The 5′-(CCCTAA)_nT₅(TTAGGG)₄-3′ (n = 4, 3, 2, 1, 0) and relevant sequences (Table 1) were purchased from Sangon Biotech (Shanghai, China). Fluorescent 3′ FAM-labeled (TTAGGG)₂TTA was purchased from TaKaRa Biotech (Dalian, China). In all experiments, oligonucleotides or DNAs were made in 10 mM Tris–HCl (pH 7.4) buffer containing 1 mM EDTA, 150 mM K⁺ and 40% PEG 200 or no PEG, heated at 95°C for 5 min and cooled down to room temperature before use. In some experiments, oligonucleotides were labeled at the 5′ end with [γ-³²P]ATP using T4 polynucleotide kinase (Fermentas, Lithuania). Table 1.

Oligonucleotides used.

The dsDNAs (Ctrl-dsDNA and Telo-dsDNA) were obtained by polymerase chain reaction (PCR) from the pGEM plasmid. The telomere sequence (TTAGGG/CCCTAA)₄ at one or both end of the Telo-dsDNAs was introduced using (CCCTAA)₄-containing primers.

For the experiments in Figure 1, 5′-end-³²P-labeled oligonucleotides (∼3 nM, 2000 c.p.m) were loaded onto 15% polyacrylamide gel containing 40% (w/v) PEG 200 or no PEG and electrophoresed at 4°C, 8 V/cm, in 1× TBE buffer containing 150 mM KCl. Gels were autoradiographed on a Typhoon phosphor imager (Amersham Biosciences, Sweden). For the experiments in Figure 2, electrophoresis were carried out as above and the oligonucleotides were visualized with ethidium bromide (EB) or 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC). For the experiments in Figure 7, 100 ng (∼1 pmol) dsDNA or 5′-³²P-labeled ssDNA was mixed with 100 pM mjaSSB, a 76 kDa single-stranded DNA-binding protein from archaeon Methanococcus jannaschii (13), loaded onto 8% polyacrylamide gel containing 40% (w/v) PEG 200, and electrophoresed at 4°C, 10 V/cm, in 1× TBE buffer containing 150 mM KCl. Gels for dsDNA were stained with EB. Samples prepared in PEG were run in PEG-containing gels. The DNAs were visualized by autoradiography or EB staining. Figure 1.

Autoradiograph of gels showing electrophoresis mobility of G4Cn and their reference oligonucleotides G4Tn and G4CnR (n = 4, 3, 2, 1, 0) prepared in 150 mM K⁺ solution in the absence or presence of 40% (w/v) PEG 200. (A) The G4Tn formed quadruplex with a poly-T tail as indicated by their increased mobility relative to the size-matched references. (B) Structures formed in the absence of PEG. (C) New structural isoforms (asterisks) formed in G4Cn in the presence PEG, which are suggested to be quadruplex by their mobility relative to that of G4Tn and G4CnR. Q indicates the quadruplex formed by G4Tn and D the hairpin duplex by G4CnR, respectively. The calculated percentage of new species in G4C4 is 70%. Detailed sequence information is given in Table 1.

The CD spectra of different oligonucleotides (5 μM) were collected from 320 to 220 nm on a CD6 spectropolarimeter (HORIBA Jobin Yvon, France) at 25°C with 1 mm pathlength cylinder quartz cuvette. CD-melting profiles were recorded at 265 or 295 nm while temperature was increased at the rate of 1°C/min. Buffer blank correction was made for all measurements.

Fluorescence polarization measurements were carried out on a Spex Fluorolog-3 spectrofluorometer (HORIBA Jobin Yvon, France) at room temperature (20°C). (TTAGGG)₂TTA-FAM (50 nM) was incubated with each of the indicated oligonucleotide (100 nM) for 5 h at room temperature. Measurements were carried out using excitation and emission wavelength at 480 and 520 nm, respectively. For each sample, 10 parallel measurements with integration time of 2 s were averaged. Buffer blank was subtracted. Polarization values were calculated according to the equation r = (I_VV − GI_VH)/(I_VV + GI_VH), where the first subscript indicates the orientation of the excitation polarization and the second of the emission polarization (V for vertical and H for horizontal). G is a calibrating factor related to the instrument settings calculated from G = I_HV/I_HH.

The UV melting profiles were obtained on a DU-640 UV-VIS spectrophotometer (Beckman, Fullerton, CA, USA) equipped with a digital circulating water bath. The absorbance of oligonucleotides at 1 μM was monitored at 260 nm while temperature was simultaneously measured using a thermal probe immersed in the sample cell. The average heating rate was about 1°C/min.

Atomic force microscopy imaging was conducted with a Picoscan atomic force microscope (Molecular Imaging, Tempe, AZ, USA) (14). PCR product purified by gel electrophoresis was dissolved in 10 mM Tris (pH 7.4) containing 2 mM Mg²⁺ and 40% PEG 200 or no PEG. About 5 μl of DNA solution was dropped onto freshly cleaved ruby muscovite mica substrate (Digital Instruments, Santa Barbara, CA, USA). After 5 min, the mica surface was rinsed with ultrapure water and gently blew dry with nitrogen. Freshly prepared samples were mounted on AFM stage and imaged under MAC mode in air using Type II MAC Clever (spring constant = 0.95 N/m, Molecular Imaging, Tempe, AZ, USA). Typical scan rates were 1–3 Hz. The images were rastered at 512 × 512 pixels, unfiltered and flattened when needed.

To better mimic the in vivo situation where the G- and C-rich strands of telomere DNA are constrained in close proximity, we synthesized 5′-(CCCTAA)₄T₅(TTAGGG)₄-3′ (named G4C4) and similar oligonucleotides. The structures of these oligonucleotides were studied in the absence and presence of 40% (w/v) PEG 200, an agent that has been used to mimic the molecularly crowded intracellular environment (15). In G4C4, the G-rich motif has the potential to either fold into G-quadruplex or form hairpin duplex with the C-rich motif. Two sequences, G4C4R and G4T4, designed to form duplex and G-quadruplex, respectively, were used as references (Table 1). The structures of these oligonucleotides were first examined by electrophoresis using ³²P-labeled oligonucleotides (Figure 1). Gel electrophoresis showed that (TTAGGG)₄ conjugated with non-complementary sequence of various length formed G-quadruplex in the presence of K⁺ (Figure 1A). As judged from the mobility, the G4C4 formed duplex rather than G-quadruplex in the absence of PEG because it co-migrated with G4C4R that forms duplex, but migrated faster than the G4T4 that forms quadruplex (Figure 1B, lanes 1–3). In the presence of PEG, the G4C4 seems to have formed quadruplex as reflected by the appearance of a new band with mobility similar to that of the G4T4 (Figure 1C, lanes 1–3). From the intensity of the two bands, it is estimated that more than 70% of the G4C4 formed quadruplex. The formation of quadruplex became more dominant when the competition of duplex formation was reduced by decreasing the length of the complementary C-rich strand (Figure 1C, lanes 6, 9). In the absence of PEG, no quadruplex formation was observed in these oligonucleotides (Figure 1B), suggesting that quadruplex formation shown in Figure 1C was induced by PEG.

The structure of G4C4 in PEG solution was further verified by fluorescent dye staining with the reference oligonucleotides (Figure 2). EB, a dye for dsDNA, identified the duplex fraction in the G4C4, which had a same migration and staining efficiency as the duplex G4C4R (Figure 2A). BMVC is a carbazole derivative that preferentially stains the quadruplex formed by the human G-rich strand (16). When the samples were pre-stained with BMVC and resolved on gel, an alternative band was observed for the G4C4, which was as efficiently stained as the G4 and G4T4 that are known to form quadruplex. On the contrary, the duplex formed by G4C4R was not stained (Figure 2B). Post-staining of this gel with EB revealed the duplex bands of G4C4 and G4C4R below the quadruplex band of G4C4 (Figure 2C). This staining assay confirmed the observation in the gel electrophoresis that the G4C4 formed quadruplex in the presence of PEG.

The quadruplex formed by G4C4 in PEG solution was further analyzed by CD spectroscopy (Figure 3). In a separate work, we provided evidences by CD, gel electrophoresis, fluorescence spectroscopy of 2-aminopurine substituted oligonucleotides that the core sequence of the G-rich strand of human telomere DNA (G₃T₂A)₃G₃ adopts parallel-stranded conformation in 150 mM K⁺ and 40% (w/v) PEG solution (manuscript submitted), which features a negative peak near 245 nm and a positive peak near 265 nm in CD spectrum as the many reported parallel-stranded quadruplexes (17–26). Here we decomposed G4C4 into three components, G4, C4 and T₅-linker, and recorded their CD spectrum separately. The G4 sequence alone produced a typical spectrum characteristic of parallel quadruplex with a negative peak near 245 and a positive peak near 265 nm. The G4C4 displayed a spectrum that is almost identical to the spectrum overlay of its composing components, G4, C4 and T5-linker (Figure 3A). Moreover, the spectrum of G4C4 resembled that of the G4T4 that forms quadruplex, but differed from that of the G4C4R that forms hairpin duplex (Figure 3B). In the majority of publications, it has been reported that anti-parallel quadruplex is characterized by a negative peak near 260 nm and a positive peak near 295 nm, while parallel quadruplex displays a negative peak near 240 nm followed by a positive peak near 265 nm (17,27,28). The CD analysis (Figure 3) suggests that the G4C4 adopted parallel quadruplex structure in the G-rich region as the G4. However, this conclusion regarding the folding topology may not be definitive because exceptions have been reported where anti-parallel quadruplex formed by some sequences also showed positive peak near 260 nm (29–31), typical of parallel quadruplex. Figure 3.

CD spectra of G4C4 and reference oligonucleotides in solution containing 150 mM K⁺ and 40% (w/v) PEG 200. (A) CD spectra of G4C4 and its composing components. The spectrum of G4 + C4 + Linker gives the overlay of individual spectrum of G4, C4 and T₅-Linker. (B) CD spectra of G4C4, G4T4 and G4C4R. The later two oligonucleotides form quadruplex and hairpin duplex, respectively.

To examine how molecular crowding could affect the quadruplex and duplex formation separately, we studied the thermal stability of the two structures formed by G4T4 and G4C4R, respectively, in the absence and presence of PEG by thermal melting. Telomere quadruplex in K⁺ solution without PEG is characterized by a positive peak at 295 nm in its CD spectrum (9), therefore the denaturation of quadruplex of G4T4 was monitored by CD at 295 and 265 nm, respectively, in the absence or presence of PEG. The denaturation of duplex is characterized by hyperchromicity at 260 nm (33) and was monitored by UV absorbance. The results in Figure 5A shows that the quadruplex formed by G4T4 was melted at 61.9°C in the absence of PEG, but was much more stable in the presence of PEG. On the other hand, PEG destabilized the duplex formed by G4C4R, resulting in a decrease of 14.5°C in melting temperature (Figure 5B). The effects of PEG on the stability of both structures is in agreement with the data reported in a recent work (12). Figure 5.

Thermal melting profiles of (A) G4T4 and (B) G4C4R in the absence and presence of 40% (w/v) PEG 200 in 150 mM K⁺ solution. (A) CD-melting profile recorded at 265 nm in the presence or 295 nm in the absence of PEG. (B) UV melting recorded at 260 nm in the presence or absence of PEG. The numbers by the melting curves indicate the melting temperature T_m (°C) obtained in each measurement.

To better simulate the telomere DNA in chromosomes, we constructed by PCR a 1.2 kb blunt-ended double-stranded DNA (Telo-dsDNA) carrying four consecutive copies of TTAGGG/CCCTAA at one end. Normal blunt-ended double-stranded DNA (Ctrl-dsDNA) containing no telomeric repeats was used as reference. The end structures of the dsDNAs were examined by AFM. Without PEG treatment, the two dsDNAs showed a typical shape of dsDNA edge at both ends (Figure 6A and B). In the presence of PEG, the formation of quadruplex could be recognized at one end of the Telo-dsDNAs as spherical dot (Figure 6C) with a height of 2.03 ± 0.48 nm which is similar to the reported values for quadruplex under AFM (34–36). The height of 0.65 ± 0.13 nm of the duplex region also matches those reported values for dsDNA (34,37). Such structure was observed in the majority of the Telo-dsDNAs, but not seen in the PEG-treated Ctrl-dsDNA (Figure 6D). Figure 6.

Atomic force microscopic images showing quadruplex formed in 1.2 kb double-stranded DNA in solution containing 150 mM K⁺ and 40% (w/v) PEG 200. Arrowheads indicate quadruplexes. Insert at the right-bottom corner of each panel is the magnified rescan image of the DNA marked by oval. Bars = 1 μm. In (C) the average height of the quadruplexes is 2.03 ± 0.48 nm and that of duplex is 0.65 ± 0.13 nm.