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Published in final edited form as: Nat Chem. 2013 Jan 20;5(3):182–186. doi: 10.1038/nchem.1548

Quantitative Visualization of DNA G-quadruplex Structures in Human Cells

Giulia Biffi 1, David Tannahill 1, John McCafferty 2, Shankar Balasubramanian 1,3
PMCID: PMC3622242  EMSID: EMS52796  PMID: 23422559

Abstract

Four-stranded G-quadruplex nucleic acid structures have been of great interest as their high thermodynamic stability under near-physiological conditions suggests that they could form in cells. Here, we report the generation and application of an engineered, structure-specific antibody that was employed to visualize quantitatively DNA G-quadruplex structures in human cells. We explicitly show that G-quadruplex formation in DNA is modulated during cell cycle progression and that endogenous G-quadruplex DNA structures can be stabilized by a small molecule ligand. Together these findings provide substantive evidence for the formation of G-quadruplex structures in the genome of mammalian cells and corroborate the application of stabilizing ligands in a cellular context to target G-quadruplexes and intervene with their function.

Extensive structural and biophysical evidence confirms that certain G-rich DNA (and RNA) sequences can fold into non-canonical secondary structures, called G-quadruplexes. G-quadruplex structures comprise two or more G-tetrads that form when four guanines are held in a planar arrangement through Hoogsteen hydrogen-bonding with additional stabilization provided by a monovalent cation coordinated to the O6-lone pairs of each guanine1. The high thermodynamic stability of G-quadruplexes under near-physiological conditions suggests that these structures would form in genomic DNA in vivo, although this has been the topic of some debate2. DNA G-quadruplex structures have been associated with a number of important aspects of genome function including transcription, recombination, replication3-6. Computational analysis has revealed the prevalence of G-quadruplex motifs in key regulatory regions of the human genome such as promoters, gene bodies and untranslated regions (UTRs)7-10. G-quadruplex motifs are also present at the ends of chromosomes (telomeres), therefore G-quadruplex structures may also be involved in maintaining chromosome stability. Indeed, telomeric DNA G-quadruplexes have been isolated from genomic DNA using small molecule ligands11 and, moreover, the application of such small molecules leads to cell death, through the displacement of protective telomeric protein components12,13.

Accurate replication of DNA G-quadruplex motifs requires their unfolding by helicases, including BLM (Bloom syndrome protein), FANCJ (Fanconi anemia group J protein), REV1 and WRN (Werner’s syndrome protein)14-17, and genome-wide studies show that predicted G-quadruplex motifs are binding sites for the helicases ATRX (alpha thalassemia/mental retardation syndrome X-linked) and PIF118,19. Our recent genome-wide study has also shown that a stabilizing small molecule, pyridostatin (PDS), induces DNA damage at sites enriched in G-quadruplex motifs and that PIF1 is likely to resolve these structures in vivo5. An important breakthrough in the field was the visualization of G-quadruplex structures at the telomeres of ciliate macronuclei in which the millions of telomeres enabled fluorescent imaging with an antibody, and revealed the cell cycle-dependent formation of telomeric G-quadruplexes20-22. Up to now, it has remained an important challenge to visualize G-quadruplex structures in the DNA of mammalian cells.

Herein, we describe an engineered, structure-specific antibody probe that binds with high selectivity and low nanomolar affinity to DNA G-quadruplex structures. The G-quadruplex-specific antibody was then employed to visualize DNA G-quadruplex structures in the genomic DNA of human cells. We have also characterized the relationship between the cell cycle and G-quadruplex formation and have demonstrated that a small molecule G-quadruplex ligand stabilizes these structures in cells.

Results and discussion

To generate a G-quadruplex-specific antibody, we employed phage display with a library of 2.3 × 1010 different single-chain antibody clones23. Through in vitro selection for G-quadruplex structures, the best hit amongst the selected binders, called BG4, was isolated for further studies. Using ELISA (enzyme-linked immunosorbent assay) methods, we showed that BG4 has high affinity for intramolecular and intermolecular DNA G-quadruplexes (Kd 0.5 – 1.6 nM, and 2.0 nM respectively) with no detectable binding to a RNA hairpin, single-stranded or double-stranded DNA (Fig. 1). To evaluate the affinity of BG4 for different structural conformations, we investigated binding to parallel propeller (MYC, KIT1 and KIT2), anti-parallel (SPB1 and TBA), mixed parallel/anti-parallel propeller (hTELO) and intermolecular (intermolec hTELO) G-quadruplex structures (Supplementary Fig. S1). Given the similarities in binding affinities, these results indicate that BG4 is a G-quadruplex structure-specific antibody that does not have a preference for any particular structural conformation. We next characterized BG4 specificity for G-quadruplexes over other nucleic acid structures in competition experiments. Up to 50-fold excess of different competitors were pre-incubated with the BG4 antibody before assessment of binding to the MYC G-quadruplex by ELISA. In no case we observed significant inhibition of binding to the target G-quadruplex by yeast tRNA, double-stranded poly (GC)n or poly (AT)n, sonicated double-stranded salmon sperm DNA, or a RNA hairpin oligonucleotide. Competition was only achieved using a positive control KIT1 G-quadruplex oligonucleotide (Supplementary Fig. S2). Collectively, these experiments robustly support the specificity of the BG4 antibody for G-quadruplex structures.

Figure 1. Structure specificity of the BG4 antibody for G-quadruplex structures.

Figure 1

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Binding curves as determined by ELISA showing that the BG4 antibody has high affinity for intramolecular and intermolecular DNA G-quadruplex structures with negligible binding to a RNA hairpin, double-stranded and single-stranded DNA. BG4 does not show a preference for any particular structural conformation, binding with similar affinity to parallel propeller (KIT1, KIT2 and MYC), anti-parallel (SPB1 and TBA), mixed parallel/anti-parallel propeller (hTELO) and intermolecular (intermolec hTELO) G-quadruplex structures. Dissociation constants (Kd) are indicated. Error bars represent the standard error of the mean calculated from 3 replicates.

BG4 was then used to visualize DNA G-quadruplex structures in human cells. After incubation of fixed cells with BG4, sensitive detection was achieved through an amplified fluorescence signal generated by incubation with a secondary antibody then a tertiary fluorochrome-labelled antibody. All cell lines examined showed punctate nuclear staining (Fig. 2a and Supplementary Figs. S3) not observed in the absence of primary BG4 antibody (Supplementary Fig. S3). The specificity of BG4 for G-quadruplexes was confirmed by loss of signal upon pre-incubation of the antibody with excess pre-folded G-quadruplex oligonucleotides, but without signal loss upon pre-incubation with single-stranded oligonucleotides (Fig. 2b and Supplementary Fig. S3). The G-quadruplex foci also disappeared after DNase treatment (Fig. 2c), but not after RNase treatment (Supplementary Fig. S3). Moreover, the number of BG4 foci increased when cells were first transfected with pre-folded G-quadruplex oligonucleotides, but not when cells were transfected with single-stranded oligonucleotides (Fig. 2d and Supplementary Fig. S3). Taken together, these observations support the targeting and visualization of DNA G-quadruplex structures in human cells by BG4.

Figure 2. Visualization of DNA G-quadruplex structures in nuclei of human cancer cells.

Figure 2

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a, Immunofluorescence showing BG4 foci (red) in U2OS osteosarcoma cell nuclei. b, Loss of BG4 foci in U2OS cells after pre-incubation of the antibody with pre-folded G-quadruplex oligonucleotides. c, Loss of BG4 foci in U2OS cells after DNase I treatment. The dotted lines are the boundary of the nuclei. d, Increase in BG4 foci number after transfection with pre-folded G-quadruplex oligonucleotides. Nuclei are counterstained with DAPI (blue). Scale bars correspond to 20 μm. e, The graph shows the quantification of BG4 foci number per nucleus for a-d. 100-200 nuclei were counted per condition and the standard error of the mean calculated from a set of 3 replicates. These observations support the targeting and visualization of DNA G-quadruplex structures in human cells by the BG4 antibody.

To further corroborate the detection of G-quadruplex structures in the cellular genome, we next assessed their distribution at the level of individual chromosomes (Fig. 3a and Supplementary Fig. S4). To do this, we incubated BG4 with chromosomes prepared from cells treated with colcemid to block mitosis at metaphase, a stage of the cell cycle where individual chromosomes are most easily visualized. Figs. 3a iv and v clearly reveal BG4 localization at chromosomal ends confirming the presence of G-quadruplex structures at human telomeres. Furthermore, discrete BG4 foci were observed dispersed across chromosomes (Figs. 3a i, ii, iii), demonstrating that G-quadruplex structures also form outside the telomeric regions. Interestingly, in some cases, we observed symmetrical staining of sister chromatids (Fig. 3a v), which supports G-quadruplex formation within the same genomic locations in newly replicated DNA. When the number of BG4 foci was quantified in 100 individual well-spread metaphase chromosomes, the majority (58%) of chromosomes had at least one site of BG4 staining and around a third displayed multiple foci (Supplementary Fig. S4). When we scored positive BG4 staining chromosomes for telomeric distribution, we found that the majority (~75%) of BG4 foci were present outside telomeres (Supplementary Fig. S4). Nonetheless, the observation that ~25% of foci were located at telomeres is evidence of site-specific localization of the BG4 antibody to a well-characterised site of G-quadruplex formation.

Figure 3. Localization of G-quadruplex structures in chromosomes.

Figure 3

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a, Immunofluorescence for BG4 on metaphase chromosomes of HeLa cells. Discrete BG4 foci (red) were observed both within interstitial regions (i, ii, iii) and at telomeres (iv, v), a well-characterised site of G-quadruplex formation. Note the symmetrical appearance of foci in some sister chromatids (v), which supports G-quadruplex formation within the same genomic locations in newly replicated DNA. Chromosomes are counterstained with DAPI (blue), scale bars correspond to 2.5 μm. b, Absence of large co-localization of telomeric TRF2 protein (green) and G-quadruplexes (red) in U2OS cells. This suggests that endogenous G-quadruplex structures are largely present outside telomeres. Nuclei are counterstained with DAPI (blue). Scale bar corresponds to 20 μm.

When fixed cells were co-stained for BG4 and TRF2 (telomere repeat-binding factor 2), a protein localized to telomeres, the majority of BG4 foci (82.4%) did not coincide with TRF2 foci (Fig. 3b). This further suggests that endogenous G-quadruplex structures are largely present outside telomeres. It is notable that not all TRF2 foci co-localize with BG4 staining (36.8%) and not all metaphase chromosomes show telomeric BG4 staining. While these observations might indicate a differential propensity of disparate telomeres to form G-quadruplex structures, they may also be accounted by differences in antibody accessibility to the target (e.g. masking by telomere-binding proteins such as components of the protective shelterin complex).

We anticipated that G-quadruplex formation is more likely to occur during DNA replication, since the associated mechanisms necessitate that duplex strands become separated at replication forks, where single-stranded DNA may fold more easily into secondary structures15. To investigate this, we followed the formation of G-quadruplex structures during cell cycle progression. BG4 staining on synchronized cell populations showed lowest levels of G-quadruplex formation at G0/G1, a phase during which no replication occurs and cellular processes are quiescent. At the G1/S checkpoint, a time when cells prepare for entry into S phase, the period when DNA replication occurs, there was a ~ 2.5-fold increase in the number of BG4 foci. During S phase the number of BG4 foci was maximal (~ 4.8-fold more than at G0/G1), which is consistent with the replication-dependent formation of G-quadruplex structures (Figs. 4a, b). To confirm that the increase in G-quadruplex structures observed during S phase was indeed dependent on DNA replication, we blocked cellular DNA synthesis with aphidicolin, a tetracyclic diterpene that specifically inhibits DNA polymerase α. As expected, aphidicolin treatment led to a large, over 2-fold, reduction in the number of BG4 foci (Figs. 4c, d). These experiments demonstrate that G-quadruplex structures are modulated during the cell cycle in a way that is sensitive to whether or not the DNA is being replicated.

Figure 4. Modulation of G-quadruplex structures during cell cycle progression.

Figure 4

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a, BG4 staining in synchronized MCF-7 mammary adenocarcinoma cell populations at the G0/G1 and G1/S boundaries and during S phase. Nuclei are counterstained with DAPI (blue). Scale bars correspond to 20 μm. b, Quantification of BG4 foci number per nucleus for a. 100 nuclei were counted per stage and the standard error of the mean calculated from a set of 3 replicates. c, Over 2-fold reduction in BG4 foci number after inhibition of DNA synthesis by aphidicolin treatment (5 μm2h). d, Quantification of BG4 foci number with or without aphidicolin treatment. 100 nuclei were counted per phase and the standard error of the mean calculated from a set of 3 replicates. These experiments demonstrate that G-quadruplex structures are modulated during the cell cycle and, in particular, support the replication-dependent formation of endogenous DNA G-quadruplexes.

The small molecule ligand pyridostatin has been reported to stabilize G-quadruplex structures and displace bound proteins5,11,13,24. We therefore rationalized that application of PDS to cells would result in the detection of more DNA G-quadruplex targets by the BG4 antibody. Indeed, when cells were treated with PDS, there was a marked increase (~ 2.9-fold) in nuclear staining (Figs. 5a, b) that disappeared after DNase treatment (Supplementary Fig. S5). This increase is not due to a change of BG4 binding affinity for G-quadruplex structures in the presence of PDS, since we noted no effect of PDS on BG4 binding by ELISA (Supplementary Fig. S5). These results indicate that, at pertinent sites, PDS traps G-quadruplex structures to increase the number of BG4 targets available. This confirms earlier work proposing that small molecule ligands interact directly with cellular DNA to stabilize G-quadruplexes that, if not resolved, lead to DNA damage5,13.

Figure 5. Stabilization of endogenous G-quadruplex structures by a small molecule ligand.

Figure 5

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a, Increase in BG4 foci number in U2OS cells after treatment with the G-quadruplex binding ligand pyridostatin (PDS). Nuclei are counterstained with DAPI (blue). Scale bar corresponds to 20 μm. b, Quantification of BG4 foci number per nucleus with or without PDS treatment. 200 nuclei were counted per condition and the standard error of the mean calculated from 3 replicates. After PDS treatment, a marked increase (~ 2.9-fold) in nuclear staining was observed. These results indicate that, at pertinent sites, PDS traps endogenous G-quadruplex structures to increase the number of BG4 targets available.

In summary, we have developed a highly specific DNA G-quadruplex antibody and employed it to visualize G-quadruplex structures in the DNA of human cells. We have demonstrated how the formation of G-quadruplexes is dynamically sensitive to the cell cycle, and have shown by direct visualization that a small molecule ligand traps these structures in cells. An insightful future goal would be to generate a map of the precise locations of G-quadruplex structures at a genome-wide level by sequencing. Our new findings therefore provide an important basis to help underpin recent chemical biological and genetic studies highlighting a range of potentially important biological roles of G-quadruplex structures.

Methods

Phage display selection

A single-chain antibody, BG4, was isolated from the Sanger phage display library (2.3 × 1010 single-chain antibody clones) through selection using a panel of intramolecular G-quadruplex structures. Two rounds of selection in solution were carried out using streptavidin-coated beads (Dynabeads M-280 Streptavidin, Invitrogen) with 1 μM of biotinylated G-quadruplex oligonucleotides for the first round of selection and 100 nM for the second round. The selected binders were then cloned into the pSANG10 expression plasmid for antibody production. Screening of the selected binders was performed by DELFIA (Dissociation-Enhanced Lanthenide Fluorescent Immunoassay) using an anti-FLAG europium-conjugated antibody (Sigma) and the DELFIA reagent (Perkin Elmer). Signal intensity was detected at 615 nm with a PHERAstar microplate reader (BMG, Labtech) using Time-Resolved Fluorescence detection.

Circular dichroism (CD) spectroscopy

For CD spectroscopy, 5 μM DNA G-quadruplex oligonucleotides (listed below) were annealed in 10 mM Tris HCl, pH 7.4, 100 mM KCl by slow cooling from 95 °C to 21 °C. Five scans were performed from 200 to 320 nm using a Jasco J-810 spectropolarimeter with the buffer spectrum subtracted and zero-correction at 320 nm.

Enzyme-linked immunosorbent assay (ELISA)

ELISAs for binding affinity and specificity were performed using standard methods. Briefly, biotinylated oligonucleotides (biomers.net GmbH) were bound to streptavidin-coated plates followed by incubation with BG4 (and pyridostatin for Supplementary Fig. S5), and detection was achieved with an anti-FLAG horseradish peroxidase (HRP)-conjugated antibody (ab1238, Abcam, UK) and TMB (3,3′,5,5′-Tetramethylbenzidine, HRP substrate, Roche). Signal intensity was measured at 450 nm on a PHERAstar microplate reader (BMG Labtech, Germany). Dissociation constants were calculated from binding curves using GraphPad Prism (GraphPad Software, Inc.) and error bars represent standard error means calculated from 3 replicates. G-quadruplex oligonucleotides were annealed in 10 mM Tris HCl, pH 7.4, 100 mM KCl by slow cooling from 95 °C to 21 °C.

hTELO 5′-GG(TTAGGG) 4TTAG-3′
hTELO-dup 5′-GG(TTAGGG) 4TTAG-3′/3′-C(AATCCC) 4AAT-5′
SSDNA 5′-GGCATAGTGCGTGGGCG-3′
MYC 5′-TGAGGGTGGGTAGGGTGGGTAA-3′
KIT1 5′-AGGGAGGGCGCTGGGAGGAGGG-3′
KIT2 5′-CGGGCGGGCGCGAGGGAGGGG-3′
SPB1 5′-GGCGAGGAGGGGCGTGGCCGGC-3′
TBA 5′-GGTTGGTGTGGTTGG-3′
RNA hairpin 5′-CAGUACAGAUCUGUACUG-3′
intermolec hTELO 5′-(TTAGGG) 3-3′
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For competition experiments, before assessment of BG4 binding to the MYC G-quadruplex by ELISA, BG4 was pre-incubated for 1 h at room temperature (21 °C) with 0, 1, 5, 10, 20, 50 equivalents of either KIT1 (G-quadruplex), RNA hairpin, yeast tRNA, double-stranded sonicated salmon sperm DNA, double-stranded poly(GC)n or poly(AT)n.

Cell cultures and immunofluorescence

U2OS (osteosarcoma), HeLa (cervical carcinoma), HT1080 (fibrosarcoma), MCF-7 (mammary adenocarcinoma) and MDA-MB-231 (breast carcinoma) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco), 1% L-glutamine, 10% FBS (fetal bovine serum); MRC-5 (fetal lung fibroblasts) cells in Minimum Essential Medium Eagle (Sigma), 10% FBS and MCF-10A (mammary epithelial) cells in Mammary Epithelial cell Growth Medium (Lonza) with cholera toxin (at 37 °C with 5% CO2. Cells grown on glass coverslips were fixed in 2% paraformaldehyde/PBS or in methanol: acetic acid (3:1) (Supplementary Fig. S3) and permeabilized with 0.1% triton-X100/PBS. After blocking in 2% Marvel™/PBS, immunofluorescence was performed using standard methods with BG4, anti-FLAG (#2368, Cell Signaling Technology) and anti-rabbit Alexa 594-conjugated (A11037, Invitrogen) antibodies. Coverslips were mounted with Prolong Gold/DAPI (4′,6-diamidino-2-phenylindole) (Invitrogen). For TRF2 detection, anti-TRF2 (ab13579, Abcam) and anti-mouse Alexa 488-conjugated (A11029, Invitrogen) antibodies were used. For enzyme treatments, coverslips were incubated after permeabilization with 120 U of Turbo DNase (0.12 U/μl) or 50 μg/ml of RNase A (Ambion) for 1 h at 37 °C. For pre-incubation experiments, BG4 was incubated with 20-fold excess of pre-folded DNA G-quadruplexes or single-stranded DNA oligonucleotides. Oligonucleotide transfections were performed using 200 nM pre-annealed DNA G-quadruplexes or single-stranded DNA oligonucleotides and TransIT™ Oligo Transfection Reagent (Mirus). For pyridostatin treatment, cells were incubated with 10 μM compound for 24 h. For metaphase preparations, cells were treated with colcemid (50 ng/ml) for 2 h before resuspension in 0.075 mM KCl for 30 min at 37 °C. Cells were then fixed in methanol: acetic acid (3:1) before spreading on slides. 100 well-spread metaphase chromosomes were analyzed for the number and localization of BG4 foci. For cell synchronization, MCF-7 cells were incubated for 24 h in serum-free DMEM (G0/G1), grown for 16 h in DMEM, 20% FBS, 200 μM mimosine (G1/S), and for 3 h in DMEM, 10% FBS (S phase). Cell cycle stages were confirmed using a fluorescent activated cell sorter (FACSCalibur, BD Biosciences). To inhibit DNA replication, cells were incubated with 5 μM aphidicolin for 2 h. Digital images were recorded using a DP70 camera (Olympus) on Axioskop 2 plus microscope (Zeiss), and analyzed with Volocity software (Perkin Elmer). 100-200 nuclei were counted per condition and the standard error mean calculated from 3 replicates. Frequency distribution graphs were plotted using GraphPad Prism (GraphPad Software, Inc.).

Supplementary Material

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Acknowledgements

We thank Tony Pope for his kind help with phage display, Wolf Reik, Raphaël Rodriguez and Debbie Sanders for stimulating discussions, and Cancer Research UK for funding.

Footnotes

Competing financial interests

The authors declare no competing financial interests.

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Supplementary Materials

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