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Adaptive and Specific Recognition of Telomeric G‑Quadruplexes via Polyvalency Induced Unstacking of Binding Units Jibin Abraham Punnoose,† Yue Ma,‡ Yuanyuan Li,† Mai Sakuma,‡ Shankar Mandal,† Kazuo Nagasawa,*,‡ and Hanbin Mao*,† †

Department of Chemistry and Biochemistry, Kent State University, Kent, Ohio 44242, United States Department of Biotechnology and Life Science Faculty of Technology, Tokyo University of Agriculture and Technology (TUAT), 2-14-16 Naka-cho, Koganeishi, Tokyo 184-8588, Japan

‡

S Supporting Information *

ABSTRACT: Targeting DNA G-quadruplexes using small-molecule ligands has shown to modulate biological functions mediated by G-quadruplexes inside cells. Given >716 000 Gquadruplex hosting sites in human genome, the specific binding of ligands to quadruplex becomes problematic. Here, we innovated a polyvalency based mechanism to specifically target multiple telomeric G-quadruplexes. We synthesized a tetrameric telomestatin derivative and evaluated its complex polyvalent binding with multiple G-quadruplexes by single-molecule mechanical unfolding in laser tweezers. We found telomestatin tetramer binds to multimeric telomeric G-quadruplexes >40 times stronger than monomeric quadruplexes, which can be ascribed to the polyvalency induced unstacking of binding units (or PIU binding) for G-quadruplexes. While stacking of telomestatin units in the tetramer imparts steric hindrance for the ligand to access stand-alone G-quadruplexes, the stacking disassembles to accommodate the potent polyvalent binding between the tetramer ligand and multimeric G-quadruplexes. We anticipate this adaptive PIU binding offers a generic mechanism to selectively target polymeric biomolecules prevalent inside cells.

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INTRODUCTION In guanine (G) rich DNA regions, such as telomeres and gene promoters, a non-B DNA structure, G-quadruplex, can form inside cells.1,2 A G-quadruplex consists of four DNA strands interconnected by 4 guanine residues via Hoogsteen H-bonds, in a form of G-quartets. These G-quartets can stack upon each other.3 They are further stabilized by monovalent cations, such as K+ and Na+, in the middle of the quartets.4 Since biological significance of G-quadruplex inside cells has been well established,5 efforts have been spent to target these new structures for pharmaceutical exploitations, especially for cancer treatment.6 In a particular example, telomere G-quadruplexes have shown roles in cancer development. Located in the chromosome termini, telomeres protect chromosome from end-to-end fusion, non-homologous end joining (NHEJ), and exonucleolytic degradation.7,8 The length of the telomere in somatic cells keeps decreasing after each cell division until a limit is reached whereupon cells enter apoptosis.9,10 In most cancer cells, the telomere length is maintained by enzymes such as telomerase, which imparts immortality to the cells.11 Inhibition of telomerase therefore becomes a viable approach to reduce proliferation of cancer cells. It has been proven that G-quadruplex structures formed in the G-rich 3′ overhang of the telomere can inhibit telomerase activity.12,13 Therefore, it is conceivable that by stabilizing these G-quadruplexes using small molecule ligands, inhibitory roles of telomere G-quadruplexes on telomerase activity can be strengthened. © 2017 American Chemical Society

Recent analysis has revealed 716 310 potential G-quadruplex forming sites in the human genome, especially in the telomeres and promoter regions.2,14 This provides ample targets for ligand binding. Most small-molecule ligands developed so far use generic interactions, such as π−π stacking and electrostatic attraction, to bind to G-quadruplexes.15−17 These mechanisms lead to promiscuous binding of small molecules to different Gquadruplexes. As nonspecific binding is expected to alter many gene expressions modulated by promoter quadruplexes, side effects of the small-molecule ligands will occur. To serve as effective agents to target specific G-quadruplexes, new binding mechanisms must be explored. Realizing multiple G-quadruplexes units can form in the single-stranded 3′ telomere overhang18,19 whereas only one or a few G-quadruplexes fold in the double-stranded gene promoters, we wish to use polyvalent binding to differentiate telomeric G-quadruplexes from promoter G-quadruplexes. Here, we synthesized a series of telomestatin derivatives, L2H2-6OTD, that contain one to four G-quadruplex binding rings. We evaluated binding affinities of these ligands to either monomeric or polymeric telomere G-quadruplexes. Surprisingly, we found a tetrameric telomestatin ligand binds multiple G-quadruplexes in the full-length 3′ telomeric overhang much better (>40 times) than monomeric quadruplexes from the same (telomere) or different regions such as Bcl-2. Using Received: January 18, 2017 Published: May 15, 2017 7476

DOI: 10.1021/jacs.7b00607 J. Am. Chem. Soc. 2017, 139, 7476−7484

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Figure 1. Monovalent and polyvalent interactions between G-quadruplexes (GQs) and ligands. (A) Polyvalent binding between polymeric ligand and polymeric GQ is expected to have the highest binding affinity. (B) The polyvalent ligand, L2H2-6OTD tetramer 1, was synthesized from protected form of dimer 2 in four steps.

absorbance, fluorescence, and CD signals, we revealed that telomestatin units stack in the tetramer, which hinders its association with monomeric G-quadruplex. In multiple Gquadruplexes, the energy released from the polyvalent binding between the telomestatin rings and quadruplex units overcomes the stacking of telomestatin units, leading to more efficient binding. We named this polyvalency induced unstacking of binding units as PIU (pronounced as “pure”) binding, which represents a new, adaptive mechanism to design ligands specifically targeting polymeric biomacromolecules.

whether or not ligand units or receptor units are tightly linked. As a result, the entropic penalty for the ligand binding to a receptor is lower in polyvalent binding, increasing its binding affinity.20 We rationalized that such a polyvalent binding scheme can be exploited to design ligand that can differentiate monomeric from polymeric G-quadruplexes. While the former G-quadruplexes mostly form in gene promoter regions, the latter readily exists in the 3′ human telomeric overhang which is single-stranded DNA with a consensus sequence of 5′(TTAGGG)n. It is noteworthy that, in some duplex DNA regions such as ILPR and CEB25, repetitive G-rich sequences exist.21,22 However, due to the competitive reannealing process of complementary strands in duplex DNA, formation of multiple G-quadruplexes will not be as facile as that in the 3′ single-stranded telomere overhang. To test this hypothesis, we designed and synthesized multimeric G-quadruplex ligands that are based on a protected form of telomestatin derivative L2H2-6OTD dimer 2 (Figures 1B and S1).23,24 First, N-alkylation of 2 with diiodide, derived from ethylene glycol, in the presence of potassium carbonate,

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RESULTS AND DISCUSSION Polyvalent Binding to Differentiate Monomeric and Polymeric G-Quadruplexes. Polyvalent binding such as antibody−antigen associations and cell−cell interactions is prevalent in nature. In the polyvalent binding, multiple ligands or receptors are closely connected (Figure 1A), which reduces the entropy of the reactants with respect to free ligands or receptors. However, the reduced entropy in the product of the ligand−receptor complex is not significantly different regardless 7477

DOI: 10.1021/jacs.7b00607 J. Am. Chem. Soc. 2017, 139, 7476−7484

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Journal of the American Chemical Society gave 3 in 75% yield. Then, a second N-alkylation was carried out with 2 and 3 in the presence of potassium carbonate to generate a protected form of the L2H2-6OTD dimer, whose Ns (2-nitrobenzensulfonamide) group was deprotected and resulting amino group was protected as Boc (tert-butyloxycarbonyl) to give octa-Boc protected L2H2-6OTD tetramer 4 in 60% yield (2 steps). Finally, all Boc groups were deprotected with TFA (trifluoroacetic acid) in dichloromethane to give L2H2-OTD tetramer 1 quantitatively. Polymeric G-quadruplexes can form in telomeric sequence, 5′-(TTAGGG)nTTA, in which n = 4, 8, 12, 16, and 24 are designated as 4G, 8G, 12G, 16G, and 24G constructs (Table S1), respectively. Previous investigations have indicated that Gquadruplexes in long telomeric sequences exist as beads-on-astring model without forming higher order structures.19,22,25 Due to this simplicity, long telomeric constructs such as the 24G become ideal to investigate polyvalent binding between multimeric G-quadruplexes and ligands. Ensemble average methods, such as thermal melting or spectroscopic measurements, are rather challenging to investigate such complex system due to presence of multitude of species that cannot be easily resolved. Single-molecule technique can identify every population in a solution mixture, which provides superior signal-to-noise ratio to elucidate the nature of polyvalent binding. Binding of Polyvalent Ligand to Telomeric GQuadruplexes. We therefore used single-molecule ligand binding assays26 to evaluate the binding capacity of monomeric or polymeric telomestatin derivatives (Figure 2) to monomeric

or polymeric telomeric G-quadruplexes. First, the telomeric sequence that form either monomeric, trimeric, or hexameric G-quadruplexes ((TTAGGG)4TTA (or 4G), (TTAGGG)12TTA (or 12G), or (TTAGGG)24TTA (or 24G)) was sandwiched between two dsDNA handles, which in turn was attached to two polystyrene beads trapped by the two laser foci. Moving one trap with respect to another then transduced a mechanical force to the folded structure. G-quadruplex structures in the telomeric sequence will unfold when the mechanical force is higher than the stabilization energy that holds G-quadruplex structure. Binding of a ligand to Gquadruplexes enhances the mechanical stability of the latter, increasing the force required to unfold the quadruplexes. The fraction of ligand-bound G-quadruplex was calculated by deconvoluting the force histogram of the corresponding ligand-quadruplex system in which free G-quadruplexes demonstrate a lower force (∼20 pN) population whereas the ligand-bound populations show an increased force (∼40 pN) (see Materials and Methods and Figures 2 and S8). With this setup, we directly compared the binding efficiency of 100 nM L2H2-6OTD monomer, L2H2-6OTD dimer, and L2H2-6OTD tetramer (see Figure S1 for structures) to monomeric telomere G-quadruplex in the 4G construct. We observed that monomer and dimer telomestatin derivatives bound to G-quadruplexes with similar binding efficiencies (respective bound fractions: 56.4% and 57.4%. See Figure 3A). Interestingly, the tetramer showed a dramatically reduced bound fraction of 9.7%, suggesting significantly weaker binding of the tetramer to the 4G construct. To confirm this surprising result, we performed UV melting experiments. Figure 3B showed that while the melting temperatures (Tm’s) of monomeric telomere G-quadruplex in the presence of monomer and dimer telomestatin derivatives increased by 11 and 14.7 °C, respectively, the tetramer revealed no significant increase in melting temperature. Next, we performed CD melting for the 4G fragment in the presence of monomer, dimer, or tetramer ligands (Figures S26 and S27). Consistent with the UV melting, we found tetramer did not bind with the 4G construct (no change in Tm) whereas both monomer and dimer showed the binding. These results are consistent with those from the single-molecule binding experiments. Next, we evaluated the binding of the L2H2-6OTD tetramer to multiple telomeric G-quadruplexes. We found percentages of ligand-bound high-force population in the presence of 100 nM tetramer were 9.7%, 25.0%, 29.8%, 40.8%, and 49.9% for the 4G, 8G, 12G, 16G, and 24G constructs in which up to 1, 2, 3, 4, and 6 G-quadruplexes can form respectively (Figure 3C). The increasing bound fraction of the tetramer ligand with the number of G-quadruplex units can be well explained by the polyvalent binding mechanism. With an increase in the Gquadruplex units, the polymeric telomestatin units in tetramer are expected to show stronger binding capability, resulting in increased fraction of the quadruplex-ligand complex. To further quantify the binding capacity of three telomestatin derivatives with monomeric or polymeric telomere Gquadruplexes, we constructed binding curves using the same single-molecule assays (Figure 4). The bound fraction of Gquadruplex in each case was obtained from the rupture force histograms and plotted with telomestatin concentrations. The dissociation constant for each system was retrieved by fitting the binding curve with a Langmuir isotherm.26 The accuracy of this single-molecule binding analysis has been established by the agreement of Kd values obtained here (14.2 and 13.8 nM

Figure 2. Single-molecule binding assays. (A) Telomeric G-quadruplex forming single-stranded DNA sequence is sandwiched by two duplex DNA handles which are respectively tethered to two optically trapped beads. (B) Typical force−extension (F-X) curves obtained while stretching the DNA in the presence or absence of G-quadruplex binding ligand. Unfolding of G-quadruplexes is indicated by a sudden increase in the extension. Insets show enlarged unfolding events. Scale bar represents 10 nm. (C) Histograms of unfolding force in the presence or absence of G-quadruplex binding ligand. Curves represent Gaussian fittings. 7478

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Figure 3. Binding of L2H2-6OTD derivatives with telomeric G-quadruplexes. (A) Unfolding force histograms of the telomere 4G construct without or with 100 nM L2H2-6OTD monomer, dimer, or tetramer. (B) 295 nm UV melting of the telomere 4G construct in a 5 mM K2HPO4 and 2 mM KH2PO4 buffer filled with 88 mM KCl at pH 7.4 with and without ligands. See Supporting Information for the determination of melting temperature (Tm). (A) and (B) depict 100 nM tetramer ligand does not bind to the 4G construct. (C) Unfolding force histograms of the telomere 4G, 8G, 12G, 16G, and 24G constructs with 100 nM L2H2-6OTD tetramer, which indicate that binding is more efficient when DNA constructs host more Gquadruplexes. Solid and dotted curves in (A) and (C) depict Gaussian fittings.

Figure 4. Binding curves of G-quadruplexes in telomeric sequences of varying length with L2H2-6OTD monomer (A), dimer (B), and tetramer (C). Monomer and dimer telomestatin derivatives have similar affinities irrespective of monomeric or multimeric G-quadruplexes. L2H2-6OTD tetramer shows an increased binding affinity for sequences that contain more G-quadruplexes. See Figures S20 and S21 for the binding between the tetramer ligand and other telomeric sequences (8G and 16G).

constants of telomestatin monomer and dimer are similar for telomere sequences with varying length (4G to 24G). Compared to the monomer G-quadruplex in the 4G construct, multiple G-quadruplexes in the 24G sequence showed slightly

for the monomer and dimer ligand bindings to the 4G DNA, respectively; see Figure 4) and those measured by surface plasmon resonance (10.8 and 8.3 nM for monomer and dimer ligands, respectively).27 As shown in Figure 4, the dissociation 7479

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Figure 5. Circular dichroism melting assays support the stacking of polyoxazole macrocyclic rings in the L2H2-6OTD tetramer. The melting was performed from 25 to 95 °C with 10 °C stepwise increment. (A) 50 μM L2H2-6OTD monomer in water does not show stacking of macrocyclic rings. (B) 50 μM L2H2-6OTD tetramer in water shows a ∼250 nm peak and a ∼270 nm trough at low temperatures, suggesting a different conformation that is consistent with stacking of the rings. 50 μM L2H2-6OTD monomer (C) and tetramer (D) in a 5 mM K2HPO4 and 2 mM KH2PO4 buffer (PBS) filled with 88 mM KCl at pH 7.4 show more pronounced ∼250 nm peaks and ∼270 nm troughs, which indicates that ring stacking is facilitated by salt.

These difficulties stress the unique advantage of performing single-molecule experiments in which only minute amount of long DNA is necessary. Polyvalency Induced Unstacking of Binding Units (PIU Binding). The trend in the binding affinity of telomestatin tetramer to different number of telomeric quadruplexes can be explained by polyvalent binding mechanism in which tetramer ligand prefers longer telomeric sequence that hosts more G-quadruplexes. However, it is puzzling that the tetramer does not seem to bind to monomeric G-quadruplexes as efficiently as monomer ligand. To probe whether binding of the L2H2-6OTD tetramer is dependent on G-quadruplexes of different origins, we tested G-quadruplex in the Bcl-2 promoter sequence, 5′-CTA GAG GGG CGG GCG CGG GAG GAA GGG GGC GGG AGC GGG GCT GC. The unfolding force histogram revealed significant binding of the L2H2-6OTD monomer to the Bcl-2 sequence as indicated by increased rupture force (47.5 ± 0.7 pN, Figure S22) with respect to that of the control without ligand (39.4 ± 1 pN). By contrast, the tetramer L2H2-6OTD telomestatin failed to show binding as the force histogram is similar to that of the control (38.4 ± 2 pN, Figure S22). The bound fraction and the efficiency of binding were not calculated since the Bcl-2 sequence is known to host different types of G-quadruplex by assembling various combinations of four G-rich tracts in the region,28,29 which makes it impossible to deconvolute folded

decreased binding constant for the monomer or dimer telomestatin. This can be explained by the observation that a small fraction of quadruplex-quadruplex interaction exists in the 24G construct,19 hindering the access of the ligand to the quadruplexes. Unlike the monomer and dimer analogues, the dissociation constants for the telomestatin tetramer dramatically decrease with the length of the G-rich sequence from 4G to 24G (Figures 4, S20, and S21). The binding constants are >700, 110, 58.8, 36.6, and 16.2 nM for the 4G, 8G, 12G, 16G, and 24G constructs, respectively. This result indicates that the tetramer ligand binds to multiple quadruplexes with more than 40-fold efficiency with respect to the shorter sequences that host fewer G-quadruplex units. The binding profiles were qualitatively confirmed by the CD melting experiments in which 4G-16G fragments were thermally denatured in the presence of monomer, dimer, or tetramer ligands (Figures S26−S30). From the 4G to 16G fragments, there was significant increase in melting temperature for the tetramer (Tm’s were 54.1, 69.1, 77.2, and 80.2 °C for the 4G, 8G, 12G, and 16G telomeric fragments, respectively; in comparison, the respective Tm’s were 54.6, 50.9, 50.6, and 50.1 °C without ligand), which is consistent with the trend of the binding affinity measured in Figure 4. It is noteworthy that telomeric constructs longer than 16G tracts were not commercially available. On the other hand, molecular biology strategies are challenging to synthesize long G-rich fragments with quantities sufficient for UV or CD melting experiments. 7480

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Figure 6. Mechanism of polyvalency induced unstacking of binding units (or PIU binding). (A) Proposed model of the PIU binding for the recognition of telomeric G-quadruplexes (GQs) by the L2H2-6OTD tetramer. Stacking of telomestatin units in the tetramer provides steric hindrance that reduces tetramer binding to monomeric G-quadruplex (left pathway). In the presence of multimeric G-quadruplexes (right), polyvalent interaction between quadruplex and telomestatin overcomes the stacking energy, increasing the binding affinity. The switch between the two distinct pathways for the tetramer ligand depicts an adaptive nature for the PIU binding. (B) Dissection of the PIU binding from free energy perspective. Due to the state function of the change in free energy of the PIU binding (ΔGPIU‑binding, route I) that is route independent, ΔGPIU‑binding is equivalent to the sum of the change in free energy of unstacking (ΔGunstacking, route II) and four times of the change in free energy of monomeric ligand binding (route III, 4ΔGmonobinding). Since ΔGunstacking has a magnitude (59.0 kJ/stack) larger than ΔGmonobinding (−58.8 kJ), tetramer ligand does not unstack while binding to a monomeric G-quadruplex (see (A) left). The PIU binding occurs when more G-quadruplexes are available to overcome the stacking in the tetramer ligand.

telomeric G-quadruplex as used here. Such a pocket is too small to accommodate a stack of telomestatin rings in the tetramer. Evidence for stacking comes from spectroscopic experiments. In the CD measurements, we compared 50 μM telomestatin monomer and tetramer in water from 25 to 95 °C (Figure 5A,B). We found that while monomer did not change its structure significantly up to 95 °C, the tetramer showed a peak (∼250 nm) and a trough (∼270 nm) at 25 °C that were quite different from those at 95 °C. Interestingly, the tetramer structure at 95 °C looks similar to that of the monomer. These data are consistent with stacking of the telomestatin units in tetramer at room temperature. When temperature increases, this stacking starts to break up. In contrast, the stacking is not formed in monomer. Next, we compared the monomer and tetramer in a PBS buffer with 100 mM KCl (Figure 5C,D). Here, even monomer shows signs of stacking (the ∼250 nm peak and the ∼270 nm trough), which is disassembled with

species from the rupture force histogram with broad force distribution (Figure S22). We propose that significantly reduced affinity for the binding of telomestatin tetramer to monomeric G-quadruplexes could be originated from the stacking of telomestatin units in the tetramer. Each telomestatin unit consists of six aromatic oxozoles, which likely facilitate the π−π stacking of the telomestatin rings in the tetramer structure. The stacking decreases the number of effective quadruplex-binding units in the telomestatin tetramer, reducing the binding affinity. In addition, the stacking increases steric hindrance for the telomestatin tetramer to approach G-quadruplex structure, further decreasing the binding affinity for stand-alone quadruplexes. Indeed, NMR structure16 has indicated that L2H2-6OTD telomestatin monomer sits in a pocket formed by a top G-quartet (from 5′ end) and the third loop of the same 7481

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in the telomere can be recognized rather than one or a few Gquadruplexes formed in promoters. Such a strategy provides a much-needed specificity to target G-quadruplexes in the 3′ telomere overhang.32,33 L2H2-6OTD tetramer in the current form is not ideal for in cellulo applications due to its high molar mass. The cellular uptake issue can be addressed by strategies such as conjugation of this molecule with membrane penetrating peptides34 or nanocarriers35 that can transport across the plasma membrane.

increasing temperature. For telomestatin tetramer, the stacking becomes rather strong as it can withstand temperature as high as 95 °C. This observation is consistent with the finding that addition of salt may reduce electrostatic repulsion30 between cationic groups in the telomestatin derivatives, which facilitates the π−π stacking of the polyoxazole macrocyclic rings in the tetramer. CD experiments with reduced ligand concentration (5 μM) in the same PBS buffer with 100 mM KCl confirmed the finding that stacking occurs in the tetramer but not in the monomer ligand (Figure S23). We further compared the absorption and emission spectra of the monomer, dimer, and tetramer ligands (Figure S24A,B). While the molar absorptivity increases linearly with the number of macrocyclic polyoxazole units in the ligands at absorption maximum (255 nm), the fluorescence intensity at 355 nm decreases at the same concentration of each ligand. Such an observation indicates quenching occurs in the dimer and tetramer ligands, which can be explained by the close distance of telomestatin fluorophores as a result of stacking. The fact that there is largest decrease in fluorescence intensity in the tetramer suggests that stacking of the telomestatin units is most effective in this ligand. Additional evidence of stacking comes from red-shifted fluorescence signals (Figure S24C,D). At a concentration in the range used in single-molecule assays (100 nM), tetramer, instead of monomer, showed a red-shifted fluorescence peak at ∼400 nm. This observation is consistent with the excimer formation due to the stacking of fluorophores.31 Taken together, all these results strongly support the stacking of polyoxazole rings in the tetramer telomestatin derivative. When telomestatin tetramer encounters multiple G-quadruplexes, the polyvalent interactions between the telomestatin groups and the quadruplex units come into play (Figure 6A). The additional binding energy from the polyvalent association is expected to overcome the stacking of telomestatin rings in the tetramer, increasing the binding affinity. In fact, we found binding affinity of the telomestatin tetramer and 24G complex is similar to that of the telomestatin monomer with the standalone G-quadruplex in the 4G construct (Figure 4). To understand this so-called polyvalency induced unstacking of binding units (or PIU binding) from free energy perspective (ΔGPIU‑binding), we dissected the binding into two steps (Figure 6B). First, the tetramer is unstacked into four linked monomers (route II). The four monomers then bind to four separate Gquadruplexes (route III). The change in free energy of binding at the standard state is calculated by ΔGbinding=-RTlnKbinding, where R is gas constant (8.314 J/mol.K), T is room temperature (298 K), and Kbinding = 1/Kd. As free energy is a state function, ΔGPIU‑binding = ΔGunstacking + 4ΔGmonobinding, where ΔGunstacking and ΔGmonobinding are the changes in free energy of unstacking and monomer ligand binding, respectively. ΔGmonobinding is multiplied by 4 to reflect the fact that four monomer binding events occur in route III. This calculation allowed us to estimate ΔG for each unstacking as 59.0 kJ/mol. This value is a little larger than the change in free energy of monomer binding (−58.8 kJ/mol), confirming that unstacking is not spontaneous when a tetramer encounters standalone Gquadruplexes. Only multiple quadruplex-ligand interactions provide sufficient thermodynamic driving force, leading to the adaptive PIU binding (Figure 6A). The dramatic difference in the binding affinity between the 4G (Kd > 700 nM) and the 24G (Kd ∼ 16.2 nM) DNA for the tetramer telomestatin ligand provides a concentration window (100 nM for example) at which only multiple G-quadruplexes

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CONCLUSIONS In summary, we have demonstrated a new binding model, polyvalency induced unstacking of binding units (or PIU binding) to specifically target multivalent molecules rather than standalone molecular structures. We have successfully demonstrated specific PIU binding for telomere over promoter Gquadruplexes, a rather challenging task facing the G-quadruplex community. The PIU binding represents a generic interaction that can be exploited to design ligands with more effective recognition for multimeric biological structures, including those found in cell skeleton and muscle systems (titins and myosins), polysaccharides, as well as those in misfolded proteins (amyloid) and other protein processing machineries (ubiquitins).

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MATERIALS AND METHODS

Syntheses of DNA Constructs and Telomestatin Derivatives. Syntheses of the DNA constructs that contain the 4G, 8G, 12G, and 24G sequences are described in the Supporting Information. Detailed characterizations of L2H2-6OTD monomer, dimer, and tetramer ligands are described in the Supporting Information, which includes UV melting, CD melting, and fluorescence analyses of ligands and ligand−G-quadruplex complexes. Mechanical Unfolding Experiments. The single molecule investigation was carried out in a home-built laser tweezers instrument for which the detailed description has been reported previously.36 All the experiments, unless specified otherwise, were carried out in a 10 mM Tris/100 mM KCl buffer, at pH 7.4 and 23 °C. To start the single-molecule experiments, digoxigenin-labeled DNA construct was immobilized onto a 2.10 μm polystyrene bead coated with antidigoxigenin (Spherotech, Lake Forest, IL) via digoxigenin− antidigoxigenin antibody interaction. The DNA immobilized bead and the streptavidin coated bead (Spherotech) were trapped by two laser foci and the DNA construct was then tethered between these two beads. The tethered DNA was stretched at a constant loading rate of 5.5 pN/s until it reached just below the plateau force (maximum 60 pN) and relaxed to 0 pN by moving one of the trapped beads. The force−extension (F−X) curves were recorded at 1 kHz using LabVIEW 8.2 (National Instruments Corp., Austin, TX). Data Analysis. The unfolding force at each structure was recorded from individual force−extension (F-X) curves and a force histogram was plotted for all the DNA constructs with or without ligands (Figure S8). We observed a two-Gaussian distribution in the presence of ligand, and the area of the second peak with higher force tends to increase with the concentration of the ligand (Figures S9−S19). This indicates that the second peak corresponds to the ligand bound Gquadruplex. Both the ligand-bound and ligand-free populations were deconvoluted and the cumulative count for each population was determined. In the case of the 8G, 12G, 16G, and 24G constructs, there is a small fraction of population in the high force region even in the absence of ligand. When ligand-bound fractions were calculated for DNA constructs, these small fractions were subtracted from the overall bound fraction. 7482

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countsP1,LC = X

× 100% countsO,LC = X countsP1,LC = 0 − × 100% countsO,LC = 0

(1)

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b00607. NMR, fluorescence, and CD spectra of telomestatin derivatives and histograms for single molecule characterization of the binding between G-quadruplexes and telomestatin derivatives (PDF)

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REFERENCES

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here %boundLC=X is the percentage of bound fraction at X nM or 0 nM ligand concentration, countsP1 and countsO are the cumulative counts of the bound population and overall population obtained from the rupture force histograms, respectively. The percentage bound population estimated this way does not account for the ligand-bound G-quadruplexes that are too stable to be unfolded in the range of 0−60 pN. Since dsDNA handles can melt beyond 60 pN,37,38 we set 60 pN as the upper limit in our experiment. To estimate these nonunfolded populations, we overlapped at least 15 relaxing F-X curves from the same molecule. In these curves, the F-X curve that had the longest extension at 31 pN was considered as the trace without any nonunfolded G-quadruplex structures, which was further confirmed by the number of unfolding features in the stretching F-X curves of the same molecule. All other relaxing curves were compared to this “base-line” relaxing curve at 31 pN at which the extension is equivalent to the contour length according to the WormLike-Chain model39 (see Figure S8G). The difference in extension at the 31 pN was then converted to the number of G-quadruplexes (∼9 nm per quadruplex19) that still contain in the relaxing curve. To confirm this identification method for nonunfolded populations, we overlapped all the relaxing traces of the 4G DNA construct that hosts only one telomeric G-quadruplex without ligand (Figure S8H). Only one population was observed at 31 pN that represented traces without nonunfolded G-quadruplexes. This result was expected since it is wellknown that all telomere G-quadruplexes can be unfolded in the range 0−45 pN.26 These nonunfolded populations were assigned to the ligand-bound G-quadruplexes due to their high mechanical stabilities in the eq 1 to calculate the ligand-bound fraction of telomere Gquadruplexes.

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AUTHOR INFORMATION

Corresponding Authors

*[email protected] *[email protected] ORCID

Jibin Abraham Punnoose: 0000-0003-2367-6874 Shankar Mandal: 0000-0002-2653-8760 Kazuo Nagasawa: 0000-0002-0437-948X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS H.M. is grateful to NSF CHE-1609514 and NSF CHE-1415883 for financial support. K.N. is thankful for partial support from Grants-in-Aid for Scientific Research (B) from JSPS (23310158 and 26282214) and a Grant-in-Aid for Challenging Exploratory Research from JSPS (21655060). Y.M. is grateful for financial support in the form of JSPS Predoctoral Fellowships for Young Scientists. 7483

DOI: 10.1021/jacs.7b00607 J. Am. Chem. Soc. 2017, 139, 7476−7484

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DOI: 10.1021/jacs.7b00607 J. Am. Chem. Soc. 2017, 139, 7476−7484