Recovery of the Formation and Function of Oxidized G-Quadruplexes

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Recovery of the formation and function of oxidized Gquadruplexes by a pyrene-modified guanine-tract Shuntaro Takahashi, Ki Tae Kim, Peter Podbevsek, Janez Plavec, Byeang Hyean Kim, and Naoki Sugimoto J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b01577 • Publication Date (Web): 02 Apr 2018 Downloaded from http://pubs.acs.org on April 2, 2018

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Journal of the American Chemical Society

Recovery of the Formation and Function of Oxidized GQuadruplexes by a Pyrene-modified Guanine Tract Shuntaro Takahashi,† Ki Tae Kim,§# Peter Podbevsek,⊥ Janez Plavec,⊥ Byeang Hyean Kim,§ and Naoki Sugimoto†, ‡,* †

FIBER (Frontier Institute for Biomolecular Engineering Research), Konan University, 7-1-20 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan § Department of Chemistry, Division of Advanced Materials Science, Pohang University of Science and Technology (POSTECH), Pohang 37673, Republic of Korea. Slovenian NMR Center, National Institute of Chemistry, SI-1000 Ljubljana, Slovenia

⊥

‡

FIRST (Graduate School of Frontiers of Innovative Research in Science and Technology), Konan University, 7-1-20 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan #

Author’s present address: Department of Organic chemistry, Faculty of Science, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva, Switzerland Supporting Information Placeholder

ABSTRACT: Oxidation is one of the frequent causes of DNA damage, especially to guanine bases. Guanine bases in the Gquadruplex (G4) are sensitive to damage by oxidation, resulting in transformation to 8-oxo-7,8-dihydroguanine (8-oxoG). Because the formation of G4 represses the expression of some cancer-related genes, the presence of 8-oxoG in a G4 sequence might affect G4 formation and induce cancer progression. Thus, oxidized-G4 formation must be controlled using a chemical approach. In the present study, we investigated the effect of introduction of 8-oxoG into a G4 sequence on the formation and function of the G4 structure. The 8-oxoG–containing G4 derived from the promoter region of the human vascular endothelial growth factor (VEGF) gene differed topologically from unoxidized G4. The oxidized VEGF G4 did not act as a replication block and was not stabilized by the G4-binding protein nucleolin. To recover G4 function, we developed an oligonucleotide consisting of a pyrene-modified guanine tract that replaces the oxidized guanine tract and forms stable intermolecular G4s with the other intact guanine tracts. When this oligonucleotide was used, the oxidized G4 stalled replication and was stabilized by nucleolin as with the unmodified G4. This strategy generally enables recovery of the function of any oxidized G4s and therefore has a potential for cancer therapy.

INTRODUCTION Guanine quadruplex (G4) structures are formed by four strands of guanine tracts stabilized by Hoogsteen base pairing and guanine quartet stacking interactions that are coordinated with metal ions (mainly K+ and Na+).1 Recently, G4 structures were shown to dynamically form in genomic DNAs and mRNAs in cells and to regulate all steps of gene expression: replication, transcription, and translation.2-7 Furthermore, various G4-binding proteins, such as nucleolins and heterogeneous nuclear ribonucleoproteins (hnRNPs), stabilize G4 structures and assist in regulating gene expression.8 Thus, the aberrant regulation of G4 formation affects normal gene expression, resulting in various diseases. G4 motifs are frequently found in the promoter regions of cancer-related genes.9 Expression of a cancer-related gene is normally suppressed because G4 formation reactions inhibit recruitment of RNA polymerase.2 Nonetheless, without G4s present, the cancer-related genes become activated. Therefore, controlling G4 formation is important for the diagnosis and treatment of cancers.9

Disruption of G4 structures occurs when bases in guanine tracts become damaged. Guanine oxidation is one of the most abundant lesion types due to charge-tunneling10-12 and results in 8-oxo-7,8-dihydroguanine (8-oxoG).13 8-OxoG changes the Hoogsteen face of the purine and prefers the syn configuration, which cannot form Hoogsteen hydrogen bonds in the guanine quartet.14-15 Thus, the oxidative lesions in G4 structures can perturb the formation of G4 structures.14-18 8-OxoG is caused by reactive oxygen species (ROS) in the cell.13 The respiratory-chain complexes in mitochondria are the main cellular source of ROS, which function as messengers under hypoxic conditions.19-21 Because hypoxia is a common feature of a tumor microenvironment, base lesions due to ROS production can occur. Thus, it is possible that oxidative lesions occur within G4-forming sequences in cancer cells and inhibit native G4 formation. Although the oxidized lesion can be repaired or diluted out by replication during the cell cycle of cancer cells,22 a replication error can cause a mutation from G to T at the position of 8-oxoG,23 thus disrupting the G4 formation permanently. Disruption of G4 structures may also inhibit the

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binding of nucleolins and other G4-binding proteins, resulting in higher expression of cancer-related genes.8, 24-25 In cancer cells, 8-oxoG bases have been detected in a G4-forming sequence within the promoter region of the vascular endothelial growth factor (VEGF) gene.26 The presence of 8-oxoG increases VEGF transcription in vivo.27 It has been suggested that the generation of 8-oxoG within a G4 structure in the VEGF gene induces G4 to form with the fifth guanine tract instead of the tract having 8-oxoG; this mechanism promotes transcription via base excision repair.27-29 Recently, it was found that 8-oxoG in G4 within the promoter region of the KRAS gene upregulates this gene.30 Thus, 8-oxoG bases within G4 sequences may be an effective target for cancer diagnosis and therapeutics. Nevertheless, a chemical approach to analysis of the formation and function of oxidized G4 structures has not been explored. In this study, we performed polymerase stop assays using template DNA containing an 8-oxoG oxidative lesion within the G4 sequences of VEGF and other promoter and telomere regions of cancer-related genes. Circular dichroism (CD) was used to quantitatively assess the topology dependence of G4 structures in replication.7 These results suggested that the G4 structures containing 8-oxoG have topologies different from those of the unmodified G4 structures that disrupt replication control. Nucleolin did not stabilize oxidized G4 structures or enhance the replication stalling. To recover G4 function, we utilized pyrene-modified guanine tracts (PyG3) to stabilize oxidized G4s31 because PyG3 can form (3+1) intermolecular G4 structures by replacing 8-oxoG bases. PyG3 significantly stabilized intermolecular G4 formation owing to stacking of the pyrene moiety and the formed G-quartet. As expected, the addition of PyG3 recovered the replication stalling on oxidized G4s. The intermolecular G4 structures highly resembled the unoxidized G4 structure and were recognized by nucleolin. Our approach can serve as a general strategy to restore G4 structures containing base lesions. RESULTS Structure and Stability of Oxidized G4s within Promoter Regions. G4-forming sequences within the VEGF gene were selected for this study because 8-oxoG bases were identified within the VEGF promoter region and the tertiary structure of G4 has been solved.26, 32-33 The G4 structures that have been solved in other cancer-related genes, c-Kit2 and Bcl2, were also selected although their oxidative lesions have not been identified.34-35 G4 structures within human telomeres are also targets of oxidative stress, which affects telomerase activity.14, 36 8-OxoG was introduced at each guanine within the 5′terminal guanine tract (Figure 1) because guanine bases at ends of the quartets are sensitive to oxidative stress,10 and the 8-oxoG in the middle of the quartet has a strong destabilizing effect on G4 structure.14 The wild-type sequences of VEGF and c-Kit2 G4 adopted various structures in solution that are not suitable for the NMR experiment and thermodynamic analysis of the formation of G4. Therefore, we used mutated sequences of VEGF and c-Kit2 G4s optimized for the NMR and thermodynamic measurements as “native” sequences unless stated otherwise.32, 34 All the sequences used in this study potentially form G4 structures with three quartets of 12 guanines and are listed in Table S1. We named each sequence as GXO, where X indicates the position of the replaced guanine at the 5′ terminus (Figure 1). For example, the four sequences

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of VEGF G4 are “VEGF native,” “VEGF G1O,” “VEGF G2O,” and “VEGF G3O.”

Figure 1. G4 structure and the position of 8-oxoG introduced in this study. The structure of each G4-forming sequence was analyzed by CD spectroscopy. In a buffer consisting of 10 mM Tris-HCl (pH 7.5), 8 mM MgCl2, and 50 mM KCl at 37°C, the CD spectra of the VEGF native G4 showed a large positive peak at 265 nm, indicating a parallel topology that is typical of G4 structure (Figure 2A). In VEGF G4 sequences containing 8oxoG within the 5′-terminal G-tract, the intensity of the peak at 265 nm decreased. Nonetheless, a shoulder peak around 295 nm appeared (Figure 2B–D). Similar results were observed for the native and oxidized c-Kit2 G4 sequences (Figure S1). The CD signature at approximately 295 nm is likely due to the unique configuration formed by 8-oxoG in the G4 structures. Some studies have yielded the CD spectra of human telomeric G4 containing 8-oxoG. Nevertheless, the contribution of 8oxoG was unclear because the antiparallel and mixed topologies also have positive peaks at 295 nm.14, 18 Given that 8oxoG favors the syn configuration, the parallel topology of G4 may transform to a mixed topology if the guanines adopt an anti configuration. A similar increase in a CD peak at approximately 295 nm was observed with a naphthalene diimide derivative bound to the native c-Kit2 G4 sequence.37 Therefore, the 8-oxoG moiety may also interact with either the G-quartet or the groove of the G4 structure. We next analyzed these oxidized VEGF and c-Kit2 G4 structures by 1D 1H NMR. The imino protons suggested the presence of Hoogsteen base pairing of the guanine bases (Figure S2), as indicated by the signal between 10.5 and 12 ppm.32-33 Nonetheless, the signal was too weak to determine the tertiary structure.

Figure 2. CD spectra of 10 µM (A) VEGF native G4 and its oxidized forms: (B) VEGF G1O, (C) VEGF G2O, and (D) VEGF G3O. All the experiments were performed in a buffer consisting of 10 mM Tris-HCl (pH 7.5), 8 mM MgCl2, and 50 mM KCl.


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To determine the stability of each structure, we performed CD melting assays by measuring the changes in CD intensity at 263 nm (Figures 3 and S3) to determine the melting temperatures (Tm) and thermodynamic stability levels at 37 °C (−∆G°37; Table 1). The VEGF native G4 was too stable to determine these parameters in the presence of 50 mM KCl. Even in the presence of 10 mM KCl, the VEGF native G4 showed very high stability, with a Tm value of 65.5 °C and a −∆G°37 value of 2.7 kcal mol−1. On the other hand, VEGF G1O, G2O, and G3O did not show a clear-cut melting profile, and the intensities of the CD signals were weak. These results indicated that all these oxidized sequences did not form the typical G4 structure. It is possible that VEGF G3O especially formed a different topology, according to the spectra (Figure 2D). The difference in intensity of VEGF G1O at 10 or 50 mM KCl might be due to salt concentration–dependent intermolecular interactions. Thus, we could not determine the thermodynamic parameters of oxidized VEGF G4s. In the case of c-Kit2, the CD signals were clearer than those of the VEGF G4s, and some of the thermodynamic values of c-Kit2 native and oxidized sequences were successfully obtained (c-Kit2 native in 10 mM KCl: Tm = 56.1 °C, −∆G°37 = 2.8 kcal mol−1; c-Kit2 G2O in 10 mM KCl: Tm = 48.5 °C, −∆G°37 = 1.0 kcal mol−1; cKit2 G3O in 10 mM KCl: Tm = 56.9 °C, −∆G°37 = 1.8 kcal mol−1; c-Kit2 native in 50 mM KCl: Tm = 77.4 °C, −∆G°37 = 4.6 kcal mol−1; c-Kit2 G2O in 50 mM KCl: Tm = 61.1 °C, −∆G°37 = 1.2 kcal mol−1; and c-Kit2 G3O in 50 mM KCl: Tm = 64.8 °C, −∆G°37 = 2.8 kcal mol−1). The measurements for cKit2 G1O in the presence of 10 and 50 mM KCl produced unclear melting profiles (Figures 3 and S3). It has been reported that the incorporation of 8-oxoG reduces G4 stability.14 According to the CD data, the oxidized G4 from VEGF and cKit2 formed a G4 structure with stability lower than that of the G4 structures of native sequences.

Figure 3. CD melting profiles of (A) 10 µM VEGF native (blue), VEGF G1O (red), VEGF G2O (green), and VEGF G3O (pink), and (B) 10 µM c-Kit2 native (blue), c-Kit2 G1O (red), c-Kit2 (green), and c-Kit2 G3O (pink) determined at 265 nm in the buffer consisting of 10 mM Tris-HCl (pH 7.5), 8 mM MgCl2, and 50 mM KCl. Effects of Oxidation on Replication Studied by a Quantitative Polymerase Stop Assay. To investigate the functions of oxidized G4 structures, we analyzed the replication function of G4 sequences having 8-oxoG. Recently, we found that replication efficiency of G4-forming DNA is dependent on both stability and topology of the G4.7 The newly developed analysis—called the quantitative study of topology-dependent replication (QSTR)—yields a phase diagram of a replication rate vs. G4 stability and serves to determine replication properties depending on the topology of template DNA. For the replication analysis, a fluorescein-labeled primer and template DNA

Table 1. Thermodynamic parameters of the native and oxidized G4 structures.

DNA

Salt Condition

Tm (°C)

VEGF native

10 mM KCl

65.5 ± 0.4

−∆G°37 (kcal mol−1) 2.7 ± 0.1

VEGF G1O

10 mM KCl

(n.d.)

(n.d.)

VEGF G2O

10 mM KCl

(n.d.)

(n.d.)

VEGF G3O

10 mM KCl

(n.d.)

(n.d.)

c-Kit2 native

10 mM KCl

56.1 ± 0.7

2.8 ± 0.3

c-Kit2 G1O

10 mM KCl

(n.d.)

(n.d.)

c-Kit2 G2O

10 mM KCl

48.5 ± 0.9

1.0 ± 0.1

c-Kit2 G3O

10 mM KCl

56.9 ± 4.7

1.8 ± 0.2

c-Kit2 native

50 mM KCl

77.4 ± 2.5

4.6 ± 0.5

c-Kit2 G1O

50 mM KCl

(n.d.)

(n.d.)

c-Kit2 G2O

50 mM KCl

61.1 ± 0.7

1.2 ± 0.1

c-Kit2 G3O

50 mM KCl

64.8 ± 2.2

2.8 ± 0.6

All the experiments were performed with 10 µM DNA in the buffer consisting of 10 mM Tris-HCl (pH 7.5), 8 mM MgCl2, and 10 or 50 mM KCl.

each containing oxidized G4 structures were designed as reported previously.7 The replication reaction was carried out using these DNAs and Klenow fragment DNA polymerase lacking 3′→5′ exonuclease activity (hereafter: “KF exo-”). The efficiency of the replication stall of the template DNA at G4 was analyzed by denaturing polyacrylamide gel electrophoresis (PAGE) in reaction samples obtained at each of the indicated time points. As shown in Figure 4A, a short product obtained from stalled complexes was observed in reactions containing the VEGF native G4 sequence. The product indicates that KF exostalled immediately before the formed VEGF G4 structure on the template DNA. On the other hand, the replication products from DNA containing VEGF G1O, VEGF G2O, or VEGF G3O showed very low amounts of stalled products and immediate accumulation of full-length products (Figure 4A). In the previous report, incorporation of dNMPs opposite 8-oxoG in the template was more ineffective as compared to the natural DNA template.38 Nevertheless, we were not able to observe any replication stalls at the position of 8-oxoG in the template strand because the incorporation of dNMP opposite 8-oxoG was very quick due to a high concentration of KF exo- and dNTPs and optimal temperature as compared to the conditions in the previous report. The lack of exonuclease activity might facilitate incorporation of dNMPs opposite 8-oxoG in the template strand. Similar results were obtained for template DNAs containing c-Kit2 G4 (Figure S4). For the QSTR analyses, we performed time course assays measuring full-length product formation to determine the rate constants at 37°C for overcoming the G4 structure (ks) and those for replication after overcoming the stall after replication (kf; Figure S5). The QSTR plot shows the relation between lnks and −∆G°37 (Figure 4B). The replication of the VEGF native template showed ks of 0.010 min−1 and kf of 21 min−1 at 37°C. On the other hand, the replication reactions of VEGF G1O, VEGF G2O, and VEGF G3O were very quick, and the ks values were estimated as 12,



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15, and 11 min−1 whereas the kf values were 43, 20, and 19 min−1, respectively. Figure 4B shows the QSTR plots of VEGF native and c-Kit2 native data fitted to linear regression analyses with the data from a random template whose −∆G°37 was assumed to be zero. In the overlay of the results from VEGF native and c-Kit2 native, the DNA formed the same parallel topology as previously reported.7 This finding suggested that these G4 structures share the same mechanism of replication. On the other hand, the results on VEGF and c-Kit2 fragments containing 8-oxoG did not overlay with the data on VEGF native and c-Kit2 native. This finding suggests that the oxidized G4s have a replication mechanism different from that of the native G4 structures. Therefore, generation of 8-oxoG causes changes in the G4 structure and in replication of the sequence.

length product (Figure S6). We then confirmed binding of MBP-NCL to the DNA templates by means of a quartz crystal microbalance (QCM) with immobilized nucleolin.40-42 Both native and oxidized VEGF G4 structures showed decreases in frequency when bound to MBP-NCL (Figure 5B). Analysis of the binding isotherms based on the Langmuir adsorption model indicated (Figure S7) that the dissociation constants (KD) of MBP-NCL were 500 nM for VEGF native and 680 nM for VEGF G2O at 37°C. The deviation of the KD value from the reported one (79 nM) might be due to the difference in sequence, which consisted of a 47mer region of the promoter including the G4 region with adjacent sequences.39 These results indicated that the native and oxidized G4 structures were recognized by the G4-binding protein, but had less of an inhibitory effect on replication of the oxidized structures. Because the replication efficiency of G4 depends on its topology,7 the stabilization by the G4-binding protein may be insufficient to stall replication at oxidized G4. Accordingly, the oxidative lesion inhibits the formation of an active G4 structure.

Figure 4. Effects of 8-oxoG on replication of G4 structures at 37°C. (A) Denaturing-PAGE images showing replication products from reactions containing VEGF DNA templates with or without 8-oxoG. Reactions contained 10 mM Tris-HCl (pH 7.5), 8 mM MgCl2, and 10 mM KCl with 1 µM primer, 1 µM template, 250 µM dNTPs, and 1 µM KF exo-. (B) A QSTR plot of −∆G°37 values versus the logarithms of the rate constants (ks) for the reactions that involve overcoming the stall on native (blue) and oxidized G4 templates (red).

Figure 5. Analyses of properties of nucleolin toward oxidized G4s. (A) Denaturing-PAGE images of replication products from a VEGF template with or without 8-oxoG in the presence of 5 µM MBP-NCL in a buffer consisting of 10 mM Tris-HCl (pH 7.5), 8 mM MgCl2, and 10 mM KCl with 1 µM primer, 1 µM template, 250 µM dNTPs, and 1 µM KF exo- at 37 °C. (B) Frequency changes of QCM upon binding of VEGF native or VEGF G2O to the MBP-NCL immobilized on the QCM plate. The arrows indicate the injection timing of each DNA in the buffer [10 mM Tris-HCl (pH 7.5), 8 mM MgCl2, and 10 mM KCl] at 37 °C. The concentration of DNA increased with injection of DNA as 20, 120, 220, and 1220 nM, respectively.

Nucleolin-dependent Stabilization of Oxidized G4 Structures. Enhanced stability is required to recover the function of oxidized G4 structures in the regulation of gene expression. We tested whether nucleolin, one of the G4-binding proteins, could stabilize oxidized G4 structures and regulate transcription of VEGF and other cancer-related genes in the cell.8, 24-25 Nucleolin bound and stabilized the parallel G4 structure, including VEGF, but did not show high affinity for telomeric G4, which has a different topology.39 We performed the replication assay on VEGF in the presence of recombinant nucleolin whose N-terminal region (amino acid positions 1–284) was replaced with a maltose-binding protein tag (MBP-NCL) to increase protein stability.39 In the presence of 5 µM MBPNCL, the full-length products in reactions containing template DNA with VEGF native G4 were less abundant than those observed in the absence of MBP-NCL (Figures 4A and 5A). This result suggested that MBP-NCL stabilized the VEGF native G4 structure and facilitated a stall of KF exo- at the VEGF G4 on the template DNA. On the other hand, replication of the template DNA having VEGF G1O, G2O, or G3O did not show any increases in the amounts of stalled products (Figure 5A) and did not repress the generation of the full-

Rescue of Normal G4 Structure with a Pyreneconjugated Oligonucleotide Containing a Single Guanine Tract. To stabilize the oxidized G4 structures, rather than a conventional approach, we used an alternative strategy: replacement of the oxidized guanine tract with a pyreneconjugated guanine tract (PyG3; Figure 6A). We hypothesized that this strategy would stabilize the oxidized G4 structure because oxidized G4 sequences containing five guanine tracts that have three consecutive guanines can form stable G4 structures from the four sets of unmodified guanine tracts.29 To increase the stability of the intermolecular G4 structure, we conjugated the G4 ligand, i.e., pyrene,31, 43 to the 5′-end of the trans-acting guanine tract.31, 44 We have previously reported that pyrene-based oligonucleotides can form a (3+1) intermo-


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lecular G-quadruplex.31 In this complex, the pyrene moiety possibly interacts with the formed G-quartet through πstacking, which stabilizes the G4 structure. (A)

Figure 6. Regulation of formation of a G4 structure containing oxidized lesions. (A) The schematic of intermolecular G4 formation with PyG3. The pyrene moiety of PyG3 is shown as a cyan star, and the oxidized guanine is highlighted in yellow. (B) Fluorescence spectra of PyG3 excited at 386 nm and 37°C in the absence of target G4 (black) and in the presence of VEGF native (blue), VEGF G1O (red), VEGF G2O (green), or VEGF G3O (pink). (C) Changes in the fluorescent signal of 10 µM PyG3 at 448 nm with increasing concentrations of oxidized VEGF derivatives at 37°C. The line indicates the fitted data to obtain dissociation constants (Kd). (D) Imino

and aromatic regions of 1D 1H NMR spectra of VEGF G1O, G2O, or G3O complexed with PyG3. All the spectra were recorded at 25 °C with samples containing 10 mM potassium phosphate buffer (pH 7.5) and 50 mM KCl. (E) CD spectra of 10 µM VEGF G4 oxidized derivatives with 10 µM PyG3. (F) CD melting profiles of 10 µM VEGF G4 oxidized derivatives with 10 µM PyG3 measured at 265 nm. All the assays except for NMR experiments were carried out in the buffer consisting of 10 mM Tris-HCl (pH 7.5), 8 mM MgCl2, and 50 mM KCl. The formation of the intermolecular G4 structures was determined by measuring the pyrene fluorescence emitted when PyG3 binds to a G4 structure within the target sequence.31, 44 The fluorescence of PyG3 increased in the presence of all oxidized VEGF G4 structures (Figure 6B). Because PyG3 can alone form an intermolecular G4 that may quench the pyrene fluorescence (Figure 6A), the (3+1) intermolecular G4 formation with a target G4 results in increased fluorescence.31 Thus, it was confirmed that the oxidized G4 sequence formed an intermolecular G4 structure with PyG3. In contrast, the fluorescence did not increase when pyrene was incubated with VEGF G3O. Thus, the pyrene moiety in the G-quartet must have been different in this condition. By analyzing changes in fluorescence with increasing concentrations of target G4 structures (Figure 6C), we obtained dissociation constants (Kd) at 37°C (VEGF G1O: 3.4 µM; VEGF G2O: 2.9 µM; and VEGF G3O: 2.9 µM). These data suggested that PyG3 has similar affinities for the oxidized G4 structures. Besides, 10 µM PyG3 was sufficient to bind to the oxidized G4 sequences and form intermolecular G4 structures. We next detected G4 formation by NMR and found that mixtures of PyG3 and individual VEGF oligonucleotides containing 8-oxoG give NMR spectra with narrow resonances (Figure 6D). Several sharp signals in the imino range from 10.6 to 11.6 ppm of all three VEGF GXO–PyG3 complexes are suggestive of formation of single G4 structures. The number of sharp imino resonances did not account for the 12 guanines involved in three G-quartet planes of the VEGF G4 structure. Nevertheless, several broad resonances could also be seen in the imino regions of 1H spectra of VEGF GXO–PyG3 complexes, suggesting that some guanines, possibly the PyG3 G-tract, are more dynamic and their imino protons are effectively exchanged with the solvent. The spectrum of PyG3 itself revealed mostly broad resonances devoid of any imino signals (Figure S8) thus pointing to aggregation via the pyrene moiety and ruling out self-formation of an intermolecular G4 structure. The CD spectrum of oxidized VEGF G4 structures with PyG3 showed typical parallel topologies, which were different from the spectra seen in the absence of PyG3 (Figure 6E). The behaviors observed for the fluorescence experiments and CD spectra were consistent for oxidized c-Kit2 G4s (Figure S9A–C). In the CD melting analyses, obvious melting profiles were observed for all the oxidized G4 structures (Figures 6F and S9D). As presented in Table 2, all the thermodynamic parameters in the presence of 10 µM PyG3 and 50 mM KCl were determined (VEGF G1O: Tm = 63.9 °C, −∆G°37 = 3.7 kcal mol−1; VEGF G2O: Tm = 66.9 °C, −∆G°37 = 4.4 kcal mol−1; VEGF G3O: Tm = 68.4 °C, −∆G°37 = 4.5 kcal mol−1; c-Kit2 G1O: Tm = 70.0 °C, −∆G°37 = 2.2 kcal mol−1; c-Kit2 G2O: Tm = 64.7 °C, −∆G°37 = 2.0 kcal mol−1; and c-Kit2 G3O: Tm = 77.6 °C, −∆G°37 = 2.8 kcal mol−1). The stability of VEGF GXO–PyG3 complexes in the physiological salt condition (including 140 mM KCl and 12



mM NaCl) was comparable to that of oxidized VEGF G4 rescued by the fifth G track, showing Tm of above 80 °C; this situation can occur in damaged DNA in the cell (Figure S10). Therefore, our pyrene-modified DNA can behave in a way that rescues the oxidized G4 as effectively as the fifth G track does. From the structural view point, CD spectral data suggested that all VEGF GXO–PyG3 complexes showed the typical spectra associated with parallel topologies of the native structures (Figure 6E), whereas the G4 rescued by the fifth G track showed a mixed topology, suggesting that the biological functions of these rescued complexes may be similar to those of native G4 structures. Table 2. Thermodynamic parameters of oxidized G4 structures in the presence of PyG3. DNA

Tm (°C)

VEGF G1O + PyG3

63.9 ± 0.3

−∆G°37 mol−1) 3.7 ± 0.2

VEGF G2O + PyG3

66.9 ± 0.1

4.4 ± 0.1

VEGF G3O + PyG3

68.4 ± 0.1

4.5 ± 0.1

c-Kit2 G1O + PyG3

70.0 ± 1.6

2.2 ± 0.1

c-Kit2 G2O + PyG3

64.7 ± 0.3

2.0 ± 0.1

c-Kit2 G3O + PyG3

77.6 ± 0.1

2.8 ± 0.1

(kcal

All the experiments were carried out with 10 µM DNA and 10 µM PyG3 in the buffer consisting of 10 mM Tris-HCl (pH 7.5), 8 mM MgCl2, and 50 mM KCl. Regulation of Replication of Oxidized G4 by PyG3. We confirmed that the oxidized G4 structures recovered their biological functions after the addition of PyG3. Here, we investigated replication of DNA containing an oxidized G4 in the presence of 10 µM PyG3. First, we assayed replication of VEGF G2O because 8-oxoG in the middle quartet has the largest destabilizing effect on G4. PAGE analyses revealed short products indicating that the replication complex stalled on VEGF native G4 (Figure 7A). The full-length product constituted 44% of all the reaction products after 1 min, which was less than that observed for VEGF native G4 (65%). Replication stalling was also enhanced with VEGF G1O, VEGF G3O, and all the oxidized c-Kit2 sequences (Figure S11). The replication assay involved DNA containing the wild-type VEGF G2O and c-Kit2 G2O sequences; as a result, the amount of stalled products increased (Figure S12). Thus, PyG3 recovered the formation of functional G4 structures of VEGF and c-Kit2, which effectively inhibited the processivity of KF exo-. As a control, UG3T2, which did not contain a pyrene moiety, did not stall replication (the full-length product constituted 72% of all the products; Figure 7A). Thus, the guanine tract of PyG3 acted as a targeting moiety for the oxidized G4, and the pyrene moiety stabilized the intermolecular G4 structure. The QSTR plots of VEGF GXO and c-Kit2 GXO in the presence of PyG3 showed a different trend as compared to oxidized G4 structures in the absence of PyG3 (Figure 7B). Those plots overlaid well with the results on VEGF native and c-Kit2 native G4 structure. These results indicated that the mechanism of inhibition of replication by oxidized G4 with PyG3 was different from that without PyG3

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but similar to that of native G4s.7 As illustrated in Figures 6C and S8C, the oxidized G4 structures with PyG3 formed parallel topologies similar to those of native G4 structures. Therefore, PyG3 not only stabilized oxidized G4 structures but also recovered their biological functions in replication. After that, we verified whether PyG3 recovered the G4 structure formation by VEGF sequences having 8-oxoG in their guanine tracts. The replication of template DNA containing VEGF G5O, VEGF G8O, or VEGF G11O yielded stalled products in the presence of PyG3 (Figure 7C). The stalled product from VEGF G11O was longer than those from the others because the intermolecular G4 formed from three guanine tracts at the 5′ terminus of template DNA. Thus, PyG3 was able to recover G4 formation regardless of the oxidation site. Furthermore, we tested whether PyG3 recovered G4 formation reactions of telomere and Bcl2 G2O structures. We observed increased replication stalling for both (Figure 7D). These results indicated that PyG3 formed an intermolecular G4 structure in various oxidized G4 sequences. Because PyG3 showed versatility in controlling structures of oxidized target G4s, the specificity for a given oxidized G4 structure might be low. To increase the sequence specificity of PyG3, we attached a guide sequence at the 3′ terminus of PyG3, which could hybridize with the sequence adjacent to and upstream of the G4 region.45-46 The replication assay was conducted with 1 µM of the modified PyG3, named PyG3-g (Figures S13A–C). According to Figure 7E, the product resulting from stalled replication complexes was clearly seen. On the other hand, the addition of PyG3 resulted in less stalling of replication because the concentration used was lower than that in the above experiments (10 µM; Figure 7A). In addition, we evaluated the pattern of dissociation of PyG3-g from the target DNA and found that the dissociation was negligible, suggesting that PyG3-g could be functional for an extended period during the cell cycle of cancer cells, for ~24 hours (Figure S13D). Because the synthesis of PyG3-g was straightforward, this approach can be used to recover any oxidized G4 structure. Finally, we determined whether the intermolecular G4 structure was recognized by nucleolin. In the replication assay using VEGF G2O template DNA in the presence of PyG3, the full-length product was less abundant (36% of all the reaction products) as compared to reactions without MBP-NCL (Figure 7A). This result means that the intermolecular G4 was recognized by MBP-NCL, and the latter had a stabilizing effect on the G4 structure. By means of the QCM, we also confirmed that the complex of VEGF G2O and PyG3 caused large frequency changes after injection into the cell with immobilized MBP-NCL as compared to the control condition, which did not contain PyG3 (Figure 7F). KD of the binding of the complex to nucleolin was 250 nM (Figure S7), which was comparable to its affinity for VEGF native (KD = 500 nM). The slight improvement in the affinity of MBP-NCL by PyG3 may indicate structural differences between intramolecular and intermolecular VEGF G4 interactions with PyG3. It has been reported that nucleolin affinity is dependent on the structure of the G4.39 Thus, the intermolecular G4 with PyG3 can be targeted by MBP-NCL. These results indicate that PyG3 recovers the biological function of damaged G4 structures, and this action rescues gene expression control. DISCUSSION


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formation of a tumor.19-21 Because the guanine in G4 is more sensitive to oxidation than that in the duplex form,10 the oxidized G4 can be a target for cancer therapy. Our analysis here suggests that the presence of an 8-oxoG in the G4 structure destabilizes and changes the structure. The stability of G4 in vitro is believed to correlate with the functions of G4 in the cell, such as its effects on replication and transcription.4, 49 Nonetheless, a recent report suggests that a replication error due to the stalling of DNA polymerase may occur when the G4 structure is too unstable to form under physiological conditions in vitro.50 Our QSTR analysis classified the effects of G4 topology on replication according to the analysis of the slope of the lnks and −∆G°37 values.7 Because lnks is proportional to −∆G‡/RT (∆G‡: activation free energy, R: gas constant, and T: temperature), ∆lnks equals −∆∆G‡/RT. Thus, the slope of the QSTR plot (∆lnks/−∆∆G°37) can be expressed as ∆∆G‡/RT∆∆G°37. This relation means that the difference in the slope of the QSTR plot denotes the difference in activation energy for resolving the G4 structure by KF per stability of the structure. The linear form of the oxidized G4s in the QSTR plot differed from that of the native G4s (Figure 4B). The slope of the linear form of the oxidized G4s was −0.71, whereas that from native G4s was −2.93. Based on the comparison with the slopes, resolving the native G4 structures via the processive activity of KF led to an activation free energy barrier (∆G‡37) ~4-fold higher than that for resolving oxidized G4s. We previously determined that both parallel G4s, including native G4s in this study and antiparallel G4s have ∆G‡37 ~3-fold higher than that for mixed G4.7 Consequently, ∆G‡37 for resolving the structure of oxidized G4 allowed for higher processivity of KF in Figure 7. Recovery of the functions of oxidized G4s by PyG3. (A) Images of denaturingcomparison with other G4s havPAGE gels showing replication of VEGF G2O in the presence or absence of 10 µM PyG3, ing parallel, antiparallel, or 10 µM UG3T2, and/or 5 µM MBP-NCL. Stalled products are highlighted with red squares. mixed topologies. Therefore, the (B) All the QSTR plots were adapted from Figure 5. The green plot shows oxidized G4 with mechanism of resolving an oxiPyG3. (C) The mechanisms of G4 formation by VEGF G5O, VEGF G8O, or VEGF G11O dized G4 is biophysically differwith PyG3, and denaturing-PAGE images showing replication products; “o” indicates 8ent from that of native G4s; this oxoG. (D) Images of denaturing-PAGE gels showing replication of telomere and Bcl2 G2O situation causes genetic dysfuncstructures in the presence or absence of PyG3. (E) Images of denaturing-PAGE gels showing tion owing to altered G4 funcreplication products from reactions containing 1 µM PyG3 or 1 µM PyG3-g. (F) Frequency tion, e.g., replication stalls and changes of QCM after binding of VEGF G2O with PyG3 (blue) to MBP-NCL immobilized transcription regulation. on the QCM plate. The frequency changes by VEGF native without PyG3 (pink, same as in PyG3 was able to recover the Figure 5B) are also presented as a reference. The arrows indicate the points of DNA injecstructure and stability of a G4 tions for each concentration (20, 120, 220, and 1220 nM). All the reactions were conducted containing the oxidative lesion, in a buffer consisting of 10 mM Tris-HCl (pH 7.5), 8 mM MgCl2, and 10 mM KCl at 37°C. via formation of the intermolecuThe letters “F,” “S,” and “P” on the left side of the gel images denote a “Full-length prodlar G4. QSTR analysis can be uct,” “Stalled product,” and “Primer,” respectively. applied to replication of the in-

An oxidative DNA lesion is a common form of damage and is important for the formation of genetic mutations. Given that mitochondria consume a considerable amount of oxygen when producing ATP; reactive oxygen species (ROS) are generated during this process. The generated ROS can be exported through aquaporin to the mitochondrial membrane.47 The sustained exposure to ROS under oxidative stress causes initiation of cancer as a result of increasing numbers of DNA mutations or DNA damage, genome instability, and cell proliferation.48 Furthermore, hypoxic conditions facilitate the formation of ROS in the mitochondria of malignantly transformed cells, with the result being that ROS also lead to malignant trans-



termolecular G4. The QSTR plot suggested that the plot of the oxidized G4 complexed with PyG3 fitted the line of native G4 but not the lines of oxidized G4s (Figure 7B). This result is quite reasonable because the intermolecular G4 structure with PyG3 and oxidized G4s formed a typical parallel G4 structure (Figures 6C and S6). This QSTR structure indicated that the recovery in topology and stability of G4 was followed by the intermolecular G4 formation with PyG3, which allows oxidized VEGF and c-Kit2 G4 to function in its native form by forming a parallel topology. Of note, in the presence of PyG3, the QSTR plots of VEGF G2O and G3O did not fit well the line (linear relation) of native G4s compared to VEGF G1O (Figure 7B). Similarly, c-Kit2 G1O and c-Kit2 G2O were found to deviate when compared to c-Kit2 G3O. Those slopes were −1.80 for VEGF G2O and VEGF G3O and −3.90 for cKit2 G1O and c-Kit2 G2O. Compared to the values of native G4s (−2.93), these values indicate that VEGF G2O and VEGF G3O have a 0.6-fold ∆G‡37 for resolving the structure by KF; c-Kit2 G1O and c-Kit2 G2O showed a 1.3-fold ∆G‡37. The difference in the effect of PyG3 on ∆G‡37 values implies that the pyrene moiety may interact with the replaced G-tract containing 8-oxoG. As shown in the fluorescence experiment, VEGF G3O yielded significantly lower intensity of the fluorescent signals (Figure 6B). This kind of additional interaction may affect the resolving mechanism and cause the difference in efficiency of a replication stall. Thus, QSTR analysis is useful for evaluating how the function of G4 in replication is recovered. Because the mechanism of the interaction is simple, there are many advantages in using PyG3 for oxidized G4s. PyG3 allows for adding modifications at each terminus. As we reported previously,46 a G4 ligand can localize to a specific G4 if a scaffold sequence adjacent to the G4 is attached. We observed here that PyG3-g is more efficient in stalling replication than intact PyG3 is (Figure 7E). To control the gene expression, it is necessary to target the oxidized G4 sequence within the duplex region. Because the stability of the oxidized G4 should be lower than that of the unoxidized one, the region may form a duplex with the complementary sequence, which does not allow for hybridization between the template strand and the guide sequence of PyG3-g. To promote the intermolecular G4 formation by PyG3 in the duplex, attachment of a peptide nucleic acid (PNA) to the terminus of PyG3 may be a reliable approach. PNA can invade and open the duplex and hybridize the target sequence in the duplex.51 This technique has been already applied to induce G4 in a duplex.52 It is easy to attach PNA to PyG3. Thus, a highly specific G4 recovery can be implemented because the PNA moiety invades the sequence neighboring the G4 sequence and enables the oxidized G4 sequence to freely interact with the PyG3 moiety. The oligonucleotide delivery to a cancer cell nucleus is a challenge for therapeutic applications. In the field of the siRNA technology, the development of a carrier of functional nucleotides and the technology of endosomal escape in cancer cells have dramatically advanced.53-54 Because PyG3 and PyG3-g have a molecular size similar to that of siRNAs, the development of a technology for delivery should be useful for the pyrene-modified oligonucleotides. Once an oligonucleotide is released into the cytosol, the oligonucleotide will spontaneously move into the nucleus as long as the oligonucleotide has an adequate modification such as a phosphorothioate backbone and conjugation with a nucleus localization signal.5556 Because the chemistry of the formation of a G-quadruplex is

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highly resistant to such modifications,57-58 PyG3 can be a useful platform to control the formation of oxidized G4 in various cells. Furthermore, our concept of formation of an intermolecular G4 can be generally applied to any other base lesion that destabilizes the G4 structure. For example, DNA polymerase tends to incorporate adenine opposite 8-oxoG.23 This action results in a mutation from G to T and destabilizes G4 formation thereby permanently affecting the gene expression. Given that the intermolecular G4 formation with PyG3 depends on the stability of the destabilized G4, the recovery of G4 formation may be achieved for not only oxidized sequences but also mutated ones. Actually, in the presence of PyG3, the stability of T mutants of VEGF (VEGF GXT) recovered, and replication reactions were effectively stalled by the recovery of G4 formation reactions (Figure S14). These results imply that the oxidation of G4 can disrupt G4 formation not only short-term but also for an extended period, and this formation reaction can be recovered by the PyG3 technique. Our QSTR analysis indicates that PyG3 changed the topology of the target G4 sequence. It has been reported that the antiparallel G4 strongly stalls replication and can effectively induce genetic instability even though the stability of the structure is quite low.7, 50 In the present study, PyG3 drove the topology into the parallel form. This kind of topological control may regulate the processivity of replication that causes epigenetic arrangement of genetic information.50 Thus, the technology based on the trans-acting guanine tract can serve as a novel tool to tune genetic information via intermolecular G4 formation. In conclusion, the oxidized G4 showed low stability and different biological functions because of changes in the structure compared to the native structures. Our pyrene-conjugated oligonucleotides could stabilize and recover the topology of the oxidized G4, which enabled it to become biologically active and to function as the native ones. The combination of the quantitative evaluation of biophysical properties and design of an oligonucleotide-based drug are promising approaches and can be applied to therapeutics for genetic diseases such as cancers.

METHODS AND MATERIALS Materials. dNTPs were purchased from Takara Bio (Japan). Ligands were dissolved in Milli-Q water or dimethyl sulfoxide. Other reagents were purchased from Wako Pure Chemical Industries (Japan) and used without further purification. Oligonucleotides. HPLC-purified fluorescein (FAM)labeled primer and template DNAs were purchased from Japan Bio Service (Japan). All DNA sequences used in this study for replication assays are listed in Table S1. PyG3 was synthesized as reported previously.31 Recombinant Proteins. The gene encoding Klenow Fragment (KF) was amplified from E. coli JM109 genomic DNA by polymerase chain reaction (PCR). PCR was carried out with PrimeSTAR DNA polymerase (Takara Bio) and primers (5′-GGGACCATATGGTGATTTCTTATGACAACTACG-3′ and 5′-GGGAGAATTCTTAGTGCGCCTGATCCCAG-3′) purchased from Eurofin Genomics (Japan). The amplified DNA fragments were digested with NdeI and EcoRI and cloned into the pMal-p5x vector (New England Biolabs). To


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express KF exo- (D355A, E357A) without the 3′→5′ exonuclease activity, the constructed plasmid was mutated using a QuikChange Mutagenesis Kit (Stratagene). E. coli EG2523 (New England Biolabs) was transformed with the mutated vector. The cells were cultured in the Luria-Bertani (LB) medium containing ampicillin at 37 °C to optical density at 600 nm (OD600) of approximately 0.5, and KF expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and further culturing for 3 hours. The cultured cells were harvested and lysed by sonication. The soluble fraction of the lysate was loaded on the column packed with amylose resin (New England Biolabs). After treatment with the Factor Xa protease to cleave the MBP-tag, KF exo- was purified on a HiTrap Heparin column followed by a HiLoad Superdex 200 column (GE Healthcare). Purified KF exo- was dialyzed against a buffer consisting of 50 mM Tris-HCl (pH 7.2), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, and 50% glycerol and was stored at −30 °C until use. The concentration was determined by UV absorbance at 280 nm with a molar extinction coefficient of 58,790 M−1 cm−1. The gene of truncated human nucleolin was constructed for expression as a fusion with maltose-binding protein (MBPNCL) in E. coli by Eurofins Genomic Service (Tokyo, Japan). The truncated genes were subcloned into pMal-c5x (New England Biolabs, UK) at the NdeI and BamHI sites. The resulting plasmid was transfected into E. coli ER2523 (New England Biolabs). Transformants were cultured in the LB medium including ampicillin, and expression of the nucleolin construct (MBP-NCL) was induced with the addition of 1 mM IPTG. The cells were harvested and lysed by sonication. MBPnucleolins were purified from the soluble fraction of the extract using amylose resin (New England Biolabs), and the eluate was loaded on a HiLoad 16/600 Superdex 200 pg column (GE Healthcare, USA), followed by further purification on a HiTrap Heparin HP column (GE Healthcare). MBP-NCL was eluted with a gradient of NaCl. Fractions were collected and dialyzed against a buffer consisting of 20 mM sodium phosphate (pH 7.0), 100 mM NaCl, and 1 mM Na2EDTA. Purified MBP-NCL was dialyzed against another buffer (50 mM TrisHCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, and 50% glycerol) and stored at −30 °C until use. The concentration was determined from the UV absorbance at 280 nm using a molar extinction coefficient of 86,460 M−1 cm−1. Replication Assays. Before addition of KF exo-, the primer and template DNA were annealed in the replication reaction buffer: 10 mM Tris-HCl (pH 7.5), 8 mM MgCl2, 1 µM KF exo-, 1 µM template DNA, 10 mM KCl, and 250 µM dNTPs. In the presence of 10 µM PyG3, the KCl concentration was adjusted to 50 mM. After preparation of the solution, the mixtures were incubated at 37 °C. The reaction was quenched with the addition of 10 mM EDTA and 80% (wt) formamide. Products were separated by PAGE in a gel containing 8 M urea at 200 V and 70 °C for 1 hour in TBE buffer. The gel images were captured using a Fluoreimager FLA-5100 (Fujifilm) before and after staining with SYBR Gold (Thermo Fisher Scientific). Band intensities were determined in the NIH ImageJ software. The amount of full-length product (P) was quantified as the intensity ratio of the full-length product band to all bands. The kinetic model was assumed to be the two-step sequential model:

→ → where P0 is the starting state of the reaction, Ps is the state immediately after dissolution of the stall caused by noncanonical structure is removed, Pf is the state after replication is complete, ks (min−1) is the rate constant of dissolution of the stall from the reaction start by KF, and kf (min−1) is the rate constant of the full-length product after the replication stall is resolved. Rate constants were calculated via a global fit in Dynafit (Biokin). A CD Melting Assay. For melting analyses, 10 µM DNA with or without 10 µM PyG3 was added to the buffer consisting of 10 mM Tris-HCl (pH 7.5), 8 mM MgCl2, and 10 or 50 mM KCl. The melting analyses were performed using a JASCO J-1500 equipped with a temperature control system. Samples were cooled from 90 to 0 °C at −1.0 °C min−1, and then the temperature was increased from 0 to 90 °C at 0.5 °C min−1. To determine thermodynamic parameters, the CD melting curves were normalized and analyzed by curve fitting in Kaleida Graph (Synergy Software). CD Spectrum Acquisition. For CD spectroscopy, 10 µM DNA with or without 10 µM PyG3 was added to the buffer consisting of 10 mM Tris-HCl (pH 7.5), 8 mM MgCl2, and 10 or 50 mM KCl. Samples were heated to 95 °C for 3 min and cooled to 0 °C at −1.0 °C min−1, and then the temperature was increased from 0 to 90 °C at 0.5 °C min−1. The CD spectra were recorded by means of a JASCO J-1500 at 10 °C intervals from 0 to 90 °C.

NMR Spectroscopy. Oligonucleotides were dissolved in 10 mM K2HPO4 buffer (pH 7.5) with the addition of 50 mM KCl and 10% 2H2O. The final DNA concentrations of individual oligonucleotides in a 600 µL mixture were 100 µM except for VEGF GXO–PyG3 mixtures, which were at a 1:2 ratio: 100 µM VEGF GXO and 200 µM PyG3. All the samples were annealed prior to transfer into NMR tubes. All the spectra were acquired at 25 °C on an Agilent VNMRS 800 MHz NMR spectrometer equipped with a cold probe. QCM Measurements. These measurements of the binding of DNAs to MBP-NCL were performed using an AFFINIX Q4 (Initium, Japan). MBP-NCL was dialyzed against 50 mM HEPES-NaOH (pH 7.5) containing 10 mM NaCl at 4 °C and reacted with EZ-Link™ Sulfo-NHS-Biotin (1:1 molar ratio) (Thermo Fisher Scientific, USA) for 1 hour. The reaction was quenched with the addition of 1/100 volume of 1 M Tris-HCl buffer (pH 7.5), then dialyzed against 50 mM HEPES-NaOH (pH 7.5) containing 10 mM NaCl at 4 °C, and next purified on a PD-10 column (GE Healthcare). The biotinylated MBP-NCL was immobilized in the NeutrAvidin-coated QCM cell. After immobilization, the solution in the cell was replaced with replication buffer (50 mM KCl). DNA solutions (100 µM) were annealed in the same buffer and injected into the QCM cell, and frequency changes (∆F) were recorded at 37 °C. To determine the dissociation constant (Kd), ∆F values were plotted against the concentration of the injected DNA. Each series of plots was fitted to the equation of the Langmuir adsorption model59:



∆ × [] ( + []) where ∆Fmax is the maximal change of frequency at an infinite concentration of DNA. ∆ =

Regarding the dissociation assay of PyG3-g, the DNA complexed with a biotinylated primer and the VEGF G2O template was immobilized on the NeutrAvidin-coated QCM cell at 25 °C. After rinses with the buffer consisting of 10 mM Tris-HCl (pH 7.5), 8 mM MgCl2, and 50 mM KCl, 2 µM PyG3-g was injected. The excess amount of PyG3-g was removed by rinsing the QCM cell with the same buffer, and we recorded the frequency change at 25 °C for ~24 h.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Sequences used in this study, CD spectra, NMR spectra, CD melting data, denaturing PAGE results, and the time course analyses of the replication products (Table S1 and Figures S1–S14)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Tel.: +81 78-303-1457. Fax: +81 78-303-1495.

Notes The authors declare that they have no competing financial interests.

ACKNOWLEDGMENT We thank Ms. M. Kakuda, Ms. J. Inoue, Ms. A. Matsuyama, and Dr. A. Haghparast for help with the experiments. This work was supported by the Grants-in-Aid for Scientific Research on Innovative Areas “Chemistry for Multimolecular Crowding Biosystems” from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan and the Japan Society for the Promotion of Science (JSPS) (KAKENHI Grant No. JP17H06351). Additional support was provided by the JSPS A3 Foresight Program “Asian Chemical Probe Research Hub,” the MEXT-Supported Program for the Strategic Research Foundation at Private Universities (2014–2019), Japan, the Hirao Taro Foundation of Konan Gakuen for Academic Research, the Okazaki Kazuo Foundation of Konan Gakuen for Advanced Scientific Research, the Chubei Itoh Foundation, and the Shimadzu Science Foundation. BHK thanks NRF of Korea (2015M3A9B8029067) for the financial support.

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Recovery of the Formation and Function of Oxidized G-Quadruplexes

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