Pyrene Excimer Fluorescence as a Probe for Parallel G-Quadruplex

Bioconjugate Chem. 2007, 18, 1213−1217

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Pyrene Excimer Fluorescence as a Probe for Parallel G-Quadruplex Formation Huihe Zhu and Frederick D. Lewis* Department of Chemistry, Northwestern University, Evanston, Illinois 60208. Received September 8, 2006; Revised Manuscript Received February 16, 2007

The formation and properties of G-quadruplex structures from short single-strand oligonucleotide conjugates possessing two to four guanines and a 5′-terminal pyrenebutanol are reported. The 4-G conjugate forms a stable G-quadruplex under low or high potassium ion concentrations, whereas the 3-G conjugate forms a stable G-quadruplex only in the presence of high potassium. The 2-G conjugate fails to form a stable G-quadruplex even at low temperature and high potassium concentration. Both pyrene monomer and excimer fluorescence are observed for the G-quadruplex structures, whereas only monomer fluorescence is observed for the single-strand conjugates. Thus, pyrene excimer fluorescence can be used as a probe for the formation of G-quadruplex structures. The excimer/monomer intensity ratios for the G-quadruplex structures are dependent upon both the temperature and potassium or lithium salt concentration. The salt effect is attributed to a change in the structure of the hydrophobic pyrene chromophores, which are assembled on the 5′-face of the G-quadruplex as a consequence of electrostriction.

INTRODUCTION

Scheme 1

Pyrene excimer fluorescence has been widely employed as a probe for the structure of biopolymers possessing covalently attached pyrene chromophores in aqueous solution (1). Complementary oligodeoxynucleotides (ODNs) possessing pendant pyrene chromophores or pyrene-derived base surogates in both strands display either monomer or excimer fluorescence, depending upon the proximity of the pyrenes (2-5). ODNs possessing a single 5′- or 3′-terminal pyrene have been used to investigate the strand polarity (parallel vs antiparallel) of duplex and triplex structures (6, 7). ODNs possessing bases modified with covalently attached pyrene groups have also been prepared and the fluorescence of their duplex structures studied (2, 8-12). These studies have established that the proximity of two pyrenes is a necessary but not sufficient condition for the observation of excimer fluorescence. Ground-state conformations which disfavor the formation of a sandwich-type excimer structure for the electronically excited pyrene dimer give rise to monomer or broadened monomer fluorescence rather than excimer fluorescence (1). The pyrene excimer/monomer ratio in oligomeric pyrene-containing C-nucleosides increases with the length of the oligomer (13). Guanine-rich ODNs can assemble to form G-quadruplex structures based on stacked arrays of G-quartets connected by Hoogsteen-type base pairing (Scheme 1a) (14, 15). G-quadruplexes can be formed by the association of four ODNs possessing a single poly(dG) domain, dimerization of ODNs possessing two poly(dG) domains, or intramolecular folding of ODNs possessing four poly(dG) domains. Quadruplexes formed via intermolecular association in the presence of potassium generally have parallel structures (16, 17), whereas hairpin dimers and intramolecular quadruplexes are highly polymorphic (18). Merkina and Fox (19) have recently reported the formation and kinetic stability of intermolecular G-quadruplex structures formed by the conjugate 5′-d(TGGGG)-FL (FL ) fluorescein), which possess a covalently attached FL dye. Reduction in the FL fluorescence intensity of these ODNs upon quadruplex * Corresponding author. E-mail: [email protected]. Voice: +1847-491-3441. Fax: +1-847-467-2184.

a

a (a) Hoogsteen base pairing in a G-quartet; (b) parallel G-quadruplex structure formed by intermolecular association; (c) pyrene-modified poly(dG) sequences.

formation is attributed to self-quenching of the four dye molecules when assembled on the same face of the Gquadruplex structure (Scheme 1b); however, FL self-quenching

10.1021/bc060279u CCC: $37.00 © 2007 American Chemical Society Published on Web 05/04/2007

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does not result excimer fluorescence (13). It occurred to us that replacement of FL with pyrene might result in the formation of quadruplex structures with high excimer/monomer fluorescence ratios. We report here the study of intermolecular G-quadruplex formation using short poly(dG) sequences possessing a 5′pyrenebutanol fluorescent probe (Scheme 1c, 1-3). Whereas the single-strand ODNs display structured monomer fluorescence, the quadruplex structures display a mixture of monomer and excimer fluorescence, both of which are assigned to parallel quadruplex structures. Thus, the appearance of excimer fluorescence can be used as a probe for parallel G-quadruplex formation. The ratio of excimer/monomer fluorescence intensity is dependent upon both the temperature and salt concentration. These observations are consistent with the presence of a mixture of pyrene geometries which either favor or disfavor excimer formation. Increased excimer fluorescence at high salt concentrations is attributed to a hydrophobic salt effect on the pyrene chromophore ground state-geometry.

EXPERIMENTAL PROCEDURES Materials. 1-Pyrenebutanol was converted to its phosphoramidite by reaction with 2-cyanoethyl diisopropyl chlorophosphoramidite in tetrahydrofuran in the presence of diisopropylethylamine. The conjugates 1-3 were prepared by means of conventional phosphoramidite chemistry using a Millipore Expedite oligonucleotide synthesizer and reagents obtained from Glenn Research following the procedure of Letsinger and Wu (20) as implemented by Lewis et al. (21). Following synthesis, conjugates 1-3 were isolated by reverse phase (RP) HPLC and repurified as needed. HPLC analyses and purifications were performed using a Phenomenex HyperClone column (250 × 4.6 mm). Molecular weights were determined by means of MALDI-TOF mass spectrometry using a Perseptive Biosystems Voyager Pro DE spectrometer with a 2,4,6-trihydroxyacetophenone (THAP)/ammonium citrate matrix. ODN 1: calcd. 1236.96; found: 1239.21. ODN 2: calcd. 1566.17; found: 1568.73. ODN 3: calcd. 1895.37; found: 1897.37. Electronic Spectra. UV spectra were performed with a Perkin-Elmer Lambda-2 UV spectrophotometer equipped with a Peltier temperature programmer to automatically increase the temperature at the rate of 0.5 °C/min. Fluorescent spectra were obtained using a Spex FluoroMax Fluorometer with temperature control provided by a Quantum Northwest TC 125 Peltier controller. Fluorescent spectra were collected after samples were deoxygenated by purging with dry nitrogen gas for 15 min. The spectra were excited at 336 nm and monitored from 360 to 600 nm. The integration time was 0.1 s, and slits for both excitation and emission were 5.0 nm. Temperature-dependent fluorescent spectra were collected with heating or cooling rate of 40 °C/h. All spectra were obtained on freshly prepared samples within 30 min of preparation. The concentration of single-strand sequences was determined from the extinction coefficient of 1-pyrenebutanol (340 nm ) (5.0 ( 0.04) × 104 M-1 cm-1 in THF solution). CD Experiments. Circular dichroism (CD) spectra and thermal denaturation experiments were recorded using a Jasco J-715 circular dichroism spectrometer equipped with a Jasco Peltier temperature controller. Spectra were recorded at room temperature approximately 30 min after sample preparation in a 1 cm path length cuvette using either 1.0 × 10 -6 or 5.0 × 10 -6 M single-strand concentration. Spectra were obtained from the sum of five scans over the 200-400 nm wavelength range with a scan rate of 100 nm m-1, a bandwidth of 2.0 nm, and a response time of 2 s. The spectra were normalized by subtraction of the background scan obtained with buffer solution. Temper-

Figure 1. Absorption spectra of the pyrenebutanol in methanol solution and conjugate 2 in aqueous 0.1 M KCl, 10 mM potassium phosphate.

ature-dependent experiments were performed in the range 1090 °C with a heating rate of 40 °C/h, monitoring the CD value (mdeg) at 265 nm. Electrophoresis. Samples for gel electrophoresis were equilibrated for 1 day at 4 °C prior to electrophoresis for 2 h at 150 V at 4 °C using a 15% nondenaturing polyacrylamide gel (PAGE) that contained 100 mM KCl in 1 × TBE, 10 mM potassium phosphate electrophoresis buffer. The fluorescence of the gel was observed over a UV transilluminator (312 nm), and then the gel was stained with Stains-All (Sigma).

RESULTS The synthesis, purification, and characterization of conjugates 1-3 are described in the Experimental Procedures. The UV spectra of pyrenebutanol in methanol and conjugate 2 in aqueous buffer with 0.1 M KCl are shown in Figure 1. The spectrum of 2 in water is similar to that obtained in buffer. The longwavelength π,π* band of pyrenebutanol is red-shifted and broadened in the conjugate spectrum. The short-wavelength bands of the conjugate are assigned to overlapping absorption of the bases and pyrene. The spectra of 1 and 3 have band structures similar to that of 2, but differ in the ratio of band intensities, as a consequence of the different number of dG bases. The CD spectra of 1-3 in water and in 0.01 M phosphate with 0.1 M KCl at room temperature are shown in Figure 2. No CD peaks are observed for 1, either with or without KCl at room temperature or at 5 °C. The CD spectrum of 2 in water is weaker than that with salt, whereas added salt has little effect on the CD spectrum of 3. In addition to the short-wavelength bands characteristic of the parallel G-quadruplex structure at 265 and 240 nm, weaker long-wavelength CD bands having vibrational structure similar to that for pyrene UV absorption (Figure 1) are observed for 2 and 3 (Figure 2b). The intensity of the pyrene CD band for 2 increases as the salt concentration is increased from 0.1 to 0.5 M KCl (data not shown). The temperature dependence of the CD spectra of 2 (0.1 M KCl) and 3 (no added salt) are shown in Figure 3. The spectra of 3 are independent of temperature, whereas those of 2 display a marked decrease in CD intensity between 60 and 70 °C, which is largely reversible upon cooling (data not shown). The fluorescence spectra of 1-3 in water and in 0.1 M KCl are shown in Figure 4. The spectra of 2 and 3 with salt and of 3 without salt display a mixture of structured pyrene monomer fluorescence and broad, red-shifted pyrene excimer fluorescence. Only monomer fluorescence is observed for conjugates 1 and

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Figure 2. Circular dichroism spectra of 1 × 10-6 M conjugates 1-3 in (a) in water and (b) in 10 mM potassium phosphate with 0.1 M KCl.

Figure 3. Temperature-dependent circular dichroism spectra of conjugates 2 and 3. Heating rate: 1.5 min/°C. (a) 5 × 10-6 M conjugate 2 in 10 mM potassium phosphate with 0.1 M KCl and (b) 1 × 10-6 M conjugate 3 in water.

2 in water. The effect of added KCl and LiCl salts on the ratio of excimer to monomer fluorescence intensity for conjugate 2 is shown in Figure 5. The ratio Iex/Im increases with added KCl to a value of 1.4 at 0.2 M KCl but does not increase further at higher KCl concentrations. The Iex/Im ratio also increases upon addition of LiCl to a solution of 2 in 0.1 M KCl. Addition of LiCl results in values of Iex/Im somewhat smaller than that for 0.2 M KCl. The effects of increasing temperature on the total fluorescence of 2 (with salt) and 3 (in buffer) are shown in Figure 6. The Iex/Im ratios obtained for both heating and cooling are shown in Figure 7. Heating of 2 results in a decrease in both monomer and excimer fluorescence intensity (Figure 6a). The ratio Iex/Im remains fairly constant up to ca. 50 °C but decreases at higher temperatures (Figure 7a). Cooling to 0 °C is necessary for recovery of the Iex/Im ratio. Heating of 3 results in a larger decrease in Im than Iex (Figure 6b), resulting in an increase in Iex/Im with increasing temperature (Figure 7b). Cooling results in lower values of Iex/Im than those observed at the same temperatures in the heating cycle. Excimer fluorescence can also be used to visually detect G-aggregate formation using nondenaturing PAGE under a UV transilluminator, the eye being more sensitive to pyrene excimer

Figure 4. Room-temperature fluorescence spectra of 1 × 10-6 M conjugates 1-3 in different salt conditions: (a) in water and (b) in 0.1 M KCl, 10 mM potassium phosphate.

Figure 5. Salt concentration dependence of the pyrene excimer/ monomer fluorescence intensity ratio for conjugate 2 (LiCl added to a solution containing 0.1 M KCl).

vs monomer fluorescence. Electrophoresis of conjugates 2 and 3 (3.5 × 10-5 M or 0.3 mM in 0.1 M KCl) yields a single strongly fluorescence band, the band for 2 having a higher migration rate. In contrast, the faster-moving band from 1 cannot be detected visually without the addition of a staining agent (data not shown).

DISCUSSION The ODNs d(TGGGT) and d(TGGGGT) are known to form stable parallel G-quadruplex structures in the presence of potassium ions (17, 22). Parallel quadruplex formation has not been reported for d(TGGT); however, hairpin dimer and intramolecular G-quadruplex structures possessing only two G-quartets have been reported (14, 23). The presence of the terminal thymines in the d(TGnT) ODNs inhibits the formation of higher aggregates. The conjugates 1-3 are derived from these ODNs by replacement of the 5′-dT by pyrenebutanol (Scheme 1b,c). The 200-300 nm region of the CD spectra of 2 and 3 in the presence of KCl (Figure 2b) are identical, respectively, to those reported for d(TGGGT)4 and for a G-quadruplex in which the 3′-thymines are connected by a branched linker which enforces a parallel structure (24, 25). The structure of 3 is similar to that of the conjugate d(TGGGG)-FL (FL ) fluorescein) recently studied by Merkina

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Figure 6. Temperature-dependent fluorescent spectra of conjugates 2 and 3. Heating rate: 1.5 min/°C. (a) 5 × 10-6 M 2 in 10 mM potassium phosphate with 0.1 M KCl and (b) 1 × 10-6 M 3 in 10 mM potassium phosphate.

Figure 7. Excimer/monomer fluorescence intensity ratios for conjugates 2 and 3. Heating and cooling rate: 1.5 min/°C. (a) 5 × 10-6 M 2 in 10 mM potassium phosphate with 0.1 M KCl and (b) 1 × 10-6 M 3 in 10 mM potassium phosphate.

and Fox (19). They report that the quadruplex formed by this conjugate is resistant to melting in the presence of 5 mM potassium. Stabilization of the FL-labeled quadruplex in sodium buffer when compared to an unlabeled quadruplex was attributed to stacking of the fluorophore on the exposed face of the terminal G-quartet, as observed for the complexes formed between G-quadruplexes and other planar hydrophobic organic molecules (26). We observe no change in the CD spectrum of 3 upon heating to 90 °C in the presence or absence of 10 mM potassium (Figure 3b), in accord with the high thermal stability observed for d(TGGGGT) or d(TGGGG)-FL (13, 17). The failure of 2 to form a stable quadruplex structure under in water (Figure 2a) and the observation of “melting” of the high salt CD spectrum at ca. 60 °C (Figure 3a) is consistent with previous reports of the diminished stability of d(TGGGT) vs d(TGGGGT) (17). The failure of 1 to form a G-quadruplex structure even at high salt concentrations indicates that the hydrophobic association of four pyrenes is not sufficient to stabilize an intermolecular G-quadruplex structure possessing only two G-quartets. The fluorescence spectra of 2 and 3 (Figure 4) consist of mixtures of monomer and excimer fluorescence, even though the CD spectra (Figure 2) are indicative of the formation of

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stable parallel G-quadruplex structures. It is possible that monomer and excimer fluorescence arise from different structures. However, PAGE gels for 2 and 3 display a single band, as visualized using pyrene excimer fluorescence. Low pyrene excimer/monomer fluorescence ratios have previously been observed upon duplex and triplex formation with pyrene endlabeled ODNs (6, 7). A high excimer/monomer fluorescence ratio has been observed for oligomeric pyrene-containing C-nucleosides (13) and for an intramolecular G-quadruplex having pyrenes attached to the 3′- and 5′-ends of the sequence d(GGTTGGTGTGGTTGG) (27). However, only broadened monomer emission is observed for several duplexes bearing pyrenes as pendant groups on multiple adjacent dU bases (9, 10). Studies of the fluorescence of conformationally constrained pyrene cyclophanes indicate that a full sandwich-like structure is required for the observation of long-wavelength excimer fluorescence, whereas structures with restricted overlap display small shifts or only broadened monomer emission (1, 28). We assume that the four pyrene chromophores occupy the 5′-face of the G-quartets formed by 2 and 3, so as to minimize hydrophobic interactions of both the G-quartet and pyrenes with water. However, it is likely that some of the pyrene chromophores have ground-state geometries that disfavor formation of a sandwich excimer with a neighboring pyrene upon electronic excitation. Evidence for the clustering of pyrenes on a single face of the G-quadruplex is provided by the effect of added salts on the excimer/monomer fluorescence ratio (Figure 5). Both added KCl and LiCl cause an increase in the Iex/Im ratio, even though 0.01 M KCl is sufficient to effect Gquadruplex formation, as evidenced by the CD spectra. Since LiCl is unable to stabilize G-quadruplex structures (29, 30), its influence on Iex/Im most likely is a consequence of a hydrophobic salt effect on the structure of the assembled pyrenes. LiCl and KCl have similar hydrophobic effects on the solubility of nonpolar molecules such as benzene in aqueous solution (31). Solvation of small ions by water results in collapse of the water molecules around the ions (32). This phenomenon, known as electrostriction, increases the energy required for creation of a space for a hydrophobic molecule such as pyrene and would force the four pyrenes attached to the quadruplex to occupy a smaller volume, plausibly favoring excimer formation. In summary, evidence based on circular dichroism spectra indicates that both 2 and 3 form parallel quadruplex structures in which the appended pyrenes occupy the 5′-face of the quadruplex. Conjugate 1 fails to form a stable quadruplex structure even at low temperature in the presence of KCl. Conjugate 3, which possesses a GGGG sequence, forms a stable quadruplex even in the absence of added salt and does not dissociate upon heating to 90 °C. In the presence of KCl, conjugate 2 forms a quadruplex structure that is stable at room temperature but undergoes thermal dissociation above 60 °C. The fluorescence spectra of the quadruplexes formed by conjugates 2 and 3 display both monomer and excimer bands, whereas single-strand conjugates display only monomer fluorescence. Thus, excimer fluorescence can be used as a probe for intermolecular quadruplex formation, both in solution and in visualizing PAGE gels. Upon heating, the excimer/monomer band ratio for 2 deceases sharply above 50 °C, concomitant with thermal dissociation of the quadruplex. In contrast, the excimer/monomer ratio for 3 increases with temperature, indicative of a change in pyrene aggregate structure in a manner that favors the face-to-face geometry required for pyrene excimer fluorescence. The effect of added salts (KCl or LiCl) on the pyrene excimer/monomer ratio is attributed to electrostriction, which confines the hydrophobic pyrenes to a small volume.

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ACKNOWLEDGMENT Funding for this project was provided by a grant from the National Science Foundation (CHE-0400663).

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