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Nucleic Acids ResNucleic Acids ResnarNucleic Acids ResearchNucleic Acids Research0305-10481362-4962Oxford University Press 1739563910.1093/nar/gkm098Structural BiologyQuadruplex ligands may act as molecular chaperones for tetramolecular quadruplex formation Cian Anne De Mergny Jean-Louis *Laboratoire de Biophysique, Muséum National d′Histoire Naturelle USM 503, INSERM UR 565, CNRS UMR 5153, 43 rue Cuvier, 75231 Paris cedex 05, France*To whom correspondence should be addressed +33 1 40 79 36 89+33 1 40 79 37 05mergny@mnhn.fr4 2007 29 3 2007 29 3 2007 35 8 2483 2493 22 12 2006 3 2 2007 3 2 2007 © 2007 The Author(s)2007This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.G-quadruplexes are a family of four-stranded DNA structures, stabilized by G-quartets, that form in the presence of monovalent cations. Efforts are currently being made to identify ligands that selectively bind to G-quadruplex motifs as these compounds may interfere with the telomere structure, telomere elongation/replication and proliferation of cancer cells. The kinetics of quadruplex–ligands interactions are poorly understood: it is not clear whether quadruplex ligands lock into the preformed structure (i.e. increase the lifetime of the structure by lowering the dissociation constant, koff) or whether ligands actively promote the formation of the complex and act as quadruplex chaperones by increasing the association constant, kon. We studied the effect of a selective quadruplex ligand, a bisquinolinium pyridine dicarboxamide compound called 360A, to distinguish these two possibilities. We demonstrated that, in addition to binding to and locking into preformed quadruplexes, this molecule acted as a chaperone for tetramolecular complexes by acting on kon. This observation has implications for in vitro and in vivo applications of quadruplexes and should be taken into account when evaluating the cellular responses to these agents.
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INTRODUCTION
G-quadruplexes are a family of secondary nucleic acids structures stabilized by G-quartets that form in the presence of monovalent cations (1,2). The level of interest in these structures has recently increased due to hypotheses that G-quadruplex structures play roles in key biological processes (3–9) and recent demonstrations of their existence in vivo (10–12). G-quadruplexes may have applications in areas ranging from supramolecular chemistry and bio- and nanotechnology (13–15) to medicinal chemistry [for recent reviews: (2,16,17)]. Efforts have been made to identify ligands that selectively bind to G-quadruplex motifs, as they may interfere with telomere structure, elongation and replication, oncogene expression and proliferation of cancer cells (18,19). These compounds may be of natural origin [such as cryptolepine (20), berberine (21) and telomestatin (22,23)] or synthetic [such as BSU1051 (18), RHPS4 (24), TMPyP4 (25), pyridine or phenanthroline dicarboxamides (26,27), triazines (28), PIPER (29,30) or bi- and trisubstituted acridines (31,32)]. It is important to understand the rules that govern the formation of G-quadruplexes and determine the stability and folding kinetics of the structures. Unfortunately, with a few notable exceptions (29), little is known concerning these parameters. Han et al. presented the first example of a small ligand (a perylene derivative, PIPER) that drives the assembly of bimolecular G-quadruplex structures (29). The presence of 10 µM PIPER accelerates the assembly of varied dimeric G-quadruplexes an estimated 100-fold (29). However, the analysis of the results was complicated by the existence of a number of different G-quadruplex structures, including dimeric and tetrameric species. Furthermore, the end product structure and molecularity were different in the presence and in the absence of the ligand, indicating that the molecule favors some conformers over the others and displaces the thermodynamic equilibria.

Parallel-stranded, tetramolecular quadruplexes (Figure 1A) offer a unique opportunity to study kinetic effects for several reasons. First, our understanding of their kinetic properties increased, thanks to the seminal work by Wyatt et al. that described the properties of simple short segments such as T2G4T2 (33). More recently, we (34,35) and others (36) analyzed the kinetics of quadruplex formation with short sequences. From a kinetic point of view, the association reaction strongly depends on strand concentration with an experimentally determined order approaching four (33,34,36); an exception being the four-stranded structures formed by the sugar-modified oligonucleotides known as locked nucleic acids (37). Second, the kinetic inertia of these complexes, rather than being a hindrance, facilitates the study of the uncoupling of the folding and unfolding phenomenon. One may deconvolute the effect of a parameter and determine whether this factor mainly affects association or dissociation—or both. We and others successfully used this property to analyze the effects of temperature, sequence, ionic strength, introduction of a modified backbone or modified quartet on the kinetics of G-quadruplex formation. Third, in contrast to structures adopted by bi- and intramolecular quadruplexes, which are highly polymorphic, tetramolecular complexes are structurally well defined. They are formed by short oligodeoxynucleotides bearing a block of three of more guanines. In this configuration (also called G4-DNA), all strands are parallel and all guanines are in the anti conformation (Figure 1A). Although direct evidence for the formation of tetramolecular structures in vivo is currently lacking, these quadruplexes have been proposed to play a role during meiosis (38,39).
Figure 1. Quadruplexes and quadruplex ligands. (A) Schematic of a quadruplex (left) and tetramolecular G-quadruplex formation/dissociation (right). This scheme simply presents the initial and final states of the reaction studied here, and assumes terminal stacking of the ligand; it does not claim to reflect the pathway of association. Are quadruplex ligands (red ovals) simply able to lock the preformed structure (i.e. increase the lifetime of the structure by lowering its dissociation constant koff), or do ligands actively promote the formation of the complex and act as quadruplex chaperones by increasing its association constant kon? (B) Formula of the quadruplex ligand 360A.



In this report, we unambiguously demonstrate that a well-characterized quadruplex ligand is able to accelerate the association of single strands into quadruplexes. Therefore, this molecule acts as a molecular chaperone for the formation of tetramolecular quadruplexes. This observation has implications for in vitro and in vivo applications of quadruplexes and should be taken into account when interpreting the cellular responses to these agents.

EXPERIMENTAL PROCEDURES
Oligonucleotides and compounds
Oligonucleotides were synthesized by Eurogentec (Seraing, Belgium). Concentrations of all oligodeoxynucleotides were estimated using published sequence-dependent extinction coefficients (40). Compounds 360A, 307A, BRACO19, BSU1051 and 12 459 were kind gifts from P. Mailliet (Sanofi-Aventis, Vitry/Seine, France). These compounds were stored at 1 or 2 mM in DMSO, further dilution being made in ddH20. TMPyP4 and PIPER were purchased from Calbiochem and solubilized in ddH20 and dilute acetic acid, respectively. Telomestatin was a kind gift of Dr K. Shin-ya; its isolation and purification was previously described (22). Telomestatin was stored at 1 mM at −20°C in the dark in 50% DMSO/50% MeOH; fresh dilutions were made for each series of experiments. The formulae of all tested ligands are shown in Supplementary Figure S1; some of their properties are shown in Supplementary Table S1.

Gel electrophoresis
Non-denaturing gel electrophoresis allows separation of single-stranded oligonucleotides from tetramolecular G-quadruplex structures (34,35). The oligonucleotides (5–200 µM final strand concentrations) were heat denatured in a buffer containing 10 mM lithium cacodylate (pH 7.2) and 110 mM KCl (or NaCl for the oligonucleotide T2G5T2). Immediately after denaturation, oligonucleotides were mixed with the compounds (5 × concentrate solutions) to reach the indicated final concentrations, in a volume of 10–20µl, and incubated at 4°C for times varying from 2 min to several days.

For the association of these short oligonucleotides, at concentrations ranging from 50 to 500 µM, UV shadow at 254 nm was a convenient method to quantify with fair precision the amount of single-strand versus G-quadruplex. The use of dyes, like methylene blue, SYBR green I or gold (Molecular Probes) and acridine orange, are often misleading as stainings and quantum yields may vary when bound to single-strands versus G-quadruplexes (not shown). About 1 nmol of oligonucleotide was loaded with 10% final sucrose concentration on a non-denaturing gel containing 20% acrylamide-bisacrylamide (19:1), 20 mM KCl and 1 × TBE. About the same amount (1 nmol) of oligothymidylate markers (dT6, dT12 or dT24) were also loaded on the gel. The TG3T sample incubated without any potassium and heat denatured just before loading was used as a control for single-strand migration. One should note that the migration of the oligothymidylate markers (short 5′dTn oligonucleotides) does not necessarily correspond to single-strands (41): these oligonucleotides were chosen here to provide an internal migration standard, not to identify single-stranded or higher-order structures. The gel was electrophoresed at 4°C in 20 mM KCl, 1 × TBE. Bands were detected by UV-shadow at 254 nm using a fluorescent silica screen (Whatman).

When using the UV-shadow approach to quantitate, one must assume that the extinction coefficients of the oligonucleotide in single-stranded and quadruplex forms are identical. Absorbance spectroscopy confirmed that these two coefficients were indeed very close. Unfortunately, compound 360A also absorbs light in the same wavelength range, and its molar extinction coefficient at 260 nm (40 000 M−1 cm−1) cannot be neglected as compared to TG3T (47 700 M−1 cm−1). 360A binding may lead to an overestimation of the fraction of oligonucleotide in the G-quadruplex structure. Nevertheless, when the same amounts were loaded on a gel, we did not observe huge variations of band intensity from induced G-quadruplex with or without any compound. Moreover, we confirmed and quantified the induction of G-quadruplex by 360A using radioactivity.

For induction of G-quadruplexes at low oligonucleotide concentrations, 20 pmol of TG3T was radiolabeled in the 5′ position using 10 µCi of γ-32P-ATP and 5 units of T4 polynucleotide kinase (New England Biolabs) in a buffer containing 70 mM Tris-HCl (pH 7.6), 10 mM MgCl2 and 5 mM DTT for 40 min at 37°C. The short radiolabeled oligonucleotide was gel-purified using standard protocols and precipitated by 2.5 M ammonium acetate and a large excess of ethanol. Finally, ammonium acetate was removed by lyophilization to avoid a possible effect of NH4+ on the oligonucleotide association kinetics. The purified, labeled 32P-TG3T was then mixed with unlabeled phosphorylated oligonucleotide to reach a final strand concentration of 5–200 µM. The amount of radiolabeled oligonucleotide was negligible compared to the quantities of unlabeled oligonucleotide added. Complex was separated from single-strand using non-denaturing gel electrophoresis (acrylamide:bis, 19:1; 20% in 1 × TBE, 20 mM KCl at 4°C) and the radioactive signal was detected after overnight exposure to a phosphorimager screen (Molecular Dynamics) at −20°C and scanning with a Typhoon apparatus (Amersham Bioscience). Under certain conditions, two retarded bands are observed in the presence of a ligand. These two species correspond to the migration of the tetramolecular quadruplex without ligand, and to the quadruplex + ligand, respectively (the latter being more retarded than the former). This behavior may be found when using sub-saturating amounts of ligand.

Absorbance measurements
Comparison of the absorbance spectra of the oligonucleotide under conditions where it should be totally unfolded (at t = 0) and completely folded allowed us to generate an isothermal differential absorbance spectrum. The shape of this curve is specific for quadruplexes (Supplementary Figure S3) (42). Isothermal and melting experiments were recorded as previously described (34). For isothermal experiments, starting from completely unfolded strands, absorbance was recorded at regular time intervals (150 s) at 245 (or 240), 295 and 488 nm. Oligonucleotide strand concentration was 100 µM for TG3T and 300 µM for PO4-TG3T in a buffer containing 10 mM lithium cacodylate (pH 7.2) and 110 mM KCl. After overnight association, a UV-melting experiment was performed on the same samples with a heating gradient of 0.5°C/min, recording the absorbance at 240, 295 and 490 nm. This melting temperature depends on the heating rate (34,43).

Analysis of quadruplex formation
Kinetic experiments followed by UV-absorbance or gel electrophoresis allowed determination of the fraction of quadruplex formed as a function of time. In the absence of ligand, experimental points were fitted to a kinetic model using Kaleidagraph 3.6.4 according to published procedures (34,35) and assuming an experimental order of four. However, in the presence of non-saturating amounts of G4 ligand, kinetic curves could not be fitted with this model, as a concomitant decrease in free-ligand concentration leads to a more complex profile. Therefore, a comparison of the kon values (in M−3 s−1) in the absence or presence of the ligands was not possible except when the ligand was present in large excess. In that case, the free ligand concentration was considered to be invariant during the time course of the kinetics experiment. However, one may always compare the experimental time required for 50% quadruplex formation (t1/2) under a given set of conditions.

RESULTS
Presentation of the system
All oligonucleotides studied here contain a single short block of three to five guanines and may form a tetramolecular complex. We chose TG3T as a model sequence. Several other sequences, such as T2G5T2 and the human telomeric oligonucleotides TTAG3 and TTAG3T, were also tested. Formation of these quadruplexes is slow, even in potassium (34); therefore, ligands that increase the kinetics of association are relatively easy to identify.

Comparison of various DNA ligands
Formation of a tetrameric quadruplex may be evidenced by non-denaturing gel electrophoresis. In the absence of ligand and potassium, no quadruplex is formed, and a single band is obtained on the gel, corresponding to the migration of a single-stranded species (Figure 2, lane 1). When 110 mM K+ is present, a partial conversion of the single-strand to the quadruplex was observed (Figure 2, lane 2). Increasing the concentration of ethidium bromide, a ligand that has a high affinity for duplexes, but a weak affinity for quadruplexes (44–46) had no effect on the quadruplex:single-strand ratio, even at 100 µM (Figure 2, lanes 15–18). On the other hand, increasing concentrations of a variety of known G-quadruplex ligands had rather diverse effects: BSU1051 (Figure 2, lanes 32–35) and 12 459 (Figure 2, lanes 28–31) had limited (if any) effect on the relative amounts of single-strands over G-quadruplex species; telomestatin (Figure 2, lanes 7–10) and TMPyP4 (Figure 2, lanes 11–14), altered slightly the relative intensities of the bands. BRACO19 (Figure 2, lanes 19–22) induced the formation of a tetramolecular quadruplex, whereas PIPER (lanes 36–39) led to the formation of a higher molecular weight species with very low mobility; however, significant amounts of single-strands were present even at the highest ligand concentration. In contrast, in the presence of the pyridine dicarboxamide derivatives, 360A or 307A, known for their high affinity and selectivity for quadruplexes (26,47,48), only quadruplex was observed (Figure 2, lanes 3–6 and 23–26). The 360A (or 307A)/quadruplex complex was slightly retarded compared to the quadruplex formed in the absence of any ligand (Figure 2, lane 2), and a similar phenomenon was observed for telomestatin (lanes 8–10), BRACO19 (lanes 20–22) and 12459 (lanes 28–31). This slightly altered migration is probably due to the molecular weight/charge contribution of the ligand to the complex, which suggests that the quadruplex–ligand complex is maintained during electrophoresis (this may not be the case for BSU1051, ethidium bromide or TMPyP4). For PIPER, the migration is drastically modified and, although we cannot exclude that this simply results from PIPER binding to the tetramolecular quadruplex, it is more likely that higher molecular weight species are induced in the presence of this compound as already reported (29).
Figure 2. Effects of various compounds on the association of a tetramolecular G-quadruplex from the oligonucleotide TG3T. These molecules are known to bind to G-quadruplexes (360A, telomestatin, TMPyP4, BRACO19, BSU1051, 307A, PIPER and 12 459, formulae shown as Supplementary Figure S1) or duplexes (ethidium bromide (EtBr)). The oligonucleotide TG3T was heat denatured before incubation at 100 µM (strand concentration) in 10 mM lithium cacodylate (pH 7.2), 110 mM KCl buffer (at 4°C for 4.5 h) with or without any compound at the indicated concentrations. Samples (1 nmol of oligonucleotide loaded) were loaded on a 20% non-denaturing acrylamide gel containing 20 mM KCl and 1× TBE, and detection was performed using UV-shadow at 254 nm. Oligonucleotide markers (M) were short 5′dTn sequences. A sample incubated without any potassium and heat denatured just before loading was used as a control for single-strand migration (ss) on the gel (lanes 1), whereas incubation without any compound indicated position of the migration of the tetramolecular G-quadruplex (G4) (lanes 2). Some gel-shifts can be observed depending on the ligand used (G4 + L). G4 + L* stands for the very slow mobility product formed with PIPER.



Kinetics of quadruplex formation in the presence of 360A
In the experiment presented in Figure 3A, solutions of TG3T were incubated for various amounts of time, between 5 min and 9 days, before separation of single-strand from quadruplex on a non-denaturing gel. In the absence of the ligand, a significant but still incomplete conversion into quadruplex was observed after 9 days; half conversion required approximately 1 day at 100 µM strand concentration. In contrast, addition of various concentrations of 360A had a dramatic effect on the association kinetics. In the presence of 50 µM 360A, half conversion required only 5 min, and the single-stranded species completely disappeared after a few days (Figure 3B). The effects of intermediate concentrations, 10 and 25 µM 360A, are shown in Figure 3, panels C and D, respectively. The relative amount of the single-stranded species was determined from a quantitative analysis of the band intensities; the unfolded fraction is plotted versus time in Figure 3E. Starting from a completely unfolded oligonucleotide in each case, the differences between curves at 0, 10, 25, and 100 µM are striking, especially for short incubation times.
Figure 3. Association kinetics of TG3T with various concentrations of 360A ligand. (A-D) Non-denaturing gel electrophoresis was used to separate single strand from quadruplex, and species were detected by UV-shadow at 254 nm. Incubations were carried out in 10 mM lithium cacodylate (pH 7.2), 110 mM KCl at 4°C for the indicated time at a 100 µM strand concentration with or without 360A at the indicated concentration. Time is given in days (d), hours (h), and minutes (′). * no time point at 5' for panel A. (E) Quantification of the single-stranded fraction using ImageQuant software. Black triangles: without compound; blue squares: with 10 µM 360A; green crosses: with 25 µM 360A; red squares: with 50 µM 360A.



Influence of a 5′ phosphate on quadruplex association and dissociation
The single strands and complexes in the gels presented in Figures 2 and 3 were revealed by a relatively insensitive technique, UV-shadowing. In order to study quadruplex formation at lower strand and ligand concentrations, a more sensitive approach was required. We visualized the oligonucleotides on some of the gels by methylene blue staining or by the use of fluorescent dyes such as SYBR or acridine orange. Unfortunately, the former technique is not sensitive enough, whereas the fluorescent dyes have different quantum yields and/or affinity for quadruplex and single-strands, complicating a quantitative analysis (data not shown). For these reasons, we followed quadruplex formation using a 5′-radiolabeled oligonucleotide. Our initial experiments were done by mixing a 5′ phosphate radiolabeled oligonucleotide, mixed with a large excess of unlabeled, non-phosphorylated oligonucleotide. We noticed that the amount of quadruplex formed as determined by the radioactive-labeling method leads to an underestimation of the total quadruplex, indicating that the radiolabeled strand was underrepresented in the complex as a result of the extra 5′ phosphate group (data not shown). In agreement with the results of Uddin et al. (49), it appeared that the presence of one or two extra negative charges per strand affected the stability of the complex and/or association rate of the quadruplex formation. Figure 4 and Supplementary Figure S4 present a comparison of the kinetics of association and dissociation of the G-quadruplexes formed by 5′-OH and 5′-phosphate TG3T oligonucleotide at various salt concentrations. As shown in Figure 4A and B, quadruplex formation was hampered by the presence of the phosphate group. Higher strand concentrations were required to form a quadruplex overnight and, at 110 mM ionic strength, the corresponding association rate constant was 29 times lower for the oligonucleotide bearing a terminal phosphate [kon = 67 × 106 and 2.3 × 106 M−3 s−1 for TG3T and PO4-TG3T, respectively; for a discussion and comparison of these absolute values, see (34,35)]. However, the difference of kon values between the 5′-phosphate and the 5′ OH oligonucleotides was slightly decreasing when working at higher salt concentrations (Supplementary Figure S4B), confirming that an electrostatics phenomenon is taking part in this unfavorable effect. This effect was also confirmed by non-denaturing gel electrophoresis, as higher strand concentrations were required to form similar amounts of quadruplexes for the phosphorylated oligonucleotide compared to the non-phosphorylated (Figure 4C and 4D), as revealed by UV-shadow analysis. For oligonucleotides bearing a 5′ phosphate, this analysis was also performed by radioactivity, using a 32P-radiolabeled oligonucleotide mixed with the 5′phosphate oligonucleotide. A quantitative analysis of the gel experiments (revealed by radioactivity or UV-shadow) is presented in Supplementary Figure S4A. Similar results were obtained when amounts were quantitated using UV-shadow or radioactivity, arguing that the fraction of radiolabeled oligonucleotides involved accurately reflects the total amount of quadruplex formed. The association constants for PO4-TG3T (kon = 2 × 106 and 1.4 × 106 M−3 s−1 calculated using values from UV-shadow and radioactivity, respectively) are in excellent agreement with the absorbance spectroscopy results (kon = 2.3 × 106 M−3 s−1) and confirm that the kinetics of association are negatively affected by the presence of a terminal phosphate. The association rate constant of the 5′-OH oligonucleotide determined using radioactively labeled oligonucleotide was calculated to be 4 × 107 M−3 s−1, which is also in good agreement with the value found by absorbance spectroscopy (considering 30% precision is given on these values). Association constant values are summarized in Table 1.
Table 1. Association constants found

Oligonucleotide sequencea	5′end	360Ac	kond (M−3 s−1)	T1/2e (°C)	Method	Reference	
TGGGT	OH	–	5.2 × 107	48	UV-absorbance	(34)	
TGGGT	OH	–	6.7 × 107	50	UV-absorbance	This study	
TGGGT	OH	–	4.0 × 107	n/a	Gel; UV shadow	This study	
TGGGT	Pb	–	2.3 × 106	49.5	UV-absorbance	This study	
TGGGT	Pb	–	2.0 × 106	n/a	Gel; UV shadow	This study	
TGGGT	Pb	–	1.4 × 106	n/a	Gel; radioactivity	This study	
TGGGT	Pb	50 µM	3.4 × 1012	n/a	Gel; radioactivity	This study	
TGGGT	Pb	100 µM	1.4 × 1013	n/a	Gel; radioactivity	This study	
TTAGGG	OH	–	1.8 × 107	50	UV-absorbance	(34)f	
TTAGGGT	OH	–	2.1 × 106	55	UV-absorbance	(34)f	
TTGGGGGTT	OH	–	6.1 × 107	>90	UV-absorbance	(34)f	
a5′ to 3′ direction. bP for 5′-phosphate group. cConcentration of Quadruplex ligand (360A) added.—: no ligand. din 110 mM KCl (or NaCl for T2G5T2), 10 mM lithium cacodylate pH 7.2 buffer. Values with ±30% accuracy. ein 110 mM KCl, 10 mM lithium cacodylate pH 7.2 buffer, with a temperature gradient of 0.5°C/min. fAlso see Supplementary Figure S6. NA: not applicable (this apparent melting temperature cannot be determined in a gel).


Figure 4. Effect of a terminal phosphate on G-quadruplex association and dissociation. Results obtained with TG3T and PO4-TG3T are shown in the left and right parts of the figure, respectively. All experiments were performed in 10 mM lithium cacodylate (pH 7.2), KCl 110 mM at 4°C. (A and B) Association kinetics of G-quadruplex formation followed by absorbance at 295 nm (black squares) and 245 nm (red triangles) for (A) TG3T and (B) PO4-TG3T at 100 µM and 300 µM strand concentration, respectively; kon values were calculated using the fourth-order model (34,35). (C and D): Association kinetics of G-quadruplex formation followed by non-denaturing gel electrophoresis (20% acrylamide in 1× TBE, 20 mM KCl at 4°C) at the time points indicated for (C) TG3T at 100 µM strand concentration and (D) PO4-5′dTG3T at 200 µM total strand concentration (mixture of 32P-TG3T labeled at the 5′ end by 32P-γ-ATP and non-radioactive PO4-TG3T). Migration of the associated (G4) and dissociated (ss) forms are indicated; species were revealed using UV-shadowing at 254 nm for non-radioactive species or by detection of the radioactive signal. (E and F) Thermal denaturation of preformed (E) [TG3T]4 and (F) [PO4-TG3T]4 G-quadruplexes (strand concentrations as in panels A and B respectively) followed by absorbance at 295 nm (black squares) and 240 nm (red triangles) at a heating gradient of 0.5°C/min.



Although there were minor differences, both methods indicate that the presence of a 5′ terminal phosphate has a negative impact on the kinetics of association of a tetramolecular quadruplex. In contrast, the presence of a phosphate group had a limited effect on the thermal stability of the quadruplex. As shown in Figures 4E and 4F, the mid-point of the thermal melting transition (T1/2) was 50 for the 5′-OH oligonucleotide and 49.5°C for the 5′PO4-TG3T oligonucleotide. Even if, at lower salt concentrations (Supplementary Figure S4C), a more significant 2°C difference was recorded, suggesting again a role of the negative charges in the destabilization of the structure, these results indicate that the presence of a terminal phosphate significantly affects the association kinetics, but has rather little effect on the thermal stability or dissociation kinetics of the quadruplex.

Kinetics of 5′PO4-TG3T quadruplex formation in the presence of 360A
The addition of a terminal phosphate slows, but does not prevent, quadruplex formation. It is, therefore, possible to follow quadruplex formation with oligonucleotides radioactively labeled at the 5′ terminus with 32P and to determine whether a ligand affects association kinetics. In the gel presented in Figure 5A, no significant quadruplex formation was observed after 21 h at 20 µM strand concentration in the absence of a ligand. At this concentration, 50% quadruplex formation (t1/2) would take years, and the reverse reaction would not be negligible. A 10 fold-higher strand concentration leads to partial quadruplex formation, see Figure 4D). Addition of 360A leads to faster association (Figure 5A, right lanes), confirming UV-shadow analysis at higher strand and ligand concentrations. The rate acceleration is quantified in Figure 5B: 0, 22, 42 and 76% quadruplex formation was obtained after 21 h in the presence of 0, 5, 10 and 20 µM 360A, respectively. At the highest ligand concentration tested, t1/2 was 80 min.
Figure 5. Compound 360A promotes formation of tetramolecular G-quadruplex [PO4-TG3T]4 at 20 µM strand concentration. (A) Association kinetics of the G-quadruplex formed by PO4-TG3T at 20 µM total strand concentration (mixture of 32P-TG3T and non-radioactive PO4-TG3T) alone or in presence of 5–20 µM 360A in 10 mM lithium cacodylate (pH 7.2), 110 mM KCl at 4°C, followed by non-denaturing gel electrophoresis (20% in 1× TBE, 20 mM KCl at 4°C) at the time points indicated. Position of migration of the quadruplex (G4+L) and single-stranded (ss) forms are shown. (B) Quantification of the single-stranded fraction (black diamonds: PO4-TG3T alone; dark red triangles: +5 µM 360A; red triangles: +10 µM 360A; and orange triangles: +20 µM 360A). Data cannot be fitted by the classical fourth-order model (34).



Kinetics of quadruplex formation in the presence of a large molar excess of 360A
Quantitative analysis of the previous gel suggested that the presence of a quadruplex ligand lead to an initial increase in the apparent association rate. However, the effects were less pronounced at longer times: the quantitative difference between 4.3 h and 21 h incubations was relatively small (Figure 5B). We reasoned that, as more and more quadruplexes were formed, the ligand, which was not in huge molar excess, would be progressively titrated out. In order to simplify the modeling of ligand action, one must work under experimental conditions for which the free ligand concentration can be considered invariant during the reaction. To approach this condition, we chose to lower the oligonucleotide strand concentration to 5 µM and to increase the ligand concentration to 50 or 100 µM (Figure 6). Assuming a maximum of 2 binding sites per quadruplex (Supplementary Figure S5), complete quadruplex formation would decrease free ligand concentration by no more than 2.5 µM, a negligible amount relative to the ligand concentration. Figure 6A demonstrates that, unsurprisingly, no quadruplex formation was obtained after one week at 5 µM strand concentration in the absence of 360A. In contrast, 50 or 100 µM ligand lead to near-complete quadruplex formation over the same period. Results were quantitated and are presented in Figure 6B. At 100 µM ligand, t1/2 was 140 min. It was possible to fit these curves using the same mathematical model we and others used for tetramolecular quadruplex formation (34). Apparent kon values of 3.4 × 1012 M−3 s−1 and 1.4 × 1013 M−3 s−1 were found for 50 and 100 µM ligand concentrations, respectively. The association rate constant determined in the absence of any ligand for the same 5′ phosphate oligonucleotide at a much higher strand concentration (200 or 300 µM) was 1.4–2.3 × 106 M−3 s−1; at 5 µM, in the absence of ligand, no quadruplex formation was observed. The comparison of these rate constants allows us to conclude that the presence of 50 or 100 µM ligand increases the rate constant 2 or 21 million-fold, respectively. Due to the order of 4 chosen for the fits, this impressive—but somehow misleading—difference corresponds to a 125–275-fold apparent increase in strand concentration.
Figure 6. Induction of the tetramolecular G-quadruplex [PO4-TG3T]4 by a large excess of 360A. (A) Association kinetics of PO4-TG3T incubated at 4°C at a final strand concentration of 5 µM in absence or presence of 360A at 50 and 100 µM; incubations are performed in 10 mM lithium cacodylate (pH 7.2), 110 mM KCl. Products were resolved on a 20% acrylamide gel; electrophoresis was performed at 4°C in buffer containing 1× TBE and 20 mM KCl. (B) Quantification of the fraction of 32P-TG3T single-strand (black squares: without ligand; blue triangles: +360A 50 µM; red squares: +360A 100 µM). The fourth-order model for tetramolecular G-quadruplexes association was applied and kon values calculated for complex formation in the presence of large excess of ligand are given (34).



360A affects the kinetics of other quadruplexes
We analyzed in detail the effect of 360A on a single sequence, TG3T. To prove that the effect of this ligand was not restricted to this peculiar sequence, we analyzed its impact on the association kinetics of three other sequences, T2AG3, T2AG3T and T2G5T2 (Supplementary Figure S6). In all cases, a significant increase in the amount of quadruplex formed was observed in the presence of 360A compared to the same conditions for oligonucleotide alone, as revealed by UV-shadow. This indicates that this ligand was able to increase the kinetics of association of various tetramolecular quadruplexes.

DISCUSSION AND CONCLUSION
The number of identified G4 ligands has grown rapidly over the past few years. A range of molecules recognize the telomeric quadruplex (18,23,24,31,45,50–63). The compound 360A belongs to a family of pyridine dicarboxamide compounds that exhibit high selectivity toward quadruplex DNA relative to other DNA structures (26,47,48). Thanks to the relatively slow kinetics of tetramolecular quadruplex formation and dissociation, it was possible to perform fluorescence titration experiments with the same oligonucleotide in quadruplex or single-stranded form (Supplementary Figure S5). Little or no variation of 360A fluorescence emission was found when an excess of the single-strand was added, whereas a near-complete quenching was obtained in the presence of the tetrameric quadruplex. These results were not unexpected, considering the equilibrium dialysis profile previously found for this molecule (48).

It is a general assumption that small molecules bind to preformed nucleic acid structures. In this article, we demonstrate that 360A may actively induce the formation of a tetramolecular quadruplex, acting as a chaperone for the association of the four strands. Furthermore, this effect was not restricted to a unique sequence; quadruplex formation by several different sequences was accelerated by the compound. The presence of a 3′ terminal guanine or a TpG was not required, as evidenced by data generated on the TTAGGG sequence. During this study, we also demonstrated that introduction of a 5′-terminal phosphate through radioactive labeling affected the kinetics, presumably due to the additional negative charges. The 5′ phosphate group slowed the association rate with little effect on thermal stability (Table 1). In principle, the presence of these extra charges could also influence the binding affinity of the ligand; however, 360A bound to both 5′-OH and 5′-phosphate quadruplexes, and affected favorably the kinetics of both quadruplexes.

Under the conditions of our studies, 360A did not exert a catalytic effect: one 360A molecule accelerates the formation of a single quadruplex complex and remains bound. The ligand remains attached to the newly formed quadruplex, due to its high affinity. This conclusion was reached using a molar quadruplex:ligand ratio above one (Figure 3C). Another interesting bit of information is that, contrary to the case of bimolecular quadruplexes, a single complex is formed; this is supported by the fact that (i) we observed a single band on the gel, (ii) the CD T1/2 values (Supplementary Figure S2B) and (iii) the isothermal difference spectra (Supplementary Figure S3B) are similar. Furthermore, the overall architecture of the quadruplex induced by the presence of the ligand is probably similar to the structure of the quadruplex formed alone, as the CD signatures of both quadruplexes were similar (Supplementary Figure S2A) and the migration of quadruplexes were not very different (with the exception of PIPER, and taking into account the fact that the ligand somewhat retards the complex due to its positive charges and molecular weight) (Supplementary Figure S6).

Of course, the observation that a ligand increases the association rate constant does not imply that this molecule has no effect on the dissociation rate constant. In fact, 360A did affect the apparent melting temperature of the quadruplex (Supplementary Figure S2B), indicating that 360A also leads to a decrease in koff (i.e. an increase in the lifetime and thermal stability of the complex). This is in agreement with other studies demonstrating that a quadruplex ligand affects the dissociation of a quadruplex (64,65). Several questions remain unanswered, and two main questions are the purpose of our ongoing studies: (i) Do these observations hold for different ligands? The gels shown in Figure 2 suggest that other molecules such as a trisubstituted acridine also affect the association process, although to a lesser extent than the bis quinolinium pyridine dicarboxamide derivatives 360A and 307A. We will determine how general this process is and to which parameters it could be related (charge, shape, affinity). (ii) Are other types of quadruplexes also affected, such as bi- and intramolecular complexes? These complexes, which might be more biologically relevant than the parallel-stranded, tetramolecular complexes studied here, will be more complicated to analyze because of their faster and more complicated kinetics of association and dissociation.

SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.

[Supplementary Material]
 ACKNOWLEDGEMENTS
We thank Patrick Mailliet and Eliane Mandine (Sanofi-Aventis, Vitry, France), Jean-François Riou (Université de Reims), Laurent Lacroix, Barbara Saccà, Patrizia Alberti, Anne Bourdoncle, Julien Gros and Samir Amrane (Museum, Paris) for helpful discussions. Telomestatin was a kind gift from Prof. K. Shin-ya (U. Tokyo). This work was supported in part by an ARC grant (n°3365) and an EU FP6 ‘MolCancerMed’ grant (LSHC-CT-2004-502943 to J-L.M.). Funding to pay the Open Access publication charges was provided by INSERM.

Conflict of interest statement. None declared.
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REFERENCES
1 Williamson JR   G-quartet structures in telomeric DNA Annu. Rev. Biophys. Biomol. Struc 1994 23 703 730 
2 Davis JT   G-quartets 40 years later: from 5′-GMP to molecular biology and supramolecular chemistry Angew. Chem. Int. Ed 2004 43 668 698 
3 Henderson E  Hardin CC  Walk SK  Tinoco I Jr  Blackburn EH   Telomeric DNA oligonucleotides form novel intramolecular structures containing guanine-guanine base pairs Cell 1987 51 899 908 3690664 
4 Oka Y  Thomas C.A. Jr   The cohering telomeres of Oxytricha Nucleic Acids Res 1987 15 8877 8898 3120149 
5 Sundquist WI  Klug A   Telomeric DNA dimerizes by formation of guanine tetrads between hairpin loops Nature 1989 342 825 829 2601741 
6 Williamson JR  Raghuraman MK  Cech TR   Monovalent cation induced structure of telomeric DNA: the G-quartet model Cell 1989 59 871 880 2590943 
7 Zahler AM  Williamson JR  Cech TR  Prescott DM   Inhibition of telomerase by G-quartet DNA structures Nature 1991 350 718 720 2023635 
8 Siddiqui-Jain A  Grand CL  Bearss DJ  Hurley LH   Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription Proc. Natl. Acad. Sci. USA 2002 99 11593 11598 12195017 
9 Grand CL  Powell TJ  Nagle RB  Bearss DJ  Tye D  Gleason-Guzman M  Hurley LH   Mutations in the G-quadruplex silencer element and their relationship to c-MYC overexpression, NM23 repression, and therapeutic rescue Proc. Natl. Acad. Sci. USA 2004 101 6140 6145 15079086 
10 Schaffitzel C  Berger I  Postberg J  Hanes J  Lipps HJ  Plückthun A   In vitro generated antibodies specific for telomeric guanine quadruplex DNA react with Stylonychia lemnae macronuclei Proc. Natl. Acad. Sci. USA 2001 98 8572 8577 11438689 
11 Duquette ML  Handa P  Vincent JA  Taylor AF  Maizels N   Intracellular transcription of G-rich DNAs induces formation of G-loops, novel structures containing G4 DNA Genes Dev 2004 18 1618 1629 15231739 
12 Paeschke K  Simonsson T  Postberg J  Rhodes D  Lipps H   Telomere end-binding proteins control the formation of G-quadruplex DNA structures in vivo Nat. Struct. Mol. Biol 2005 12 847 854 16142245 
13 Li JJ  Tan W   A single DNA molecule nanomotor Nano Lett 2002 2 315 318 
14 Alberti P  Mergny JL   DNA duplex-quadruplex exchange as the basis for a nanomolecular machine Proc. Natl. Acad. Sci. USA 2003 100 1569 1573 12574521 
15 Bourdoncle A  Estévez Torres A  Gosse C  Lacroix L  Vekhoff P  Le Saux T  Jullien L  Mergny JL   Quadruplex-based molecular beacons as tunable DNA probes J. Am. Chem. Soc 2006 128 11094 11105 16925427 
16 Maizels N   Dynamic roles for G4 DNA in the biology of eukaryotic cells Nature Struct. Mol. Biol 2006 13 1055 1059 17146462 
17 Oganesian L  Bryan TM   Physiological relevance of telomeric G-quadruplex formation: a potential drug target Bioessays 2007 29 155 165 17226803 
18 Sun D  Thompson B  Cathers BE  Salazar M  Kerwin SM  Trent JO  Jenkins TC  Neidle S  Hurley LH   Inhibition of human telomerase by a G-quadruplex-interactive compound J. Med. Chem 1997 40 2113 2116 9216827 
19 Mergny JL  Hélène C   G-quadruplex DNA: A target for drug design Nature Med 1998 4 1366 1367 9846570 
20 Guittat L  Alberti P  Rosu F  Van Miert S  Thetiot E  Pieters L  Gabelica V  De Pauw E  Ottaviani A    Interactions of cryptolepine and neocryptolepine with unusual DNA structures Biochimie 2003 85 535 547 12763313 
21 Franceschin M  Rossetti L  Dambrosio A  Schirripa S  Bianco A  Ortaggi G  Savino M  Schultes C  Neidle S   Natural and synthetic G-quadruplex interactive berberine derivatives Bioorg. Medicinal Chem. Lett 2006 16 1707 1711 
22 Shin-ya K  Wierzba K  Matsuo K  Ohtani T  Yamada Y  Furihata K  Hayakawa Y  Seto H   Telomestatin, a novel telomerase inhibitor from Streptomyces anulatus J. Am. Chem. Soc 2001 123 1262 1263 11456694 
23 Kim MY  Vankayalapati H  Shin-ya K  Wierzba K  Hurley LH   Telomestatin, a potent telomerase inhibitor that interacts quite specifically with the human telomeric intramolecular G-quadruplex J. Am. Chem. Soc 2002 124 2098 2099 11878947 
24 Gowan S  Heald R  Stevens M  Kelland L   Potent inhibition of telomerase by small-molecule pentacyclic acridines capable of interacting with G-quadruplexes Mol. Pharmacol 2001 60 981 988 11641426 
25 Han FXG  Wheelhouse RT  Hurley LH   Interactions of TMPyP4 and TMPyP2 with quadruplex DNA. Structural basis for the differential effects on telomerase inhibition J. Am. Chem. Soc 1999 121 3561 3570 
26 Pennarun G  Granotier C  Gauthier LR  Gomez D  Hoffschir F  Mandine E  Riou JF  Mergny JL  Mailliet P    Apoptosis related to telomere instability and cell cycle alterations in human glioma cells treated by new highly selective G-quadruplex ligands Oncogene 2005 24 2917 2928 15735722 
27 De Cian A  DeLemos E  Mergny JL  Teulade-Fichou MP  Monchaud D   Highly efficient G-Quadruplex recognition by bisquinolinium compounds J. Am. Chem. Soc 2007 129 1856 1857 17260991 
28 Riou JF  Guittat L  Mailliet P  Laoui A  Petigenet O  Megnin-Chanet F  Hélène C  Mergny JL   Cell senescence and telomere shortening induced by a new series of specific G-quadruplex DNA ligands Proc. Natl. Acad. Sci. USA 2002 99 2672 2677 11854467 
29 Han HY  Cliff CL  Hurley LH   Accelerated assembly of G-quadruplex structures by a small molecule Biochemistry 1999 38 6981 6986 10353809 
30 Kern JT  Kerwin SM   The aggregation and G-quadruplex DNA selectivity of charged 3,4,9,10-perylenetetracarboxylic acid diimides Bioorg. Med. Chem. Lett 2002 12 3395 3398 12419369 
31 Harrison RJ  Cuesta J  Chessari G  Read MA  Basra SK  Reszka AP  Morrell J  Gowan SM  Incles CM    Trisubstituted acridine derivatives as potent and selective telomerase inhibitors J. Med. Chem 2003 46 4463 4476 14521409 
32 Burger AM  Dai FP  Schultes CM  Reszka AP  Moore MJ  Double JA  Neidle S   The G-quadruplex-interactive molecule BRACO-19 inhibits tumor growth, consistent with telomere targeting and interference with telomerase function Cancer Res 2005 65 1489 1496 15735037 
33 Wyatt JR  Davis PW  Freier SM   Kinetics of G-quartet-mediated tetramer formation Biochemistry 1996 35 8002 8008 8672504 
34 Mergny JL  De Cian A  Ghelab A  Saccà B  Lacroix L   Kinetics of tetramolecular quadruplexes Nucleic Acids Res 2005 33 81 94 15642696 
35 Mergny JL  De Cian A  Amrane S  Webba da Silva M   Kinetics of double-chain reversals bridging contiguous quartets in tetramolecular quadruplexes Nucleic Acids Res 2006 34 2386 2397 16682446 
36 Petraccone L  Pagano B  Esposito V  Randazzo A  Piccialli G  Barone G  Mattia CA  Giancola C   Thermodynamics and kinetics of PNA-DNA quadruplex-forming chimeras J. Am. Chem. Soc 2005 127 16215 16223 16287312 
37 Petraccone L  Erra E  Randazzo A  Giancola C   Energetic aspects of locked nucleic acids quadruplex association and dissociation Biopolymers 2006 83 584 594 16944520 
38 Sen D  Gilbert W   Formation of parallel four-stranded complexes by guanine-rich motifs in DNA and its applications for meiosis Nature 1988 334 364 366 3393228 
39 Sen D  Gilbert W   A sodium-potassium switch in the formation of four-stranded G4-DNA Nature 1990 344 410 414 2320109 
40 Cantor CR  Warshaw MM  Shapiro H   Oligonucleotide interactions. 3. Circular dichroism studies of the conformation of deoxyoligonucleotides Biopolymers 1970 9 1059 1077 5449435 
41 Kejnovska I  Kypr J  Vorlickova M   Oligo(dT) is not a correct native PAGE marker for single-stranded DNA Biochem. Biophys. Res. Commun 2007 353 776 779 17196168 
42 Mergny JL  Phan AT  Lacroix L   Following G-quartet formation by UV-spectroscopy FEBS Lett 1998 435 74 78 9755862 
43 Petraccone L  Erra E  Esposito V  Randazzo A  Galeone A  Barone G  Giancola C   Biophysical properties of quadruple helices of modified human telomeric DNA Biopolymers 2005 77 75 85 15614794 
44 Guo Q  Lu M  Marky LA  Kallenbach NR   Interaction of the dye ethidium bromide with DNA containing guanine repeats Biochemistry 1992 31 2451 2455 1547228 
45 Koeppel F  Riou JF  Laoui A  Mailliet P  Arimondo PB  Labit D  Petigenet O  Hélène C  Mergny JL   Ethidium derivatives bind to G-quartets, inhibit telomerase and act as fluorescent probes for quadruplexes Nucleic Acids Res 2001 29 1087 1096 11222758 
46 Rosu F  Pauw ED  Guittat L  Alberti P  Lacroix L  Mailliet P  Riou J-F  Mergny J-L   Selective interaction of ethidium derivatives with quadruplexes Biochemistry 2003 42 10361 10371 12950163 
47 Lemarteleur T  Gomez D  Paterski R  Mandine E  Mailliet P  Riou J-F   Stabilization of the c-myc gene promoter quadruplex by specific ligands inhibitors of telomerase Biochem. Biophys. Res. Com 2004 323 802 808 15381071 
48 Granotier C  Pennarun G  Riou L  Hoffschir F  Gauthier LR  DeCian A  Gomez D  Mandine E  Riou JF    Preferential binding of a G-quadruplex ligand to human chromosome ends Nucl Acid Res 2005 33 4182 4190 
49 Uddin MK  Kato Y  Takagi Y  Mikuma T  Taira K   Phosphorylation at 5′ end of guanosine stretches inhibits dimerization of G-quadruplexes and formation of a G-quadruplex interferes with the enzymatic activities of DNA enzymes Nucleic Acids Res 2004 32 4618 4629 15333694 
50 Wheelhouse RT  Sun D  Han H  Han FX  Hurley LH   Cationic porphyrins as telomerase inhibitors: the interaction of tetra (N-methyl-4-pyridyl) porphyrin with quadruplex DNA J. Am. Chem. Soc 1998 120 3261 3262 
51 Fedoroff OY  Salazar M  Han H  Chemeris VV  Kerwin SM  Hurley LH   NMR-based model of a telomerase inhibiting compound bound to G-quadruplex DNA Biochemistry 1998 37 12367 12374 9730808 
52 Perry PJ  Read MA  Davies RT  Gowan SM  Reszka AP  Wood AA  Kelland LR  Neidle S   2,7-disubstituted amidofluorenone derivatives as inhibitors of human telomerase J. Med. Chem 1999 42 2679 2684 10411488 
53 Harrison RJ  Gowan SM  Kelland LR  Neidle S   Human telomerase inhibition by substituted acridine derivatives Bioorg. Med. Chem. Lett 1999 9 2463 2468 10498189 
54 Neidle S  Harrison RJ  Reszka AP  Read MA   Structure-activity relationships among guanine-quadruplex telomerase inhibitors Pharmacol. Ther 2000 85 133 139 10739868 
55 Caprio V  Guyen B  Opoku-Boahen Y  Mann J  Gowan SM  Kelland LM  Read MA  Neidle S   A novel inhibitor of human telomerase derived from 10H-indolo[3,2-b]quinoline Bioorg. Med. Chem. Lett 2000 10 2063 2066 10999471 
56 Mergny JL  Lacroix L  Teulade-Fichou MP  Hounsou C  Guittat L  Hoarau M  Arimondo PB  Vigneron JP  Lehn JM    Telomerase inhibitors based on quadruplex ligands selected by a fluorescent assay Proc. Natl. Acad. Sci. USA 2001 98 3062 3067 11248032 
57 Alberti P  Schmidt P  Nguyen CH  Hoarau M  Grierson D  Mergny JL   Benzoindoloquinolines interact with DNA quadruplexes and inhibit telomerase Bioorg. Med. Chem. Lett 2002 12 1071 7074 11909720 
58 Shi DF  Wheelhouse RT  Sun DY  Hurley LH   Quadruplex-interactive agents as telomerase inhibitors: Synthesis of porphyrins and structure-activity relationship for the inhibition of telomerase J. Med. Chem 2001 44 4509 4523 11741471 
59 Heald RA  Modi C  Cookson JC  Hutchinson I  Laughton CA  Gowan SM  Kelland LR  Stevens MFG   Antitumor polycyclic acridines. 8. Synthesis and telomerase-inhibitory activity of methylated pentacyclic acridinium salts J. Med. Chem 2002 45 590 597 11806711 
60 Gowan SM  Harrison JR  Patterson L  Valenti M  Read MA  Neidle S  Kelland LR   A G-quadruplex-interactive potent small-molecule inhibitor of telomerase exhibiting in vitro and in vivo antitumor activity Mol. Pharmacol 2002 61 1154 1162 11961134 
61 Rossetti L  Franceschin M  Bianco A  Ortaggi G  Savino M   Perylene diimides with different side chains are selective in inducing different G-quadruplex DNA structures and in inhibiting telomerase Bioorg. Med. Chem. Lett 2002 12 2527 2533 12182853 
62 Read M  Harrison RJ  Romagnoli B  Tanious FA  Gowan SH  Reszka AP  Wilson WD  Kelland LR  Neidle S   Structure-based design of selective and potent G quadruplex-mediated telomerase inhibitors Proc. Natl. Acad. Sci. USA 2001 98 4844 4849 11309493 
63 Maraval A  Franco S  Vialas C  Pratviel G  Blasco MA  Meunier B   Porphyrin-aminoquinoline conjugates as telomerase inhibitors Org. Biomol. Chem 2003 1 921 927 12929629 
64 Rosu F  Gabelica V  Shin-ya K  DePauw E   Telomestatin induced stabilization of the human telomeric DNA quadruplex monitored by electrospray mass spectrometry Chem. Commun 2003 34 2702 2703 
65 Green JJ  Ladame S  Ying L  Klenerman D  Balasubramanian S   Investigating a quadruplex-ligand interaction by unfolding kinetics J. Am. Chem. Soc 2006 128 9809 9812 16866537

