Nucleic acids targeted to drugs: SELEX against a quadruplex ligand

Biochimie 93 (2011) 1357e1367

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Research paper

Nucleic acids targeted to drugs: SELEX against a quadruplex ligand Amandine Renaud de la Faverie a, b,1, Florian Hamon c,1, Carmelo Di Primo a, b, Eric Largy c, Eric Dausse a, b, Laurence Delaurière a, b, Corinne Landras-Guetta c, Jean-Jacques Toulmé a, b, ***, Marie-Paule Teulade-Fichou c, **, Jean-Louis Mergny a, b, * a

Université de Bordeaux, Laboratoire ARNA, F-33000 Bordeaux, France INSERM, U869, IECB, F-33600 Pessac, France c Institut Curie, Section Recherche, CNRS UMR176, Centre Universitaire Paris XI, Bât. 110, F-91405 Orsay, France b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 April 2011 Accepted 24 May 2011 Available online 31 May 2011

A number of small molecules demonstrate selective recognition of G-quadruplexes and are able to stabilize their formation. In this work, we performed the synthesis of two biotin-tagged G4 ligands and analyzed their interactions with DNA by two complementary techniques, FRET and FID. The compound that exhibited the best characteristics (a biotin pyridocarboxamide derivative with high stabilization of an intramolecular quadruplex and excellent duplexequadruplex specificity) was used as bait for in vitro selection (SELEX). Among 80 DNA aptamer sequences selected, only a small minority (5/80) exhibited G4-prone motifs. Binding of consensus candidates was confirmed by SPR. These results indicate that G4 ligands that appear highly specific when comparing affinities or stabilization for one quadruplex against one duplex, do not only bind quadruplex sequences but may also recognize other nucleic motifs as well. This observation may be relevant when whole genome or transcriptome analysis of binding sites is seeked for, as unexpected binding sites may also be present. Ó 2011 Elsevier Masson SAS. All rights reserved.

Keywords: Quadruplexes Unusual nucleic acids structure Aptamer SELEX SPR DrugeDNA interactions

1. Introduction Quadruplex ligands bind to a family of DNA secondary structures called G-quadruplexes, which result from the stacking of several G-quartets (typically 2e5), each quartet being composed of four coplanar guanines [1,2]. The level of interest in these structures has increased due to the recent demonstration that G-quadruplex structures play roles in key biological processes [3e6]. Quadruplexes have now been studied in organisms from Escherichia coli to humans. It is estimated that over 376,000 G-quadruplexes are present in the human genome [7,8]. Small molecules that stabilize G-quadruplex structures often exhibit interesting biological activities such as antiproliferative properties. These ligands may interfere with telomere conformation and telomere elongation, opening new possibilities for cancer

* Corresponding author. INSERM, U869, IECB, 33600 Pessac Cedex, France. Tel.: þ33 (0) 540 003 034; fax: þ33 (0) 557 571 015. ** Corresponding author. CNRS UMR 176 ; Bat. 110, Institut Curie, Orsay, France. *** Corresponding author. Université de Bordeaux, Laboratoire ARNA, F-33000 Bordeaux, France. E-mail addresses: [email protected] (J.-J. Toulmé), mp.teulade-fichou@ curie.u-psud.fr (M.-P. Teulade-Fichou), [email protected] (J.-L. Mergny). 1 These authors contributed equally. 0300-9084/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2011.05.022

treatment [9]. Since their initial discovery in 1997 [10], the number of known G-quadruplex ligands has grown rapidly over the past few years [11]. While first generation compounds exhibited a relatively limited duplex vs. quadruplex selectivity, a variety of scaffolds lead to an exquisite selectivity toward G4 structures over duplexes (for ex [12]). On the other hand, a still underachieved objective is to design a ligand exhibiting specific binding for one quadruplex topology over the others. To understand the specificity of recognition, one approach is to quantify binding to a number of independent sequences, either in parallel or in succession. Besides the now classical competition dialysis test [13], we have recently developed two assays that allow testing 20 or more sequences ([14], and Tran et al., published in the same issue). However, if scaling up the process would perhaps allow screening a hundred or so motifs, this number is far from the possible sequence diversity of G4-forming sequences e and even less for random sequence motifs. To obtain a more general picture of the sequence preference of these compounds, we reasoned that in vitro evolution of nucleic acid sequences (the so called SELEX method) could provide interesting information. Indeed up to 1014 different sequences (i.e., structures) can be screened at once through SELEX for their specific binding property to a given target. Aptamers have been raised against a wide range of molecules, including nucleic acid hairpins [23]. By starting from a random pool

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of sequences (in here, a random window of 30 nucleotides was chosen), one may hope to select the motifs that bind more tightly to a G4 ligand. Such selection would be facilitated by working with a biotin-labeled G4 ligand, as [15]. In this work, we selected two unrelated G4-binding scaffolds i.e., pyridodicarboxamide and a copper terpyridine, prepared their biotin derivatives and analyzed their quadruplex stabilization and selectivity via FRET melting and FID experiments. The most promising compound, PDC-biotin, was then used as a bait to fish DNA ligands via the SELEX approach. A number of candidate sequences were identified, and binding to the ligand (with or without biotin) was confirmed by SPR.

concentrated to dryness under vacuum. The residue was taken back in 50 ml of CH2Cl2 and 50 ml of H2O. The white precipitate was filtered, washed with CH2Cl2 and H2O (3x each). This purification afforded biotin N-hydrosuccinimidyl ester 1 (2.10 g, 6.15 mmol, 75% yield) as a white powder (Scheme 3). m.p.: 203e204  C; 1H NMR (300 MHz, CDCl3 þ 1 drop DMSOd6): d ¼ 6.43 (s, 1H), 6.37 (s, 1H), 4.33e4.27 (m, 1H), 4.17e4.13 (m, 1H), 3.05 (q, J ¼ 11.4 Hz, J ¼ 6.9 Hz, 1H), 2.86e2.79 (m, 5H), 2.68 (t, J ¼ 7.2 Hz, 2H), 2.58 (d, J ¼ 12.6 Hz, 1H), 1.70e1.59 (m, 3H), 1.56e1.36 (m, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d ¼ 170.3, 169.0, 167.7, 61.0, 59.2, 55.3, 30.0, 27.9, 27.6, 25.5, 24.3 ppm; LRMS (ESI-MS): 364.1 [M þ Na]þ.

2. Material and methods

2.1.2. Tert-butyl-4,7,10-trioxa-1,13-tridecanamine carbamate 2 In a 250 mL round-bottomed flask, 4,7,10-trioxa-1,13tridecanamine (4.00 ml, 18.34 mmol, 1.0 eq.) was dissolved in ethanol (100 ml) to afford a colorless solution. Tert-butyl phenyl carbonate (3.39 ml, 18.34 mmol, 1.0 eq.) was added and the mixture was refluxed for 20 h. Reaction mixture was concentrated under vacuum and dissolved in H2O (150 ml). The solution was slightly acidified (pH w 4e5) with 1 M HCl and extracted with CH2Cl2 (3  150 ml). The aqueous phase was strongly basified with concentrated aq. NaOH (pHw12e13) and extracted with CH2Cl2 (3  150 ml). The organic phases were combined, dried and concentrated under vacuum. The residue was purified by flash chromatography on silica gel (CH2Cl2/MeOH/NH4OH: 80/17.5/2.5) affording 2 (2.88 g, 9.00 mmol, 49% yield) as an oil. 1 H NMR (300 MHz, CDCl3): d ¼ 5.12 (s, 1H), 3.65e3.55 (m, 12H), 3.24e3.20 (m, 2H), 2.82 (m, J ¼ 6.5 Hz, 2H), 1.75e1.70 (m, 4H), 1.43 (s, 9H) ppm; 13C NMR (75 MHz, CDCl3): d ¼ 156.2, 79.0, 70.7 (2 peaks), 70.3 (2 peaks), 69.6 (2 peaks), 39.7, 38.7, 33.3, 29.7, 28.6 ppm; LRMS (ESI-MS): 321.3 [M þ H]þ.

2.1. Synthesis description Firstly, the N-hydrosuccinimide ester of biotin 1 was prepared in presence of coupling agent (EDCi and DMAP) in DMF and then precipitated from a dichloromethaneewater mixture (1:1) with a 75% yield. The monoprotection of 4,7,10-trioxa-1,1,3tridecanamine was achieved in presence of tert-butyl phenyl carbonate in ethanol under reflux. After workup, 2 was isolated with 49% yield. 1 and 2 were efficiently coupled in DMF and the BOC-protecting group was removed in acidic conditions affording compound 3 with a 92% yield over the 2 steps (Scheme 1). On the other hand, 4-chloropyridine-2,6-dicarboxylic acid 4 was obtained with a 75% yield from chelidamic acid by reaction in phenylphosphonic acid followed by a slow hydrolysis. 4 was coupled with quinolin-3-amine in DMF in presence of EDCi and HOBt to afford PDC-Cl 5 with a 78% yield by precipitation from the reaction mixture. The nucleophilic substitution between 3 and 5 in DMSO afforded 6 with 63% yield. Methylation with iodomethane in DMF, afforded PDC-biotin derivative with 87% yield (Scheme 2). The original Cu-ttpy-biotin was prepared in two steps starting from bromethyl derivative Br-ttpy [16]. First, nucleophilic substitution of Br-ttpy by 3 in presence of triethylamine in DMF, afforded ttpy-biotin with 19% yield. Then, ttpy-biotin previously dissolved in dichloromethane, was deposited in a vial and a solution of copper nitrate in acetonitrile was carefully layered [16]. A slow precipitation at the interface provided Cu-ttpy-biotin, which was recovered by filtration with 50% yield (Scheme 3). 2.1.1. Biotin N-hydrosuccinimidyl ester 1 In a 100 mL round-bottomed flask, biotin (2.00 g, 8.19 mmol, 1.0 eq.) and N-hydroxysuccinimide (1.04 g, 9.00 mmol, 1.1 eq.) were dissolved in hot DMF (30 ml) affording a colorless solution. DMAP (5 mg) and EDCi (1.73 g, 9.00 mmol, 1.1 eq.) were added and solution was stirred overnight at RT. Reaction mixture was

2.1.3. N-biotin-4,7,10-trioxa-1,13-tridecanamine 3 In a 100 ml flask, 1 (461 mg, 1.35 mmol, 1.0 eq.) and 2 (433 mg, 1.35 mmol, 1 eq.) were dissolved in DMF (15 ml) and stirred at RT for 16 h. The mixture was concentrated under vacuum. The residue was dissolved in HCl/MeOH ([HCl] 3 M) and stirred at RT for 16 h. After basification with NaOH (3 M), mixture was slowly concentrated under vacuum. The residue was purified by flash chromatography on silica gel (CH2Cl2/MeOH/NH4OH (25%): 80/17.5/2.5) affording 3 (555 mg, 1.24 mmol, 92% yield) as a colorless oil, that solidifies at standing. 1 H NMR (300 MHz, CDCl3): d ¼ 7.01 (s, 1H), 6.64 (s, 1H), 5.84 (s, 1H), 4.50 (m, 1H), 4.31 (m, 1H), 3.64e3.56 (m, 12H), 3.33 (m, 2H), 3.14 (m, 1H), 2.92e2.72 (m, 4H), 2.52 (d, J ¼ 12.6 Hz, 2H), 2.21 (t, J ¼ 6.5 Hz, 2H), 1.78e1.67 (m, 8H), 1.44 (m, 2H) ppm; 13C NMR (75 MHz, CDCl3): d ¼ 173.5, 163.9, 70.5, 70.3, 70.0, 69.8, 69.7, 69.5, 62.0, 60.1, 55.9, 39.5, 37.2, 35.7, 31.2, 28.8, 28.1, 25.6 ppm; LRMS (ESI-MS): 447.2 [M þ H]þ.

Scheme 1. a) N-hydrosuccinimide, EDC, DMAP, DMF, RT, 16 h, 75%; b) tert-butyl-phenyl-carbonate, ethanol, reflux, 20 h, 49%; c) DMF, RT, 16 h; d) HCl, MeOH, RT, 16 h, 92% over 2 steps.

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Scheme 2. a) Phenylphosphonic dichloride, 120  C, 2 h; b) H2O, RT, 1 h, 75% over 2 steps; c) quinolin-3-amine, EDCi, HOBt, CH2Cl2/DMF, RT, 16 h, 78%; d) 3, NEt3, DMSO, 90  C, 16 h, 63%; e) CH3I, DMF, 40  C, 16 h, 87%.

2.1.4. 4-chloropyridine-2,6-dicarboxylic acid 4 In a 50 mL round-bottomed flask, phenylphosphonic dichloride (3.06 ml, 21.8 mmol, 4.0 eq.) was added to 4-oxo-1,4dihydropyridine-2,6-dicarboxylic acid (1.0 g, 5.46 mmol, 1.0 eq.) and the reaction mixture was heated at 120  C for 2 h under an inert atmosphere. After cooling, 5 ml H2O were added dropwise. The mixture was stirred vigorously for 1 h and concentrated to dryness under vacuum. The residue was suspended in water and filtered, washed with water (2x), CH2Cl2 (2x) and Et2O (3x) affording 4 (831 mg, 4.08 mmol, 75% yield).

m.p.: 211e212  C; 1H NMR (300 MHz, DMSO-d6): d ¼ 8.23 (s, 2H) ppm; 13C NMR (75 MHz, DMSO-d6): d ¼ 164.6, 150.0, 145.3, 127.3; LRMS (ESI-MS): 224.1 [M þ Na]þ. 2.1.5. PDC-Cl 5 In a 100 ml flask, 4-chloropyridine-2,6-dicarboxylic acid 4 (275 mg, 1.351 mmol, 1.0 eq.) and quinolin-3-amine (487 mg, 3.38 mmol, 2.5 eq.) were suspended in CH2Cl2/DMF. HOBt (41.3 mg, 0.338 mmol, 0.25 eq.) and EDCi (647 mg, 3.38 mmol, 2.5 eq.) were added successively and the mixture was stirred for 16 h at RT. The

Scheme 3. a) 3, NEt3, DMF, 60  C, 4d, 19%; b) CuNO3, CH3CN, CH2Cl2, 4  C, 24 h, 50%.

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yellow mixture was homogeneously turning pink with formation of a precipitate. The reaction mixture was filtered and solid was carefully washed with H2O (3x) and CH2Cl2 (3x). The solid was dried under vacuum affording 5 (480 mg, 1.06 mmol, 78% yield) as a pale yellow powder. m.p.: >230 C; 1H NMR (300 MHz, DMSO-d6): d ¼ 11.48 (s, 2H), 9.37 (s, 2H), 8.98 (s, 2H), 8.46 (s, 2H), 8.07e8.04 (m, 4H), 7.74 (t, J ¼ 7 Hz, 2H), 7.65 (t, J ¼ 7 Hz, 2H) ppm; LRMS (ESI-MS): 452.1 [M  H]. 2.1.6. PDC-PEG-biotin 6 In a 50 ml flask, 5 (50 mg, 0.11 mmol, 1.0 eq.), 3 (98 mg, 0.022 mmol, 2.0 eq.) and triethylamine (0.15 ml, 0.11 mmol, 1.0 eq.) were dissolved in anhydrous DMSO (1.5 ml) and reaction mixture was stirred overnight at 90  C under argon atmosphere. After the addition of H2O, a precipitate appeared which was filtered off. The solid was suspended in a mixture of absolute EtOH and Et2O which was evaporated to dryness. The resulted product was purified by chromatography on neutral alumina using CH2Cl2/EtOH (98/2) as mobile phase affording 6 (60 mg, 0.069 mmol, 63% yield) as a white powder. m.p.: 225e226  C; 1H NMR (300 MHz, CDCl3): d ¼ 11.29 (s, 2H), 9.36 (s, 2H), 8.96 (s, 2H), 8.04 (d, J ¼ 8.1 Hz, 4H), 7.77e7.48 (m, 8H), 6.42 (s, 1H), 6.36 (s, 1H), 4.28 (m, 1H), 4.10 (m, 1H), 3.58e3.46 (m, 10H), 3.41e3.34 (m, 2H), 3.05 (m, 3H), 2.79 (dd, J ¼ 12.3 Hz, J ¼ 4.8 Hz, 1H), 2.55 (d, J ¼ 12.9 Hz, 1H), 2.03 (t, J ¼ 7.2 Hz, 2H), 1.85 (t, J ¼ 6 Hz, 2H), 1.64e1.42 (m, 6H), 1.28 (m, 2H) ppm; 13C NMR (75 MHz, CDCl3): d ¼ 171.8, 163.1, 162.7, 156.3, 146.1, 144.2, 131.9, 128.6, 128.3, 127.9, 127.7, 127.1, 124.1, 69.8, 69.6, 69.5, 68.1, 67.6, 61.0, 59.1, 55.4, 35.7, 35.2, 29.4, 28.5, 28.2, 28.0, 25.3 ppm; LRMS (ESIMS): 864.6 [M þ H]þ.

(t, J ¼ 6.0Hz, 2H), 1.78e1.67 (m, 8H), 1.44 (m, 2H) ppm; LRMS (ESIMS): 768.5 [M þ H]þ. 2.1.9. Cu-ttpy-biotin In a vial, copper nitrate (24 mg, 0.128 mmol, 3.0 eq.) in acetonitrile (1 ml) was carefully added dropwise over ttpy-biotin 8 (33 mg, 0.043 mmol, 1.0 eq.) in CH2Cl2 (1 ml) to form two immiscible layers, that was then kept at 4  C for 4 days. The green solid which formed at the interface was filtered and carefully washed with acetonitrile, CH2Cl2 and Et2O affording Cu-ttpy-biotin (19 mg, 0.021 mmol, 50% yield) as a green powder. LRMS (ESI-MS): 845.3 [MeNO3 þ OH]þ; HRMS (ESI-MS): m/z calculated for C42H53N8O8SCu: 892.3003; found 892.3022. HPLC: 1 peak at 4.8 min in a multistep gradient H2O (TFA 0.5%)/CH3CN. 2.1.10. Characterization 1 H and 13C spectra were recorded at 25  C on a Bruker Avance 300 using TMS as internal standard. Deuterated CDCl3 and DMSO-d6 were purchased from SDS. The following abbreviations are used: singlet (s), doublet (d) and multiplet (m). Low resolution mass spectrometry (ESI-MS) was recorded on a micromass ZQ 2000 (waters). High resolution mass spectrometry was provided by the I.C.S.N. (Gif/ Yvette, France). Melting points were taken on a Kofler melting point apparatus and are uncorrected. Silica gel chromatography was carried out with Merck silica gel (Si 60, 40e63 mm). Reagents and chemicals were purchased from SigmaeAldrich unless otherwise stated. Solvents were purchased from SDS. Alumina gel chromatography was carried out with Merk aluminiumoxid (70e230 mesh ASTM) 90 aktiv neutral which was deactivated with addition of water (7%) corresponding approximately to an activity grade III. 2.2. Absorbance measurements

2.1.7. PDC-biotin In a 50 ml flask, 6 (30 mg, 0.035 mmol, 1.0 eq.) was dissolved in anhydrous DMF (0.13 ml) and iodomethane (0.4 ml) was added dropwise. The reaction mixture was stirred at 40  C overnight under argon atmosphere. After concentration under vacuum, a precipitate was induced by addition of absolute EtOH. The precipitate was filtered off and dried with Et2O affording PDCbiotin (35 mg, 0.030 mmol, 87% yield) as an orange powder. m.p.: 139e140  C; 1H NMR (300 MHz, CDCl3): d ¼ 11.82 (s, 2H), 10.15 (s, 2H), 9.68 (s, 2H), 8.54 (d, J ¼ 8.1 Hz, 4H), 8.24 (m, 2H), 8.08 (m, 2H), 7.73 (m, 2H), 7.59 (bs, 1H), 6.41 (m, 1H), 4.77 (s, 6H), 4.29 (s, 1H), 4.11 (s, 1H), 3.57e3.35 (m, 17H), 3.05 (m, 3H), 2.78 (m, 1H), 2.01 (s, 2H), 1.86 (m, 2H), 1.66e1.34 (m, 6H), 1.27 (s, 2H) ppm; LRMS (ESI-MS): 446.9 [M  2I]2þ; 1020.7 [M þ H  I]þ; HRMS (ESI-MS): m/z calculated for C47H59N9O7SI: 1020.3303; found 1020.335, HPLC: 1 peak at 14.9 min in a multistep gradient H2O (TFA 0.5%)/CH3CN. 2.1.8. Ttpy-biotin 8 In a 50 ml flask, 40 -(4-bromo-methyl-phenyl)-2,20 :60 ,200 -terpyridine (90 mg, 0.22 mmol, 1.0 eq.), triethylamine (0.315 mL, 2.24 mmol, 0.1 eq.) and 3 (100 mg, 0.22 mmol, 1.0 eq.) in DMF (minimum amount) were heated to 60  C for 4 days. The solution was diluted in CH2Cl2 and washed twice with aq. NH4Cl (2  20 ml). The organic phases were dried over MgSO4, filtrated and evaporated. The residue was purified on silica gel with CH2Cl2/MeOH/ NH4OH (80/17.5/2.5) as eluent affording ttpy-biotin 8 (32.8 mg, 0.043 mmol, 19% yield) as a white powder. 1 H NMR (300 MHz, CDCl3): d ¼ 8.73 (bs, 4H), 8.67 (d, J ¼ 8.1 Hz, 2H), 7.89 (t, J ¼ 7.5 Hz, 4H), 7.57 (d, J ¼ 7.8 Hz, 2H), 7.36 (t, J ¼ 6 Hz, 2H), 6.96 (s, 1H), 6.21 (s, 1H), 5.36 (s, 1H), 4.45 (m, 1H), 4.28 (m, 1H), 4.03 (s, 2H), 3.64e3.53 (m, 12H), 3.32 (m, 2H), 3.11 (m, 1H), 2.92e2.84 (m, 2H), 2.74 (d, J ¼ 12.9 Hz, 1H), 2.20 (t, J ¼ 7.2 Hz, 2H), 1.93

Experiments are performed on a Uvikon XL spectrophotometer. 2.2.1. Thermal difference spectra (TDS) By measuring the difference between the absorbance spectra (between 220 and 340 nm) at high temperature (oligonucleotide is totally unfolded) and the one at low temperature (oligonucleotide is folded) one can obtain a thermal difference spectra (TDS) which provides information on the nature of the folded form [17]. Thermal difference spectrum of G4 has two positive peaks around 249 and 270 nm and one negative at 295 nm. A 1 cm path length quartz cell is used in a reaction volume of 580 mL. Oligonucleotides are prepared as a 4 mM solution in 10 mM lithium cacodylate pH 7.2, 100 mM NaCl or KCl buffer and annealed by heating to 90  C for 2 min, followed by cooling to 20  C. Scans are performed over a wavelength range of 220 (or 240)e295 nm. 2.2.2. Tm determination Absorbance is measured at 240 and 295 nm as previously described [18,19]. 2.2.3. Circular dichroism (CD) CD spectra are recorded on a JASCO-J815 spectropolarimeter as previously described [20] with a scanning speed of 500 nm min1, a response time of 1 s, 1 nm data pitch and 1 nm bandwidth. 2.3. Fluorescence 2.3.1. FRET-melting experiments FRET-melting measurements are performed as described previously [21,22]. Experiments are carried out in 96-well plates on a real time PCR apparatus M3005P Stratagene as follow: 5 min at 25  C, then increase of 1  C every minute until 95  C. Each condition is

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tested in duplicate, in a volume of 25 mL for each sample. F21T is prepared at 0.2 mM, ligands are at 1 mM and competitors at 3 or 10 mM final concentration. All are in a buffer of 10 mM lithium cacodylate pH 7.2, 100 mM NaCl or 10 mM KCl (completed by 90 mM LiCl) then heated at 90  C during 2 min and finally put in ice. 2.3.2. HT-G4-FID (High-throughput quadruplex fluorescent intercalator displacement assay) G4-FID measurements are performed as described previously [45]. TO (thiazole orange) and cacodylic acid were purchased from Aldrich and used without further purification. Stock solutions of TO (2 mM in DMSO), PDC-biotin and Cu-ttpy-biotin (500 mM in DMSO) were used for G4-FID assay. Oligonucleotides purified by reverse phase HPLC were purchased from Eurogentec (Belgium). Sequences: 22AG corresponds to the human telomeric repeat: [50 -AG3(T2AG3)3-30 ]; c-kit1 [50 -G3AG3CGCTG3AG2AG3-30 ] and c-kit2 [50 -G3CG3(CG)2AG3AG4-30 ] are two sequences of the c-kit oncogene promoter. TBA is the Thrombin Binding Aptamer sequence [50 -GGTTGGTGTGGTTGG-30 ]. ds26 is the duplex obtained with the selfcomplementary sequence [50 -CA2TCG2ATCGA2T2CGATC2GAT2G-30 ]. G4-FID measurements were performed on a FLUOstar Omega microplate reader (BMG Labtech) with a 96 wells black quartz microplate (Hellma). A temperature of 25  C is kept constant inside the microplate reader. Every ligandeoligonucleotide combination is tested in duplicate. The microplate is filled with i) Kþ or Naþ-buffer solution (qs for 200 mL) ii) 10 mL of a solution of pre-folded oligonucleotides (5 mM) and TO (10 mM for G4-DNA or 15 mM for ds26) and iii) an extemporaneously prepared 5 mM ligand solution in Kþ or Naþ-buffer (0e100 mL along the line of the microplate, i.e., from column A to column H: 0, 0.125, 0.25, 0.375, 0.5, 0.625, 0.75, 1.0, 1.25, 1.5, 2.0 and 2.5 mM). After 10 min of orbital shaking at 500 rpm, fluorescence is measured using the following experimental parameters; positioning delay: 0.5 s, 20 flashes per well, emission/ excitation (bandwidth) filters 485/520. The percentage of displacement is calculated from the fluorescence intensity (FA), using: percentage of displacement ¼ 100  [(FA/FA0)  100], FA0 being the fluorescence from TO bound to DNA without added ligand. The percentage of displacement is then plotted as a function of the concentration of added ligand. The DNA affinity was evaluated by the concentration of ligand required to decrease the fluorescence of TO by 50%, noted DC50, and determined after nonlinear fitting of the displacement curve.

2.4. SELEX In vitro selection was carried out as previously described using a DNA library with a 30 nt random region [23]. First, DNA library is heated at 70  C for 5 min, cooled at 4  C for 2 min and placed at room temperature for 15 min. Then 300 pmol of DNA library (or decreasing amounts in the successive rounds) are mixed with streptavidin beads (50 mg of Streptavidin MagneSphere Paramagnetic Particles from Promega or 500 mg of Dynabeads M-280 from Invitrogen Dynal) previously equilibrated in SE buffer (20 mM HEPES pH 7.4, 140 mM potassium acetate, 20 mM sodium acetate and 3 mM magnesium acetate) for 30 min. 2.4.1. Selection steps Candidates not retained by the beads are mixed with 10 pmol of the ligand (or decreasing amounts in the successive rounds) at room temperature in a final volume of 100 mL of SE buffer for 30 min. The ligand is beforehand mixed with beads for 15 min at room temperature. Unbound DNA is removed and the beads are washed with SE buffer and resuspended in 100 mL water. Candidates are eluted in 80 mL from the target by heating for 1 min at 75  C.

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2.4.2. Amplification, cloning and sequencing Candidates are replicated using polymerase chain reaction for 20 cycles in a final volume of 1 mL, using 100 units of AmpliTaq Gold DNA polymerase (Applied Biosystems) and P3 and P5 primers (50 d-GGGAGACAAGAATAAACGCTCAA and 50 d-GCCTGTTGTGAGC CTCCTGTCGAA respectively). PCR amplification of the recovered candidates and production of single-stranded DNA for the next round were performed according to [23,24]. The 50 extremity of the primer is made heavier with a succession of six C3 links extended with a DNA stretch of 20 nucleotides. During the PCR, this six C3 region cannot be amplified by the Taq DNA polymerase. A PCR product with two strands of unequal length is thus synthesized. Purification and separation of both strands are performed using Nanosep 10K Omega (PALL Life Sciences) devices and polyacrylamide gel, followed by 10 mM Tris HCl (pH 8)/25 mM NaCl/1 mM EDTA extraction and sodium acetate and ethanol precipitation. Cloning is performed following TOPO TA Cloning kit (Invitrogen) with E. coli One shot TOP10 (Invitrogen) and using electroporation. PCR products from positives colonies are send for sequencing to the Centre de Génomique Fonctionnelle, Bordeaux (France). 2.5. Surface plasmon resonance (SPR) binding assays The SPR experiments were carried out on a BiacoreÔ 3000 apparatus. 2.5.1. Evaluation of the SELEX populations Around 250 RU of biotinylated PDC was immobilized on a streptavidin-coated SAD200m sensor chip (XanTec, Dusseldorf) prepared as previously described [25]. Binding experiments were performed at 23  C in SE buffer (used for SELEX) containing 0.005% P20 surfactant. Free biotin was injected at 50 nM after ligand immobilization to saturate free streptavidin and to improve surface stability. The SELEX populations in SE buffer were injected at 500 nM during 3 min across the sensor chip surface, at a 20 mL/min flow rate. The regeneration of the functionalized surface was achieved with a 1 min pulse of 5 M LiCl followed by a 1 min pulse of a solution containing 40% formamide, 3.6 M urea and 30 mM EDTA. The surface was then washed with a 1 min pulse of SE buffer. The sensorgrams were double-referenced using BiaEval 4.1 software (Biacore) to remove instrument noise and buffer contribution to the signal [26]. 2.5.2. Binding assays with consensus candidates Around 30 RU of biotinylated oligonucleotide candidates 30a, 33a and 40a were immobilized on a SA streptavidin-coated sensor chip (Biacore GE Healthcare, Uppsala), prepared according to the manufacturer’s instructions. Binding experiments were performed at 23  C in P buffer (20 mM sodium phosphate pH 7, 140 mM KCl and 20 mM NaCl). Free biotin was injected to saturate free streptavidin. PDC prepared in P buffer was injected at increasing concentrations during 1 min across the sensor surface, at a 20 mL/min flow rate. The baseline level is reached after 3 min or less (not shown) which is inferior to the delay between each injection cycle (injection starts 320 s after the beginning of the cycle). 3. Results 3.1. Design and synthesis of PDC-biotin and Cu-ttpy-biotin 3.1.1. Choice of the G4 recognition scaffold and derivatization strategy A number of independent families of G4 ligands have been synthesized and/or studied by our groups (to name a few:

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dibenzophenanthrolines/quinacridines [27], pyridodicarboxamides [28], terpyridine metallo-organic complexes [16], cryptolepin [29], meridine [30] or ethidium analogs [31]). Among this plethora of compounds, the pyridodicarboxamide (PDC) bisquinolinium and terpyridine metallo-organic complexes appeared as the most attractive scaffolds due to their high affinity and selectivity, along with their rapid and convenient synthetic access [32]. We therefore decided to synthesize the biotin derivative of one representative member of each series. Previous studies on these series indicated that (at least) one position was available for functionalization. A classical design consisted in linking the biotin to the ligand via a flexible linker. The chosen linker was relatively long, to allow biotinestreptavidin interaction in the presence of a DNA bound to the quadruplex ligand. 3.1.2. Synthesis Both syntheses are extensively described in the experimental section. The original PDC-biotin derivative was prepared through a converging synthetic route. The original Cu-ttpy-biotin was prepared in two steps starting from Br-ttpy [16].

3.2. Evaluation of the biotin derivatives An essential condition to use these biotin derivatives for SELEX experiments was to demonstrate that the biotin label did not significantly affect quadruplex affinity and selectivity. To confirm this hypothesis, we analyzed the binding of the two-biotin derivatives and compared them with the non-biotinylated controls (formulas shown in Fig. 1A; FRET results summarized in Fig. 1B). FRET-melting experiments demonstrated that PDC-biotin retained a quadruplex stabilization potential equivalent to PDC (ΔTm ¼ 30.8  C and 34.1  C, at 1 mM ligand, respectively). In

Table 1 Binding properties of PDC and PDC-biotin. Compound

ΔTm (Kþ)a

S (Kþ)b

ΔTm (Naþ)a

S (Naþ)b

PDC PDC-biotin Cu-ttpy Cu-ttpy-biotin

34.1 30.8 15.3 8.6

1.02 1.09 0.78 0.73

24.9 26.2 16 6.9

0.88 0.93 0.78 0.61

a Stabilization, in  C, of the F21T apparent melting temperature, at 1 mM compound concentration in either 10 mM KCl þ 90 mM LiCl (Kþ conditions) or 100 mM NaCl (Naþ). b S is the selectivity index, corresponding to the ratio between ΔTm obtained in presence and absence of ds26, a double-stranded competitor.

contrast, Cu-ttpy-biotin is significantly less active than Cu-ttpy (ΔTm ¼ 8.6  C and 15.3  C, at 1 mM ligand, respectively), demonstrating that the functionalization of this ligand has a somewhat detrimental effect. Increasing concentrations of a non-labeled double-stranded competitor, ds26, were then added to the reaction mixture. As shown in Fig. 1B, ds26 had little or no effect on the quadruplex stabilization induced by PDC and PDC-biotin, even at the highest concentration tested (10 mM; i.e., a 50-fold excess over the quadruplex F21T sequence. S z 1 for both compounds in KCl). In contrast, Cu-ttpy-biotin exhibited somewhat degraded properties as compared to Cu-ttpy, which is already less specific than PDC (S < 1): the presence of ds26 led to a decrease in F21T stabilization by the biotinylated compound, indicating that this double-stranded sequence could act as a bait for the quadruplex ligand. Results are summarized in Table 1. 3.2.1. HT-G4-FID experiments HT-G4-FID experiments confirmed that PDC-biotin retains a high quadruplex affinity over a number of G4 structures as

Fig. 1. Binding properties of the quadruplex ligands (A) Chemical formula of PDC, PDC-biotin, Cu-ttpy and Cu-ttpy-biotin. (B) Quantitative analysis of the FRET-melting competition experiments. The stabilization in potassium is indicated for the four compounds in the absence (black bars) or presence of double-stranded (ds26) at 3 mM (gray bars) or 10 mM (light gray bars).

A. Renaud de la Faverie et al. / Biochimie 93 (2011) 1357e1367 Table 2 G4-FID results.

Table 3 Base content of the selected sequences.a

Compound Cu-ttpy-biotin Cu-ttpy PDC-biotin 360A

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DC50 Ratioa DC50 Ratioa DC50 Ratioa DC50 Ratioa

22AG.K

22AG.Na

TBA

c-kit1

c-kit2

0.48 11 0.30 23 0.41 65 0.39 30

1.19 4 0.34 21 0.84 32 0.56 20

n.d. n.d. n.d. n.d. 0.47 57 0.24 49

n.d. n.d. n.d. n.d. 0.52 51 0.19 62

n.d. n.d. n.d. n.d. 0.42 64 0.18 65

ds26 5.23 7.04

Base

A

T

C

G

Totala

Number %

540 22.5

699 29.1

541 22.6

619 25.8

2399 100

b

26.70

a 80/92 Sequences were retained for analysis. Only the random sequence window is analyzed (Most sequences are 30 nucleotide long, a few are 29 or 31 nt-long).

11.74b

DC50 is the ligand concentration (in mM) required for half displacement of TO. Different structures (five quadruplexes and the ds26 duplex) were tested. a This ratio expresses the selectivity vs. ds26 and is calculated as follows G4 DC50/dsDC50. b Estimation; n.d.: not determined.

compared to 360A (DC50 around 0.5 mM, except for 22AG.Na, see Table 2 and Supplementary Fig. S1) together with a remarkable selectivity vs. duplex DNA (DC50 > 25 mM). Conversely, Cu-ttpybiotin appeared to have a significantly lower affinity and moreover to be less selective than Cu-ttpy [16]. HT-G4-FID confirmed that PDC-biotin was the best candidate for the SELEX study. 3.3. SELEX experiments In order to perform SELEX experiments with PDC-biotin, we synthesized a DNA library containing 30 randomized nucleotides flanked by fixed regions complementary to primers for

amplification (experiment schematized in Supplementary Fig. S2). In order to minimize the capture of undesired candidates, counterselection steps are performed at every SELEX round to eliminate non-specific binders interacting with naked beads. Candidates not retained by the beads are mixed with the biotinylated ligand at room temperature. Unbound DNA is removed and the beads are washed, resuspended in water, then eluted by heating (the full protocol is presented in the experimental section). Surface plasmon resonance experiments were performed to follow the evolution of the populations after each selection round (Fig. 2). Minimal binding was observed for the sequences selected after the first round. In contrast, starting with round 3, the populations bound to the PDC-functionalized sensor chip. The highest signals were observed when populations of rounds 7 and 8 were injected across the surface. A candidate from an in vitro selection against a different target, using the same DNA library, was used as a negative control. As expected, the resulting sensorgram (Fig. 2) shows that the candidate behaves as the populations of the initial rounds. In contrast, when a well-characterized G4-forming

Fig. 2. Analysis by surface plasmon resonance of the SELEX populations binding to PDC. SELEX populations prepared at 500 nM in SE buffer were injected at 20 mL/min during 3 min across a PDC-functionalized surface. The regeneration of the surface was achieved with a 1 min pulse of 5 M LiCl followed by a 1 min pulse of a solution containing 40% formamide, 3.6 M urea and 30 mM EDTA. The first and second arrows indicate the beginning and the end of the injection, respectively.

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Table 4 Quadruplex forming potential of the selected sequence.a G4 potential

Likelyb

Possiblec

Unlikely

Total

Number %

5 6.25

15 18.75

60 75

80 100

a 80/88 Sequences were retained for analysis (the 8 remaining sequences were discarded as they did not pass QC). Only the random sequence window is analyzed (the terminal 50 and 30 motifs used for amplification do not contain G-blocks). b Fits the general consensus for G4 formation (four blocks of 2þ guanines). c The sequence contains 2e3 G-blocks of guanines, potentially allowing intermolecular quadruplex formation.

sequence, 32B3 Kras, was injected a high binding response was observed (Fig. 2). This could suggest that the SELEX populations contain candidates that are able to fold as G4 structures. 3.4. Analysis of the candidates Ninety-two sequences were cloned in E. coli using Topo TA cloning. We isolated sequences following rounds 7 and 10. PCR products from positives colonies were sent for sequencing to the Centre de Génomique Fonctionnelle, Bordeaux, France. Eighty-eight sequences were retrieved (shown in Supplementary Fig. S3); 80 sequences of them were of excellent quality (perfect match for both primer sequences with a central region of 29e31 nucleotides). We then performed a global analysis of base content. Surprisingly, we did not find an overrepresentation of guanines in the initially random region (G representing 25.8% of the bases, i.e., very close to the expected ¼ initial composition; Table 3). Had quadruplex-prone sequences emerged as a dominant motif for the selected sequences, one would have expected an enrichment of this base. We nevertheless analyzed each sequence individually to check for G4-forming potential. Out of the 80 selected sequences, only 5 exhibited a canonical quadruplex-prone motif, i.e., four blocks of at least two guanines, compatible with the formation of an intramolecular G4 structure (note that for one of these sequences, the distance between two G-blocks was larger than the general 7-nt consensus accepted for bioinformatic searches: as we previously demonstrated that longer loops could be tolerated within a G4

structure, we kept that sequence within the “likely” category (Table 4)). Nevertheless, not only G4-likely sequences were relatively rare (5/80 ¼ 6.25%), they were of modest stability, and none of the sequences are capable of forming motifs of high thermal stability, as none of them contained 4 blocks of 3 guanines. Besides these “likely” sequences, 15/80 sequences could be considered as marginally compatible with quadruplex formation, meaning that they contained clusters of 2þ guanines, but not in sufficient number (4þ) to allow intramolecular quadruplex formation. Finally, 60/80 (75%) of the sequences were very unlikely to form quadruplexes. In order to identify common traits between the sequences binding to PDC-biotin, the sequences were then aligned using Clustal [33]. Four major families were identified (Fig. 3); while the top sequences (“A” family) were theoretically compatible with G4 formation, the consensus motifs found for the three other families were clearly not susceptible to form G-quadruplexes. Mfold predictions [34] of some of these sequences suggested that intramolecular WatsoneCrick interactions of intermediate to high stability could be formed with some candidates (ΔG between 6 and 14.75 kcal/mol; examples of Mfold predicted stable secondary motifs provided in supplementary information e note that quadruplex structures are not supported by Mfold). From the alignments presented in Fig. 3 and the Mfold predictions (examples provided in Supplementary Fig. S5), 12 consensus sequences were synthesized; their sequences are shown in Fig. 4, top. Their lengths varied between 30 and 60 nucleotides, as they may encompass the random region (in red) and parts of the 50 and 30 tails used for PCR, when these motifs were likely involved in secondary structures. First, using classical methods used to evidence quadruplex formation (CD and TDS) we confirmed that they were not capable of forming G-quadruplexes, except for 10.13e30 and 7.7e30. We then confirmed that these sequences were capable of binding PDC, using SPR (Fig. 4, bottom) or fluorescence titration (not shown). Unfortunately, while the SPR profiles provided in Fig. 4 unambiguously demonstrate that these motifs interact with the PDC ligand, they did not allow a precise determination of the Kd of the complex. In the range of concentrations assayed (up to 50 mM) no saturation of the binding of PDC to the immobilized oligonucleotides was observed. This suggests either that the PDC ligand, due to its chemical nature, is able to

Fig. 3. Alignment of selected sequences. Full Clustal analysis [33] is provided as Supplementary Fig. S4; this figure illustrates four interesting conserved motifs (labeled AeD) within the random region. Family A corresponds to two related G4-prone sequences (runs of two consecutive guanines are boxed). Identity is indicated: for example, 7.01 corresponds to the first sequence isolated after the 7th selection round. Highly conserved nucleotides are shown in different colors for each motif. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 4. Analysis by surface plasmon resonance of PDC binding to candidates. Top: Sequences chemically synthesized with a biotin at the 50 end and assayed by SPR. When applicable, the corresponding family (AeD) defined in Fig. 3 is indicated on the left. Parts of the oligonucleotide corresponding to the initial random window are shown in red. Length (in nt) is indicated after the clone reference (for ex, 10.12e40 corresponds to a 40 mer sequence taken from clone #12 isolated after round 10). Bottom: Interaction analysis of three candidates with PDC. Biotinylated candidates were immobilized as described in Materials and Methods. PDC prepared in P buffer was injected at different concentrations (5e50 mM) during 1 min across the sensor chip surface. No regeneration step was required between each injection. No binding was observed using a surface functionalized with a TTC control sequence (50 d-TTCTTTCTTTCTTTCTTTCT). (A): 7.7e30 candidate. (B): 7.17e33 candidate. (C): 10.12e40 candidate. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

stack on itself once bound to the DNA motifs or that the Kd of the complex is above 50 mM. 4. Discussion and conclusion The results presented here demonstrate that one can achieve functionalization of a G4 ligand with biotin ([15] and this study) or other labels [32]. Nevertheless, one needs to check if the tag affects the properties of the ligand. As shown here, the biotin label significantly altered the properties of the Cu-ttpy moeity. Similar results were found with the fluorescent thiazole orange label [32], confirming that such modification of a small molecule may alter the properties of the G4-binding core. One nevertheless may conclude that the neutral biotin tag has a lower detrimental effect on selectivity e if any e than the positively charged TO group. Having validated the PDC-biotin ligand, we performed a SELEX experiment to identify DNA sequences that bind to this derivative. It initially came as a surprise (and for some of us, a disappointment!) that so few of the selected motifs were actually G4 prone. Analysis of individual sequences by SPR confirmed that non G4forming sequences directly interact with the G4 ligand. Despite having confirmed that PDC-biotin has a strong affinity for the human telomeric quadruplex motif in vitro, we failed to isolate clones with similar sequences. This illustrates that the SELEX strategy we use is by no mean exhaustive. On the one hand one may propose that there is a counterselection against G4-prone sequences during each amplification step even though fourstranded aptamers have been identified against a number of

proteins or small molecules [35e44]. On the other hand directed in vitro evolution relies on the selection of shapes allowing maximal interactions with the offered target. A successful SELEX will lead to the identification of a folded oligomer that establishes every possible hydrogen bond with donor or acceptor sites, and electrostatic as well as pep interaction with the available sites of the target molecule. Very generally this involves a conformational change of the aptamer upon interaction with its ligand. Indeed some aptamers are wrapped around the target molecule, that is no longer accessible in the case of small molecules like tobramycin. From this view point quadruplexes are probably not the best ligands for small molecules as they exhibit a limited flexibility. Imperfect hairpins with loops and bulges allow for better induced fit and might actually surpass four-stranded structures as far as binding is concerned. Therefore, even if a G4 ligand appears specific when challenged by specific double-stranded or singlestranded sequences (ds26 in the FRET melting assay), this does not necessarily mean that these ligands only bind to G-quadruplexes! If limited strong sites are available in perfect doublestranded DNA it is likely that a large number of competing sites are offered by tertiary RNA structures. Finally, one may argue that the method used to elute candidates from the target (heating at 75  C in water) could not be harsh enough to dissociate aptamereligand complexes of highest stability. Nevertheless, heating at a higher temperature for elution favors the non-specific background (residual candidates directed against the streptavidin and the magnetic beads surface could be eluted). Moreover, after using higher temperature for the elution, we have observed an elution of the streptavidin.

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One way to overcome this problem is to perform the elution by competition with the free target (unbiotinylated target). The limitation to use this protocol is the possibility that the free target at high concentration would inhibit the PCR amplification of the candidates. Other selection experiments will be performed using different elution schemes in order to compare the population of sequences recovered. Site-specific substitutions will be necessary to define precisely the binding pocket and confirm that the bulges and loops predicted by Mfold are relevant for recognition (one should note that Mfold does not currently support quadruplex structures and misses all G4-prone motifs). Finally it will be interesting to i) investigate whether these DNA motifs recognize other quadruplex ligands and ii) determine the outcome of a selection with a RNA library. Acknowledgments We thank members of the Mergny, Toulmé and Teulade-Fichou laboratories for helpful discussions. This work was supported by an ANR grant (G4-TOOLBOX, ANR-Blan-09-355) to J.L.M., M.P.T.F. and J.J.T. A.R.F. is the recipient of an Université Bordeaux Segalen e MESR PhD studentship. J.L.M. acknowledges support from the Région Aquitaine, the Fondation pour la Recherche Médicale (F.R.M.), and INCa and wishes to dedicate this article to the memory of G. Cherruault e a friend reaped by a Katana we still cannot always parry. Appendix. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biochi.2011.05.022. References [1] S. Neidle, S. Balasubramanian, Quadruplex Nucleic Acids. RSC Biomolecular Sciences, Cambridge, 2006, pp. 301. [2] D. Sen, W. Gilbert, Formation of parallel four-stranded complexes by guaninerich motifs in DNA and its applications for meiosis, Nature 334 (1988) 364e366. [3] L.A. Cahoon, H.S. Seifert, An alternative DNA structure is necessary for pilin antigenic variation in Neisseria gonorrhoeae, Science 325 (2009) 764e767. [4] P. Sarkies, C. Reams, L.J. Simpson, J.E. Sale, Epigenetic instability due to defective replication of structured DNA, Molecular Cell 40 (2010) 703e713. [5] M.J. Law, K.M. Lower, H.P. Voon, J.R. Hughes, D. Garrick, V. Viprakasit, M. Mitson, M. De Gobbi, M. Marra, A. Morris, A. Abbott, S.P. Wilder, S. Taylor, G.M. Santos, J. Cross, H. Ayyub, S. Jones, J. Ragoussis, D. Rhodes, I. Dunham, D.R. Higgs, R.J. Gibbons, ATR-X syndrome protein targets tandem repeats and influences allele-specific expression in a size-dependent manner, Cell 143 (2010) 367e378. [6] C. Ribeyre, J. Lopes, J.-B. Boulé, A. Piazza, A. Guédin, V.A. Zakian, J.-L. Mergny, A. Nicolas, The yeast Pif1 helicase prevents genomic instability caused by Gquadruplex-forming CEB1 sequences in vivo, PLoS Genetics 5 (2009) e1000475. [7] A.K. Todd, M. Johnston, S. Neidle, Highly prevalent putative quadruplex sequence motifs in human DNA, Nucleic Acids Research 33 (2005) 2901e2907. [8] J.L. Huppert, S. Balasubramanian, Prevalence of quadruplexes in the human genome, Nucleic Acids Research 33 (2005) 2908e2916. [9] A. De Cian, L. Lacroix, C. Douarre, N. Temime-Smaali, C. Trentesaux, J.F. Riou, J.L. Mergny, Targeting telomeres and telomerase, Biochimie 90 (2008) 131e155. [10] D. Sun, B. Thompson, B.E. Cathers, M. Salazar, S.M. Kerwin, J.O. Trent, T.C. Jenkins, S. Neidle, L.H. Hurley, Inhibition of human telomerase by a G-quadruplex-interactive compound, Journal of Medicinal Chemistry 40 (1997) 2113e2116. [11] D. Monchaud, M.P. Teulade-Fichou, A hitchhiker’s guide to G-quadruplex ligands, Organic & Biomolecular Chemistry 6 (2008) 627e636. [12] I.M. Dixon, F. Lopez, A.M. Tejera, J.P. Esteve, M.A. Blasco, G. Pratviel, B. Meunier, A G-quadruplex ligand with 10000-fold selectivity over duplex DNA, Journal of the American Chemical Society 129 (2007) 1502e1503.

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