures, DNA or RNA can adopt higher-order and functionally-useful structures, such as G4 quadruplexes. G-quadruplexes are unique four-stranded structures formed by guanine-rich nucleic acid sequences. The building blocks of G-quadruplexes are G-quartets, which are derived from the association of four guanines into a cyclic Hoogsteen hydrogen-bonding arrangement. The G-quartets stack up with one on top of another to form G-quadruplex 5,6 structures. G-quadruplexes can be basically divided into the following three primary topologies: parallel, antiparallel, and 7 hybrid-type structures. G-quadruplexes are widely dispersed in eukaryotic genomes, including telomeric DNA, rDNA and a series of gene 8 promoter regions. During the past two decades, G-quadruplex structures have attracted extensive attention because of their biological significance and potential applications in 9-11 supramolecular chemistry. An ever-increasing interest in G-
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China. E-mail: [email protected].
†Electronic
Supplementary Information (ESI) available: [Synthesis and characterization of IZCM-2 to IZCM-8, and other additional supporting figures]. See DOI: 10.1039/x0xx00000x
quadruplexes has promoted the development of rapid and simple approaches for the selective and sensitive detection of these structures. Thus, the discovery of fluorescent probes for G-quadruplexes has become an extremely active area of research.12-14 Several probes specific for G-quadruplexes have previously been reported.15-26 Nevertheless, most of these studies primarily focused on the improvement of probe selectivity for G-quadruplexes. Their detection limits are rarely described. Notably, the selectivity and detection limit are both important criteria for evaluating an ideal probe. Designing fluorescent probes for G-quadruplexes when only considering their selectivity would hamper their further application. Recently, we discovered a novel fluorescent probe called IZCM-1 for detecting G-quadruplexes by incorporating a coumarin fluorophore into the 2,4,5-triarylimidazole framework.27,28 Notably, the fluorescence quantum yield of IZCM-1 increased by approximately 100 times when binding to some G-quadruplexes. However, its detection limit is not satisfactory. The most outstanding limit of detection (LOD) value of IZCM-1 for G-quadruplexes in solution is 40 nM. When employing IZCM-1 as a staining reagent for G-quadruplexes in electrophoresis gels, the corresponding detection limit is more than 200 ng (see the ESI†). Such data are much higher than data for commercial gel EtBr stains and SYBR® dyes with detection limits of less than 10 ng for most nucleic acids. Nevertheless, EtBr and SYBR® dyes are non-selective for Gquadruplexes and other nucleic acids.29-31 It is hard to note the G-quadruplex bands when using these dyes to stain mixed nucleic acid samples that are separated in a gel matrix. Thus,
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the research gaps impelled us to synthesize new triarylimidazole fluorescent probe aiming not only to improve its detection limit but also to maintain its selectivity for Gquadruplexes. The sensitivity of commercial nucleic acid probes is caused by a high affinity for nucleic acids, in combination with a high 32 quantum yield upon binding. In studying IZCM-1, we also found significant and positive correlations between the binding affinity and fluorescence performance of IZCM-1 in the 28 presence of different G-quadruplexes. These results made us envisage the possibility of upgrading the probe´s detection limit by improving its binding affinity and fluorescence quantum yield with G-quadruplexes. In considering the structure of IZCM-1, which includes a 1-methylpiperazine group, this terminal cationic group makes an important contribution to the electrostatic interaction of IZCM-1 with Gquadruplexes. However, this group alone is likely insufficient because previous experience indicated that the presence of at least two cationic side chains to the chromophore is usually 33,34 required to gain high G-quadruplex binding potency. In addition, the nitrogen atom in the junction between the side chain and phenyl group could also be changed because the substituent connected to the chromophore sometimes 35,36 influenced the fluorescence quantum yield of a compound. Based on these considerations, we tried to modify the structure of IZCM-1, and we thereby designed a new set of disubstituted triarylimidazole fluorescent probes.
fluorescence performance on G-quadruplexes. The detailed interactions of the most outstanding probe with various Gquadruplexes, double-stranded and single-stranded nucleic acids and the corresponding photophysical changes were studied. In addition, the probe's detection limits for various nucleic acids were examined in both the solution and a gel matrix, with the aim of developing a selective and sensitive triarylimidazole fluorescent probe for G-quadruplexes.
Results The synthesis of triarylimidazole fluorescent probes
The designed probes (IZCM-2 to IZCM-8) were prepared by condensing coumarin aldehyde and the corresponding substituted diphenylethanedione moiety according to our previous studies.27,28 Their structure and purity were confirmed by 1H and 13C NMR spectrometry, HRMS spectrometry, and HPLC analysis (Scheme S1 and Figure S1-28, ESI†). Screening triarylimidazole fluorescent probes Table 1. Optical data and fluorescence quantum yields (ΦF) of disubstituted triarylimidazole fluorescent probes in buffer (10 mM Tris-HCl, pH=7.2, 100 mM KCl) and in the presence of different nucleic acids ΦF
a
λabs
λem
(nm)
(nm)
Buffer
T21
ds26
Pu22
IZCM-2
449
526
0.012
0.043
0.041
0.257
IZCM-3
459
528
0.004
0.007
0.009
0.226
IZCM-4
448
529
0.041
0.046
0.045
0.079
IZCM-5
444
529
0.050
0.084
0.084
0.454
IZCM-6
449
529
0.020
0.053
0.082
0.308
IZCM-7
448
528
0.012
0.059
0.077
0.518
IZCM-8
447
532
0.006
0.010
0.014
0.086
a
1 μM of each compound and 10 μM of each DNA were used in the determination of ΦF.
Figure 1. The structures of IZCM-1 and di-substituted triarylimidazole fluorescent probes IZCM-2 to IZCM-8. As shown in Figure 1, the designed probes (IZCM-2 to IZCM8) bear two identical substituents in comparison with IZCM-1. The substituents include an amine side chain and a carboxyl side chain, which would be helpful to understand the electrostatic impact. In addition, oxygen atoms were introduced instead of nitrogen atoms in the junctions between the side chains and phenyl groups. In this study, these designed probes were synthesized and screened for their
To identify the most promising triarylimidazole fluorescent probe, newly synthesized compounds were screened for fluorescence performance with a focus on the fluorescence quantum yield and selectivity for G-quadruplexes. Three representative oligonucleotides, including single-stranded DNA T21, double-stranded DNA ds26 and G-quadruplex DNA formed by the oligonucleotide Pu22, were employed in the assays. As shown in Table 1, these candidate probes exhibited a slight difference in their absorption and emission maximums in buffer solution. Their fluorescence emissions were weak, even in the presence of singlestranded and double-stranded DNA. By contrast, most of the candidates displayed strong fluorescence when adding Gquadruplex Pu22 except IZCM-4 and IZCM-8. It was also noteworthy that the fluorescence quantum yield values were always more pronounced for candidates bearing oxygen atoms in the junctions between side chains and phenyl groups (IZCM-5, IZCM-6 and IZCM7). Among these three candidates, IZCM-7 was the best probe in
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view of its outstanding fluorescence quantum yields upon binding to G-quadruplex Pu22. In addition, IZCM-7 displayed weaker fluorescence emissions in buffer solution or in the presence of single-stranded and double-stranded DNA relative to IZCM-5 and IZCM-6, thus showing its better selectivity towards G-quadruplexes. From among the candidates, IZCM-7 was chosen as the most promising triarylimidazole fluorescent probe for further detailed investigation. The Optical properties of IZCM-7 The detailed optical properties of IZCM-7 were then investigated by absorption and fluorescence spectroscopy in different solvents. As shown in Table 2, IZCM-7 exhibited a slight difference in the absorption maximum in different solvents and showed weak fluorescence emission in most low-viscosity liquids. Interestingly, a remarkable fluorescence enhancement of IZCM-7 arose in the highviscosity glycerol solution (60% aqueous glycerol solution), suggesting that the fluorescence intensity of IZCM-7 could be tunable. It was also noteworthy that there was a positive correlation between the fluorescence quantum yield and solvent viscosity, showing that the quantum yield of IZCM-7 increased significantly with the increasing glycerol content in the glycerolwater mixed solution. The double-logarithmic plot of the quantum 2 yield vs. the viscosity was almost linear (R = 0.9786, Figure S29, ESI†). In addition, IZCM-7 had little propensity to aggregate in buffer according to its concentration-dependent absorbance spectrum (Figure S30, ESI†). These results closely resembled those found in the IZCM-1 studies, suggesting that the variations in fluorescence emission may be caused by conformational changes in the excited state of the 2,4,5-triarylimidazole-derived chromophore, most likely based on the rotational state of the single 37 bonds around the imidazole moiety. Table 2. Optical properties of IZCM-7 in different solvents a
Solvent
η (cP )
λabs (nm)
λem (nm)
ΦF
Acetone
0.32
444
525
0.028
Dichloromethane
0.44
448
527
0.058
Chloroform
0.57
453
530
0.120
Toluene
0.59
447
520
0.033
Methanol
0.60
441
529
0.109
Ethanol
1.20
441
528
0.097
Water
1.00
448
528
0.019
60% glycerol
10.80
440
528
0.329
a
7 in high-viscosity solution, suggesting that G-quadruplexes could similarly lock IZCM-7 into an active fluorescent conformation. In addition, a significant lighting up in the fluorescence was also observed when IZCM-7 was treated with other types of Gquadruplexes, including KRAS, ILPR, THTG, bcl-2, c-kit2, c-kit3, HRAS and htg22 (Figure 2B). By contrast, negligible fluorescence enhancement was observed when titrating IZCM-7 with singlestranded DNA (Py22, T21 and A21), double-stranded DNA (ds26, 19AT, dx12 and ct-DNA), and protein BSA. This trend in fluorescence intensities could be observed by the naked eye under UV light (Figure S31, ESI†).
Figure 2. (A) The fluorescence titration of 1 µM IZCM-7 with the stepwise addition of the G-quadruplex Pu22 (arrow: 0–10 mol equiv.) in 10 mM Tris-HCl buffer, 100 mM KCl, pH 7.2. The fluorescence emissions of IZCM-7 with and without Pu22 under UV light were shown in the inner panel. (B) The fluorescence intensity enhancement of 1 µM IZCM-7 at 525 nm against the sample concentrations, λex = 450 nm.
Viscosity at 25 °C
Fluorescence spectroscopic studies of IZCM-7 interactions with nucleic acid The detailed fluorescence properties of IZCM-7 with various Gquadruplexes and other nucleic acids were explored by using a fluorescence titration assay. As shown in Figure 2A, IZCM-7 alone in buffer displayed weak fluorescence emission. With the gradual addition of G-quadruplex DNA Pu22, an emission peak at approximately 525 nm was significantly enhanced. This remarkable fluorescence enhancement closely resembled the behavior of IZCM-
Figure 3. (A) The fluorescence titration of 1 µM IZCM-7 with the stepwise addition of ct-DNA in 10 mM Tris-HCl buffer containing 1 µM G-quadruplex Pu22, 100 mM KCl, pH 7.2. (B) The fluorescence titration of 1 µM IZCM-7 with the stepwise addition of the Gquadruplex Pu22 without and with 200 µM ct-DNA in 10 mM TrisHCl buffer, 100 mM KCl, pH 7.2. Competition titrations were performed to further confirm the selective fluorescence response of IZCM-7 binding to Gquadruplexes, in which the ability of IZCM-7 to retain an enhanced
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fluorescence intensity with the addition of G-quadruplexes was 18 challenged by duplex DNA. As shown in Figure 3A, in the presence of various amounts of duplex ct-DNA, the enhanced fluorescence of IZCM-7 with G-quadruplex Pu22 was only slightly affected, even at the 200 μM duplex competitor concentration. In addition, when gradually adding the Pu22 G-quadruplex into the solution containing IZCM-7 and 200 μM ct-DNA, the enhanced fluorescence emissions were practically identical to those in the experiment without a duplex competitor (Figure 3B). Collectively, these results demonstrated the promising potential of IZCM-7 to serve as a promising G-quadruplex fluorescent probe even in a competitive environment. Table 3. The fluorescence quantum yields of IZCM-7 with different a nucleic acids and BSA protein Sample
ΦF
Relative ΦF
Tris
0.012
1
KRAS
0.595
50
ILPR
0.582
49
THTG
0.553
46
bcl-2
0.528
44
Pu22
0.518
43
c-kit2
0.479
40
HRAS
0.454
38
c-kit3
0.439
37
htg22
0.403
34
ds26
0.077
6
19AT
0.074
6
dx12
0.070
6
ct-DNA
0.022
2
py22
0.077
6
T21
0.059
5
A21
0.018
1
BSA
0.021
2
(HRAS, c-kit3, htg22). This behavior in IZCM-7 partly resembled that of IZCM-1 in its display of a selective fluorescence response towards parallel G-quadruplexes. However, fluorescence emission enhancement and quantum yield values of IZCM-7 with non-parallel G-quadruplexes were apparently upgraded. The discrepancy between parallel and non-parallel G-quadruplexes was reduced. The detection limits of IZCM-7 for G-quadruplex Encouraged by the strong fluorescence emission enhancement and improved quantum yield values of IZCM-7 with Gquadruplexes, we then investigated the detection limits associated with using IZCM-7 to identify G-quadruplexes in solution. The LOD values were calculated on the basis of the equation LOD = K × 38,39 Sb/m. The K value is generally taken to be 3 according to the International Union of Pure and Applied Chemistry (IUPAC) recommendation. The Sb value represents the standard deviation for multiple measurements (n = 20) of blank solution. The m value is the slope of the calibration curve, which is derived from the linear range of the IZCM-7 fluorescence titration curve with different nucleic acids and represents the sensitivity of this method (Figure S32, ESI†). The linear ranges of the fluorescence titration curve for IZCM-7 with most of the G-quadruplexes ranged from 0 to 400 nM. The m values were then obtained by fitting these curves and the corresponding LOD values are shown in Table 4. Clearly, the LODs of IZCM-7 for parallel G-quadruplexes (KRAS and Pu22) in solution were below 10 nM. These values were better than that of antiparallel G-quadruplex c-kit3 and hybrid-type G-quadruplex htg22, which were below 20 nM. In addition, we could not obtain the exact LOD values for double-stranded DNA ds26 because of its negligible fluorescence enhancement during titration. It was also noteworthy that the LOD values of IZCM-7 for G-quadruplex in solution were significantly improved relative to IZCM-1. The LOD values of IZCM-1 for G-quadruplex KRAS, Pu22, c-kit3 and htg22 were 13 nM, 17 nM, 118 nM and 165 nM, respectively (Figure S33, ESI†). Accordingly, the improving detection limits of IZCM-7 for Gquadruplex revealed the importance of two new amine chains and oxygen atoms in the junctions. Table 4. The detection limits and linear ranges of IZCM-7 for different nucleic acids in solution
a
1 μM of IZCM-7 and 10 μM of each sample were used in the determination of ΦF. The fluorescence quantum yield values of IZCM-7 with different nucleic acids and BSA protein are summarized in Table 3. Notably, these values were consistent with the results for fluorescence titration and competitive experiments showing that fluorescence emission enhancement was always more pronounced for Gquadruplex structures. The quantum yield values of IZCM-7 with Gquadruplexes reached 0.51 on average. Typically, the quantum yield value of IZCM-7 was up to 0.60 in the presence of KRAS Gquadruplex. These data were 50% higher than that of IZCM-1 (0.40) 28 under the same conditions. This structural modification significantly improved the quantum yield values of IZCM-7. On the other hand, the fluorescence emission enhancement and the quantum yield values of IZCM-7 with parallel G-quadruplexes (KRAS, ILPR, THTG, bcl-2, Pu22, and c-kit2) were slightly but perceptibly higher than those with non-parallel G-quadruplexes
Nucleic Acid
LOD ( nM )
Linear Range ( nM )
KRAS
3
0 - 200
Pu22
8
0 - 400
c-kit3
15
0 - 400
htg22
19
0 - 400
ds26
> 1000
nd
a
a
nd: not determined
In addition to the LOD values of IZCM-7 in solution, we were also interested in its performance in staining G-quadruplex bands after gel electrophoresis. G-quadruplex DNA (KRAS, Pu22, c-kit3 and htg22), double-stranded DNA (ds26) and single-stranded DNA (mPu22, a mutant of Pu22) were employed in the polyacrylamide gel electrophoresis (PAGE) experiment. After electrophoresis, the polyacrylamide gels were immersed in 4 µg/mL IZCM-7 staining solution for 20 minutes. As shown in Figure 4A, clear dose-response
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fluorescence bands were accompanied by an increasing concentration of G-quadruplex DNA, and the bands of doublestranded DNA and single-stranded DNA were hardly detectable. In addition, parallel G-quadruplexes (KRAS and Pu22) were much easier to detect compared with antiparallel G-quadruplex c-kit3 and hybrid-type G-quadruplex htg22. These findings were consistent with the trends observed from LOD values of IZCM-7 for nucleic acids in solution. However, the PAGE experiments were repeated by using commercial SYBR® Green I stain as a benchmark. Compared with IZCM-7, SYBR® Green I was a stain for all types of nucleic acids (Figure 4B). Most importantly, the performance of IZCM-7 in staining G-quadruplex bands was comparable to that of SYBR® Green I and was much stronger than that of IZCM-1 (Figure S34, ESI†). These results could also be observed from their detection limits as summarized in Table 5. Notably, the detection limits of IZCM-7 for G-quadruplex KRAS was identical to that of SYBR® Green I, suggesting the potential commercial value of IZCM-7 as a selective stain for G-quadruplexes.
Figure 4. The dose-response staining of G-quadruplexes (KRAS, Pu22, HRAS, and htg22), duplex DNA (ds26) and single-stranded DNA (mPu22) by (A) IZCM-7 and (B) SYBR® Green I. The conditions were as follows: 10 μL of nucleic acids was loaded on to 20% acrylamide in 1 × TBE buffer containing 100 mM KCl. The nucleic acid concentration was increased from 0.05 to 6.4 μM.
After electrophoresis, the polyacrylamide gels were immersed in a 4 µg/mL IZCM-7 staining solution for 20 minutes and SYBR® Green I was used as a benchmark. As shown in Figure 5A, clear concentration-dependent bands for G-quadruplex Pu22 were observed when applying IZCM-7 as a gel stain. The background was clean. By contrast, mixed bands were observed when applying SYBR® Green I as gel stain. It was hard to note the band of Gquadruplex Pu22 owing to the interference of double-stranded DNAs. In addition, the detection limit of IZCM-7 for G-quadruplex Pu22 under competition conditions was also 7 ng, demonstrating the practicability of using IZCM-7 as a specific stain for Gquadruplexes, even in the presence of abundant double-stranded DNA. Taking all of these results together, IZCM-7 displayed promising application prospects for the selective and sensitive Gquadruplexes in both the solution and gel matrix.
Figure 5. The dose-response staining of G-quadruplex Pu22 in the presence of mixed double-stranded DNAs (dx12, ds26, 19AT, Hairpin-4T, Hairpin-6T, Hairpin-8T and Hairpin-10T) by (A) IZCM-7 and (B) SYBR® Green I. Conditions: 10 μL of nucleic acids were loaded onto 20% acrylamide in 1 × TBE buffer containing 100 mM KCl. The concentration of G-quadruplex Pu22 was increased from 0.05 to 6.4 μM. The content of each double-stranded DNA was maintained at 200 ng. UV-Vis spectroscopic studies of IZCM-7 interactions with Gquadruplex
Table 5. The detection limits of IZCM-7 and SYBR® Green I as gel a stains for different nucleic acids LOD ( ng ) Nucleic Acid IZCM-7
SYBR® Green I
KRAS
5
5
Pu22
7
3
c-kit3
14
7
htg22
28
3
ds26
510
