A multi-functional guanine derivative for studying the DNA G-quadruplex structure

Takumi Ishizuka , Pei-Yan Zhao , Hong-Liang Bao and Yan Xu *
Division of Chemistry, Department of Medical Sciences, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan. E-mail: xuyan@med.miyazaki-u.ac.jp

Received 7th June 2017 , Accepted 28th August 2017

First published on 1st September 2017


In the present study, we developed a multi-functional guanine derivative, 8FG, as a G-quadruplex stabilizer, a fluorescent probe for the detection of G-quadruplex formation, and a 19F sensor for the observation of the G-quadruplex. We demonstrate that the functional nucleoside bearing a 3,5-bis(trifluoromethyl)benzene group at the 8-position of guanine stabilizes the DNA G-quadruplex structure and fluoresces following the G-quadruplex formation. Furthermore, we show that the functional sensor can be used to directly observe DNA G-quadruplexes by 19F-NMR in living cells. To our knowledge, this is the first study showing that the nucleoside derivative simultaneously allows for three kinds of functions at a single G-quadruplex DNA. Our results suggest that the multi-functional nucleoside derivative can be broadly used for studying the G-quadruplex structure and serves as a powerful tool for examining the molecular basis of G-quadruplex formation in vitro and in living cells.


Introduction

G-quadruplex structures recently became an attractive area of research, as they have emerged as potential targets for drug design because of their biological importance.1 These structures and their chemical and biochemical properties could be better understood by controlling G-quartet-folding topologies in a specified manner.2 For example, the syn/anti conformation of guanine residues in G-quadruplexes can modulate G-quadruplex folding.3 In previous studies, we have demonstrated that the proper substitutions of dG in the syn conformation with 8-methylguanine (8mG)4 and 8-bromo-2′-deoxyguanosine (8BrG)3 can stabilize G-quadruplex structures. We also showed that these substitutions could be performed to form a highly stable G-quadruplex structure between the probe and target sequence, and allow for more efficient targeting of cancer telomere DNA.5

To investigate the structure and function of G-quadruplexes, fluorescent probes are typically used to allow the direct observation of folding and localization of the structure. Some studies have reported the use of modified nucleobase derivatives as fluorescent probes for the detection of G-quadruplex formation.6 For example, an 8-vinyl-2′-deoxyguanosine (VdG) was used as a fluorescent probe for the visualization of G-quadruplex folding.6c

The 19F sensor has recently been used as a powerful tool for the analysis of biomolecule conformations by 19F NMR spectroscopy.719F is an ideal conformational probe owing to its high sensitivity and low background signals in biological samples. We have recently shown that the 19F sensor can be used to study the G-quadruplex structure in vitro and that its sensitivity can be used to observe telomere RNA G-quadruplex formation in living cells.8

Although these modified nucleobase derivatives have been used to study G-quadruplexes, they work independently on each function as a G-quadruplex stabilizer, a fluorescent probe, and a 19F sensor for G-quadruplexes. For example, we showed that 8mG can stabilize the G-quadruplex conformation, but the derivative cannot be used as a fluorescent probe and 19F sensor.

In the present study, we developed a multi-functional guanine derivative 8FG that simultaneously allows for (i) the stabilization of the G-quadruplex structure, (ii) the detection of the G-quadruplex structure by fluorescence, and (iii) the observation of the G-quadruplex structure by 19F-NMR in vitro and in living cells (Fig. 1a). We first evaluated the effect of 8FG on the thermal stability of the G-quadruplex structure. Next, we investigated its ability to emit fluorescence during the G-quadruplex formation. Finally, we showed that the probe acts as a 19F sensor to directly observe G-quadruplex structures in vitro and in living cells by 19F-NMR spectroscopy. These results demonstrated that the nucleoside derivative simultaneously allows for three kinds of functions at a single G-quadruplex DNA.


image file: c7an00941k-f1.tif
Fig. 1 Chemical structure of guanine derivative 8FG (a) and sequences used in this study (b). 8-Substituted 8FG shows a predominant syn conformation and induces fluorescence in the G-quadruplex, also acts as a 19F sensor for monitoring the G-quadruplex.

Results and discussion

To synthesize the multi-functional probe, a 3,5-bis(trifluoromethyl)benzene moiety was introduced into the 8-position of 2′-deoxyguanosine by Sonogashira coupling. The guanine derivative was incorporated into oligonucleotides (ODNs) using phosphoramidite chemistry (see the ESI). 8-Substituted 8FG showed a predominant syn conformation due to steric hindrance between the 8-substituent and the ribose ring, suggesting that the substitution of dG in the syn conformation of a G-quadruplex can stabilize the structure. We first examined the effect of 8FG on the stability of the intermolecular (3 + 1) human telomeric G-quadruplex that was formed by the single-repeat (ODN1) and three-repeat (ODN3) human telomeric sequences (Fig. 1b).3b,9 We found that the 8FG substitution in dG at the expected syn conformations induced an increase in the thermal stability of the G-quadruplex in comparison with the G-quadruplex formed by natural sequences. The CD spectrum of the G-quadruplex structure formed by ODN1 and ODN3 in 100 mM K+ ion solution showed a strong positive Cotton effect at 290 nm with a negative signal at 235 nm, which is characteristic of the hybrid G-quadruplex structure (Fig. 2a).3 We found that the 8FG substitution in dG at the expected syn conformations induces an increase in the thermal stability of the G-quadruplex in comparison with the G-quadruplex formed by natural sequences (Fig. 2b). Thermal stabilization was also observed by the 8FG substitution in an intermolecular G-quadruplex of human telomere DNA (ODN4 vs. ODN5, Fig. S1).10 We further investigated the effect of 8FG on the stability of an intramolecular G-quadruplex of a thrombin binding aptamer (ODN6 vs. ODN7, Fig. S2),11 which subsequently indicated that the 8FG substitution results in a significant increase in its thermal stability. The results clearly demonstrate that 8FG is a G-quadruplex stabilizer.
image file: c7an00941k-f2.tif
Fig. 2 CD spectra (a) and CD melting curves (b) of intermolecular G-quadruplexes formed by the 8FG substituted ODN1 and ODN3, natural sequences ODN2 and ODN3. (c) Fluorescence spectra of ODN1 (10 μM) and ODN3 (0–20 μM) in 100 mM KCl and 10 mM Tris-HCl buffer (pH 7.0) at 25 °C with a mole fraction variation (λex = 386 nm). Inset: fluorescence image of ODN1 only (1[thin space (1/6-em)]:[thin space (1/6-em)]0) and ODN1/ODN3 (1[thin space (1/6-em)]:[thin space (1/6-em)]1) hybrid G-quadruplex after illumination with a UV lamp (365 nm). (d) Titration plot of the fluorescence monitored at 450 nm for the (3 + 1) hybrid G-quadruplex.

We next investigated 8FG as a potential fluorescent probe for observing the G-quadruplex structure. We performed fluorescence microscopy experiments to investigate the fluorescence change when ODN1 and ODN3 formed the G-quadruplex in the presence of 100 mM K+ ion (Fig. 2c). The intensity of the emission was monitored at 450 nm (λex = 386 nm) during the G-quadruplex formation with increasing ODN3 concentration. We found that the addition of ODN3 results in a strong fluorescence, whereas a control sequence ODN3m that cannot form a G-quadruplex does not induce an increase in fluorescence intensity (Fig. 1b and Fig. S3). A clear fluorescence that derived directly from the G-quadruplex formation is visible to the naked eye (inset in Fig. 2c). Fig. 2d shows the Job plot of the fluorescence emission monitored at 450 nm. A clear inflection point around 50% indicates a 1[thin space (1/6-em)]:[thin space (1/6-em)]1 stoichiometry for the formation of interstrand G-quadruplexes by ODN1 and ODN3. This is consistent with the previous result that the three-repeat and single-repeat human telomeric sequences form interstrand G-quadruplexes.3b We observed clearly the blue fluorescence from ODN1- and ODN3-treated cells (Fig. S4). Negative samples of the cells that were treated with ODN1 or ODN3 remained virtually nonfluorescent, which is consistent with the previous report that used fluorescence imaging to visualize the G-quadruplex in live cells.12 Thus, 8FG can be used as a fluorescent probe to monitor the G-quadruplex formation. It is not absolutely clear why the formation of the G-quadruplex can induce enhanced fluorescence, because the current understanding of the excited states and electronic properties of G-quadruplex is incomplete. We speculate that the main contributor to the emission is a G-tetrad formed by 8FG of ODN1 and dGs of ODN3, or base-stacking of 8FG and other dGs. The G-quadruplex structure is expected to impact the excited-state and energy transfer properties of the system with effects on the intensity of fluorescence.

We further investigated whether 8FG can be used as a sensor for the analysis of the G-quadruplex conformation by 19F NMR spectroscopy. 19F NMR signals are strongly dependent on the structural environment of the 19F label. Thus, it should be possible to distinguish different DNA structures of the same sequence by the corresponding resonances of the different structures, such as the single strand and G-quadruplex (Fig. 3a). We performed a concentration-dependent experiment to investigate the structural behavior of ODN1 and ODN3 in the formation of the DNA G-quadruplex by 19F NMR. The single-stranded ODN1 (100 μM) showed a single signal at −62.83 ppm, whereas the addition of ODN3 to ODN1 resulted in a new signal at −62.71 ppm (Fig. 3b). The new signal was clearly observed at an ODN3 concentration of 33 μM (ODN1/ODN3 = 1[thin space (1/6-em)]:[thin space (1/6-em)]0.33), and its intensity was remarkably greater than that of the initial peak. The original signal from the single-stranded ODN1 disappeared completely when the ODN3 concentration increased to 100 μM (ODN1/ODN3 = 1[thin space (1/6-em)]:[thin space (1/6-em)]1). Each 19F NMR signal arises as a result of the unique fluorine environment; thus, the new signal confirms the formation of the interstrand G-quadruplex by ODN1 and ODN3. We performed CD spectroscopy which revealed the conformation of ODN1, suggesting that ODN1 is an unstructured single strand (Fig. S5), which is consistent with the previous report.13


image file: c7an00941k-f3.tif
Fig. 3 (a) Concept for the detection of different DNA structures by a 19F label. Two 19F resonances of different chemical shifts are expected according to the single-stranded DNA and G-quadruplex. Purple box represents 8FG. (b) 19F NMR spectra of ODN1 and ODN3 at different concentrations. ODN1 (100 μM) was mixed with ODN3 (0, 33, 66 and 100 μM). The mole fraction of ODN1 and ODN3 is shown on the right. (c) 19F-NMR spectra at different temperatures. DNA 100 μM, 10 mM Tris-HCl buffer, 100 mM KCl, pH 7.0. Temperatures indicated on the right. (d) 19F signal change of ODN4 or ODN6 in the absence and presence of KCl. Red and blue circles indicate the G-quadruplex and unstructured single strand, respectively.

We subsequently carried out a temperature-dependent experiment to monitor G-quadruplexes by 19F NMR (Fig. 3c). At 25 °C, we observed a single signal at −62.71 ppm, which was consistent with the signal from the G-quadruplex formed by ODN1 and ODN3. As the temperature increased from 25 °C to 85 °C, the intensity of the signal at −62.71 ppm for the G-quadruplex decreased, while a new signal at −62.83 ppm corresponding to the unfolded single strand appeared. We found that the intensity of the new signal increased with increasing temperature. At 85 °C, only this peak remained strongly intense, whereas the peak corresponding to the G-quadruplex (at −62.71 ppm) completely disappeared. The results suggest that the transition of the G-quadruplex and single strand can be monitored by 19F NMR spectroscopy.

Encouraged by the ability to use 19F NMR spectroscopy to monitor the conformational transition behavior of the G-quadruplex via the observation of signal changes in the 19F resonances as a function of temperature, we characterized the melting process by plotting the normalized relative peak areas of the 19F resonance signals at various temperatures (Fig. S6).

The Tm value was estimated to be 56.4 °C, indicating a stabilized G-quadruplex structure. In the present case, the NMR derived Tm value (high sample concentration) was lower compared to the CD derived Tm value (low sample concentration). Some similar results were also observed in the previous studies.7e We speculate that the 19F NMR-based determination of Tm values is sensitive to varying measurement conditions and sample situations. In the present case, the Tm values obtained in a long measurement time (each temperature for 1 h) in NMR compared to those in CD (each temperature for 1 min). Importantly, the corresponding curve from NMR provides the Tm value with good precision for DNA at concentrations that are generally too high to be accessible for CD melting analysis.

19F NMR spectroscopy was further employed to investigate the intermolecular G-quadruplex structure of human telomere DNA (ODN4) and the intramolecular G-quadruplex structure of a thrombin binding aptamer (ODN6) (Fig. 3d). In the absence of K+, one signal was detected in the 19F NMR spectra, suggesting a single-stranded DNA. One new signal was observed in the presence of K+, indicating the intermolecular and intramolecular G-quadruplex formation by ODN4 and ODN6, consistent with previously reported results.10,11 These results demonstrated that the multi-functional guanine derivative 8FG can serve as a useful 19F sensor for the observation of the G-quadruplex structure by 19F-NMR.

Finally, we investigated whether 8FG could monitor the 19F signal of the G-quadruplex inside living Xenopus laevis oocytes by in-cell 19F-NMR. In-cell NMR Xenopus laevis oocytes provide valuable information while observing the intracellular sample by NMR.14 However, the stronger background in the cellular environment often leads to complicated or poor-quality in-cell 1H NMR spectra. Because there is no natural intracellular concentration of fluorine, fluorinated DNA oligonucleotides do not suffer from high background signals. Here, we monitored the G-quadruplex formation in Xenopus laevis oocytes by the direct microinjection of the DNA oligonucleotides. An overview of the experiment is shown in Fig. 4a. To observe a 19F signal following the G-quadruplex formation, approximately 150 Xenopus laevis oocytes that were injected with 19F-labeled oligonucleotides were collected and gently settled in an NMR sample tube. The in-cell 19F-NMR spectrum was compared with the reference in vitro spectrum, enabling reliable determination of the intracellular telomere DNA conformation. Fig. 4b shows a comparison of the in vitro and in-cell NMR spectra for the ODN1 and ODN3 in their pure form (top and middle panels) and upon oocyte injection (bottom panel). ODN1 indicated a single strand and ODN1 and ODN3 showed a G-quadruplex conformation. Only one signal was observed in the bottom panel NMR spectrum, for which the chemical shift was identical to that observed for the corresponding G-quadruplex in the in vitro19F NMR spectrum (middle panel). This result reveals that the ODN1 and ODN3 formed intermolecular G-quadruplex structures inside living cells. The line width of the signal increased in the in-cell spectrum compared to that in the in vitro spectrum, partially because of the higher viscosity of the cellular environment.15 NMR signals depend on whether the molecule of interest is freely available for tumbling in solution. In general, molecules display small tumbling rates due to their sizes, intermolecular interactions, intramolecular interactions with subsets of residues, and the high viscosity environments of intracellular materials, leading to fast relaxation and broad NMR lines with reduced overall signal intensities. In addition, an inherent sample inhomogeneity due to the in-cell environment is also the cause of the broad line width of the in-cell signal.15 To further investigate the state of the intermolecular G-quadruplexes, the oocyte samples were lysed after the in-cell NMR experiment. Notably, the NMR spectral patterns for the lysates of the ODN1 and ODN3 samples were similar to those observed for both the corresponding in vitro and in-cell samples, indicating the intermolecular G-quadruplex formation (Fig. S7).


image file: c7an00941k-f4.tif
Fig. 4 (a) Schematic overview of in-cell 19F NMR experiments. For in-cell 19F NMR applications in Xenopus oocytes, DNA sample was injected into the oocyte cells. Comparison with the position of the reference in vitro spectrum provides a reliable determination of intracellular DNA conformation. (b) Comparison of 19F NMR spectra of the in vitro sample of DNA (up and middle) and in Xenopus oocytes (bottom).

Altogether, these observations suggest that the 19F signal of 8FG can clearly represent the G-quadruplex structure in living cells. These findings provide new insights into the structural behavior of telomere DNA G-quadruplexes in the cellular environment.

Conclusions

Here, we developed a multi-functional guanine derivative, 8FG, as a G-quadruplex stabilizer, a fluorescent probe, and a 19F sensor of G-quadruplex formation. First, we demonstrated that the incorporation of 8FG into the appropriate position in human telomere and thrombin binding aptamer sequences could stabilize the intermolecular and intramolecular G-quadruplex structures. Moreover, we showed that the formation of G-quadruplex structures by the 8FG-containing sequences increases fluorescence emissions significantly, suggesting that 8FG can be used as a fluorescent probe for monitoring the G-quadruplex formation. Finally, using the derivative 8FG, we directly observed the DNA G-quadruplex structure in the cellular environment, indicating that 8FG is an ideal 19F sensor for studying the DNA G-quadruplex structure in living cells. These results suggested that 8FG is a multi-functional nucleoside derivative for studying the molecular basis of the DNA G-quadruplex structure in vitro and in living cells.

Experimental section

DNA synthesis and purification

All DNAs were synthesized on the 1.0 μmole scale with an NTS DNA/RNA synthesizer (NIHON TECHNO SERVICE Co., Ltd), using the solid-phase phosphoramidite chemistry. After automated synthesis, the oligonucleotides were detached from the support and deprotected according to the manufacturer's protocol. All oligonucleotides were purified by RP-HPLC (JASCO) using an appropriate linear gradient of 50 mM ammonium formate in H2O and 50 mM ammonium formate in 1[thin space (1/6-em)]:[thin space (1/6-em)]1 acetonitrile/H2O. The fractions containing the product were collected and lyophilized to give purified oligonucleotides. The purified oligonucleotides were desalted with NAP5 columns (GE Healthcare), and identified by using a MALDI-TOF MS (negative mode).

CD measurements and analysis of CD melting profile

CD spectra were obtained using a JASCO J-820 CD spectrophotometer. The spectra were recorded using a 1 cm path length cell. Samples were prepared by heating the oligonucleotides at 95 °C for 5 min and gradually cooling them to room temperature. The melting curves were obtained by monitoring at 290 nm for ODN1, 2 and 3 or 295 nm for ODN4, 5, 6 and 7. Solutions for CD spectra were prepared as 0.3 mL samples at a 5 μM strand concentration in the presence of 100 mM KCl, 10 mM Tris-HCl (pH 7.0).

Fluorescence measurements

Fluorescence spectra were obtained using a JASCO FP-8200 spectrofluorometer. The spectra were recorded using a 1 cm path length cell. The fluorescence spectra of ODN1 (10 μM) and ODN3 (0–20 μM) were obtained in 100 mM KCl and 10 mM Tris-HCl buffer (pH7.0) at 25 °C. The excitation of the probes used in this study was achieved at 386 nm. For each sample, at least two spectral scans were accumulated over a wavelength range from 400 to 700 nm. The emission spectra were recorded at an excitation wavelength of 386 nm, and the excitation spectra were recorded at an emission wavelength of 461 nm for 8FG nucleoside. To visualize the cellular localization of 8FG-containing ODNs, HeLa cells were transfected with ODNs of 20 μM as a final concentration and incubated for 3 h. Then cells were washed twice with PBS and visualized by fluorescence microscopy (Leica TCS SP8). The excitation wavelength (λex) and the emission wavelength (λem) were 386 and 461 nm, respectively.

19F-NMR experiments

NMR experiments were performed on a Bruker AV-400 M spectrometer. Trifluoroacetic acid as an external standard (−75.66 ppm) was used. The in vitro NMR experiment with the DNA G-quadruplex (100 μM) was performed in 10 mM Tris-HCl (pH7.0), 100 mM KCl containing 10% (v/v) D2O. In-cell NMR samples were prepared by direct microinjection of 50 nL aliquot of the stock solution (5 mM: the stock solution was initially made using the method described above for the formation of the G-quadruplex structure) into the oocyte cell nucleus. Approximately 150 Xenopus laevis oocytes were injected by using an IM-30 Electric Microinjector (NARISHIGE, Tokyo). After injection, the oocytes were transferred to a disposable dish and washed carefully with oocyte stocking buffer (15 mM Tris-HCl (pH 7.6), 88 mM NaCl, 1 mM KCl, 0.4 mM CaCl2, 0.3 mM Ca(NO3)2, 0.8 mM MgCl2 and 2.4 mM NaHCO3). The injected oocytes were transferred to a Shigemi tube (Shigemi 5 mm Symmetrical NMR microtube assembly, matched with D2O, bottom length: 8 mm) and kept in a total volume of 1 mL of oocyte stocking buffer containing 10% of D2O. Samples for measurements in the extract were prepared by crushing the oocytes mechanically.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We thank Prof. N. Kenmochi, Dr T. Uechi and Dr Y. Nakajima at the Frontier Science Research Center, University of Miyazaki for in-cell NMR sample preparation and valuable comments. This work was supported by a Grant-in-Aid for Scientific Research (26288083 and 17H03091) to Y. X. and a Grant-in-Aid for Young Scientists (B) (16K17938) to T. I. from the Ministry of Education, Science, Sports, Culture and Technology (Japan). Support from the Takeda Science Foundation is also acknowledged.

Notes and references

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Footnote

Electronic supplementary information (ESI) available: Experimental data and NMR spectra. See DOI: 10.1039/c7an00941k

This journal is © The Royal Society of Chemistry 2017