Transcription Driven by Reversible Photocontrol of Hyperstable G

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Transcription Driven by Reversible Photocontrol of Hyper-stable G-quadruplexes Shinzi Ogasawara ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00216 • Publication Date (Web): 15 Oct 2018 Downloaded from http://pubs.acs.org on October 17, 2018

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Figure 1. (a) Cartoon representing a reversible regulation of hyper-stable G-quadruprex by photoisomerization of 8PVG. (b) Time course of trans to cis and cis to trans pho-toisomerization of 8PVG contained in GQ 2. Trans isomer rates ware calculated from absorbance at 413 nm. (c) Switching cycles between trans and cis by alternate illumination with 460 nm and 370 nm light for 2 min and 30 sec, respectively. (d) Normalized fluorescence intensity, (e) melting curves and (f) CD spectra of 8PVG-modified G-quadruplex (GQ 5). (g) Reversible switching between hyper-stable and non-structured state of GQ 5 monitored by ellipticity at 295 nm. 184x327mm (300 x 300 DPI)

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Figure 2. (a) Illustration of the template used in the polymerase stop assay. (b) Gel image and (c) relative efficiency for production of full-length RNA, including slipped RNA, in transcription of each template. Trans and cis forms were obtained by illuminating template DNA with 370 nm light for 30 sec and 460 nm light for 2 min before the transcription reaction. Mismatch means transcription of GQ-installed template with complementary strand for linear sequence. The transcription efficiency and standard error values were calculated from the results of three independent experiments.


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Figure 3. (a) Design of template containing two G-quadruplexes. (b) Relative efficiency for production of full-length RNA, including slipped RNA, in transcription of template containing one or two G-quadruplexes. (c) Reversible photoregulation of transcription using template containing two GQ 4. The reaction mixtures were illuminated with 370 nm light at time 0 and 40 min (black arrowheads) and with 460 nm light at the 20 and 60 min time points (white arrowheads). (d) Luciferase activity of each template DNAs containing linear sequence, two GQ 1 and two GQ 4 in rabbit reticulocyte lysate. Transcription efficiency or luciferase activity and standard error values were calculated from the results of three independent experiments.



Figure 4. (a) Illustration for the construction of plasmid installed two G-quadruplexes into downstream of the CMV promoter. (b) Fluorescence images and (c) average fluorescence intensity of zebrafish embryos injected with non-modified plasmid (n = 48), GQ 1–modified plasmid (n = 44), or GQ 4–modified plasmid (trans form; n = 46, cis form; n = 44) at 24 hours post fertilization (hpf). The average fluorescence intensity of entire embryo and standard error were calculated from the results of three independent experiments. Scale bar, 0.5 mm. (d) Spatial control of Venus expression in an embryo injected with GQ 4– modified plasmid by spot illumination with 460 nm light for 2 min at 15 hpf. Scale bar, 0.5 mm. (e) Time course of fluorescence intensity in reversible photo-activation of GQ 4–modified plasmid. The plasmid was illuminated with 370 nm light for 30 sec before injection. Red line: non-illuminated embryo, blue line: embryo illu-minated with 460 nm light for 2 min at 1 hpf, green line: embryo illuminated with 460 nm light for 2 min at 1 hpf and with 370 nm light for 30 sec at 4 hpf. 180x70mm (300 x 300 DPI)


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Transcription Driven by Reversible Photo-control of Hyper-stable Gquadruplexes Shinzi Ogasawara†,‡ †PRESTO, ‡Graduate

Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, JAPAN

School of Science, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo, 060-0810, JAPAN

Supporting Information Placeholder ABSTRACT: G-quadruplexes occur in promoter regions,

5’-untranslated regions of mRNA and telomeric regions, and they function as regulatory elements for various key biological events, such as transcription, translation, and telomere elongation. As the stability of G-quadruplexes dramatically impacts these biological processes, controlling G-quadruplex stability via external stimuli such as light enables regulation of important biological phenomena with high spatial and temporal resolution. Here, we report a method for reversible photoregulation of transcription by controlling the stability of Gquadruplexes via cis-trans photoisomerization of photochromic nucleobase (PCN). Transcription was effectively inhibited when the PCN-modified Gquadruplex was in a hyper-stable state, whereas transcription activity recovered markedly when the Gquadruplex changed to an unstable state induced by trans to cis PCN photoisomerization. Moreover, a reversibly photo-activatable plasmid was constructed by introducing PCN-modified G-quadruplexes downstream of the cytomegalovirus promoter of the pCS2 plasmid, which was used to demonstrate photoregulation of gene expression in zebrafish embryos.

KEYWORDS: G-quadruplex, transcription, gene expression, optochemical regulateon

Guanine-rich nucleic acids can spontaneously form non-canonical structures called G-quadruplexes. Gquadruplexes are stabilized by stacking of G-quartets, which are planar configurations consisting of four guanines connected to each other through Hoogsteen hydrogen bonding under physiologic conditions in the presence of monovalent cations such as K+ and Na+.1-3 Bioinformatic studies have revealed that regions of potential G-quadruplex formation are present near promoter regions in many organisms, including mammals,4-6 protozoa,7 yeast,8, 9 plants,10, 11 and

bacteria,12 suggesting that G-quadruplexes play an important role in modulating transcription. This hypothesis is supported by evidence showing that addition of small-molecule ligands13 that stabilize Gquadruplexes downregulates several genes, including CMYC,14 CKIT,15 VEGF,16 and KRAS.17, 18 Therefore, controlling the stability of G-quadruplexes located near promoter regions via external stimuli presents a promising strategy for regulating transcription. The simplest mechanism of transcription modulation by Gquadruplexes involves “arrest” (i.e., inhibition) of RNA polymerase progression due to steric hindrance. Several reports based on this mechanism described control of transcription by stabilization of G-quadruplexes inserted downstream of promoter regions of genes of interest by addition of ligands19 and proteins.20, 21 However, these methods allow for only a single off-to-on regulation event, because the reagents added as triggers cannot be removed from the system. Moreover, spatial and temporal resolution is very low. An alternative and more-ideal trigger to enable control of G-quadruplex stability is light, as the duration, location, and dose of light stimulation can be accurately and easily controlled.22 Although incorporation into nucleic acid sequences of cage compounds that can be dissociated by photo-illumination is a useful way to control the stability of G-quadruplexes using light, these methods are also irreversible, as the uncaged nucleic acid produced by light illumination cannot be re-caged in a cell.23-25 In contrast, we previously reported a method enabling reversible control of the stability of G-quadruplexes in thrombin aptamers.26 This method involves site-specific installation of photochromic nucleobases (PCNs) that reversibly isomerize following light illumination. Here, I describe a novel photo-activation method for transcription through reversible photo-control of hyperstable G-quadruplexes inserted downstream of the promoter region in a gene of interest. RESULTS AND DISCUSSION


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Photoregulation of Hyper-stable G-quadruplexes. The higher the G-quadruplex stability, the greater the inhibition of RNA polymerase progression.27 The stability of G-quadruplexes depends strongly on the number of G-quartets and their associated topology, whether of parallel, antiparallel, or parallel/antiparallel hybrid type. One of the most stable G-quadruplexes reported to date is the antiparallel type including four Gquartets (GGGGTTGGGGTGTGGGGTTGGGG), which exhibits a Tm value >95°C.27 Therefore, we used this type of G-quadruplex in our method. Moreover, since introducing PCNs into G-quadruplex increases its stability,26 we predicted that PCN-modified Gquadruplex indicates hyper-stability and can effectively regulate transcription in reversible manner with light. 8Pyrenylvinyl deoxyguanosine, 8PVG, was chosen as the PCN for in vivo use because it can be isomerized by nontoxic UV-A light.28, 29 The details of 8PVG synthesis were previously reported. 28, 29 8PVG was incorporated into the oligonucleotides shown in Table 1 using standard automated DNA synthesis protocols. Table 1. Oligonucleotide sequences used in this study.

Entry

Sequences (X = 8PVG)

GQ 1

5’-GGGGTTGGGGTGTGGGGTTGGGG-3’

GQ 2

5’-GGGGTTGGXGTGTGGGGTTGGGG-3’

GQ 3

5’-GGGGTTGGXGTGTGGXGTTGGGG-3’

GQ 4

5’-GGXGTTGGGGTGTGGGGTTGGXG-3’

GQ 5 Linear

5’-GGXGTTGGXGTGTGGXGTTGGXG-3’ 5’-TATTTGGGTTGTAACTATCGAGG-3’

Reversible photoisomerization of 8PVG introduced into the G-quadruplex was initially investigated using UV/Vis spectroscopy and HPLC. Trans to cis and cis to trans isomerization of 8PVG under 460 nm and 370 nm light reached photostationary state within 90 sec at 90% conversion and 10 sec at 93% conversion, respectively (Figure 1b). The cis form of 8PVG showed no thermal isomerization even at 60°C (Figure S2). Reversible switching was repeated 50 times by alternate illumination with 460 nm light for 2 min and 370 nm light for 30 sec; good reversibility of trans-cis photoisomerization was observed without any side reactions, as shown in Figure 1c. The drastic change in fluorescence intensity upon trans-cis photoisomerization was observed. (Figure 1d). Moreover, the fluorescence spectrum of GQ 5 shifted to long wavelength compared with that of GQ 2 when 8PVG is in the trans form, indicating stack of pyrene units. Such red shift disappeared when 8PVG was isomerized to cis form by illumination with 460 nm light. Next, we evaluated how the isomerization of 8PVG affected the structure of Gquadruplexes using circular dichroism (CD)

spectroscopy and UV-monitored thermal denaturation analysis. In the presence of K+, the native sequence (GQ 1) was characterized by a negative peak near 265 nm and a positive peak near 295 nm in the CD spectrum (Figure 1f), indicating an antiparallel G-quadruplex configuration. G-quadruplexes modified with one or two 8PVG, GQ 2–GQ 4, also exhibited an antiparallel configuration when 8PVG was in both the trans and cis forms (Figure S1). By contrast, a drastic conformational difference between the trans and cis forms was observed in GQ 5. GQ 5 showed an antiparallel G-quadruplex CD signature when 8PVG was in the trans form. However, these peaks were lost after photoisomerization to the cis form, indicating that GQ 5 cannot form a G-quadruplex when 8PVG adopts the cis form. In the melting curve of GQ 1, hypochromicity due to G-quadruplex denaturation was observed at temperatures >90C (Figure 1e). Conversely, there was no hypochromicity in the melting curve of both the trans and cis forms of GQ 5, suggesting no structural changes occurred over the temperature increase. From these results, we concluded that GQ 5 with 8PVG in the trans form is not denatured even at 100°C, whereas G-quadruplexes cannot be formed even at 20°C when the 8PVG is in the cis form . G-quadruplex formation and denaturation of GQ 5 in trans and cis form of 8PVG was also confirmed by NMR (Figure S3). Such hyperstability in the trans form was also found in GQ 3 and GQ 4 (Figure S1). However, when 8PVG was isomerized to the cis form, the Gquadruplexes became destabilized, as evidenced by the appearance of gradual hypochromicity over a broad range of temperatures. Polymerase Stop Assay of GQ-Installed Templates. Hyper-stable G-quadruplexes modified by 8PVG are the most suitable for inhibiting transcription via an arrest mechanism. We thus used a polymerase stop assay to examine the difference in transcriptional efficiency between the trans and cis forms of 8PVG installed into Gquadruplexes. Random sequences (linear) and nonmodified or modified G-quadruplexes were integrated 35 bases downstream of the T7 promoter of a synthetic template (Figure 2a). Templates were used in polymerase stop assay with complementary strand. The formation of G-quadruplex competes with the formation of duplex. Therefore, the length of transcript greatly depends on which structure is thermally stable. The results of gel electrophoresis analysis indicated that transcription of the template containing the linear sequence yielded full-length 70-nt RNA, whereas transcription of the GQ 1-containing template was only 65% efficient with longer slipped transcripts and 35-nt RNA corresponding to products arrested by the Gquadruplex (Figure 2b, c). Stabilization of GQ 1 with ligand (BRACO-19) increased the arrested products (Figure S4). In contrast, 91% of products were fulllength, including the slipped transcript, in transcription



of the template containing GQ 2 with the cis form of and the efficiency decreased to 38% when 8PVG was in the trans form. A transcription assay using short templates confirmed that the pyrenyl group of 8PVG does not affect the transcription efficiency in either the trans or cis forms (Figure S5). Therefore, the difference in transcriptional efficiency between the trans and cis forms of 8PVG in producing full-length RNA arises from differences in the stability of the G-quadruplexes. Thermal denaturation experiments revealed that the stability of GQ 2 with 8PVG in the cis form was lower than that of GQ 2 with 8PVG in the trans form (Figure S1). Once G-quadruplex is destabilized by trans to cis photoisomerization of 8PVG, G-quadruplex is unfolded by forming duplex with complementary sequence and full-length RNA is yielded. Therefore, production of full-length RNA from GQ-installed template was reduced by abolishing the ability of duplex formation using mismatch strand (i.e., complementary strand of linear sequence) (Figure 2c, Figure S6). As GQ 3, GQ 4, and GQ 5 can form hyper-stable G-quadruplexes when 8PVG adopts the trans form, production of full-length RNA was efficiently suppressed. In contrast, destabilization of those G-quadruplexes by isomerization to the cis form recovered production of full-length transcripts. In particular, GQ 4 exhibited the highest photomodulation efficiency, as production of transcripts of full-length RNA when 8PVG was in the cis form was 5.8 times higher than when 8PVG was in the trans form. The difference in suppression rate between GQ 3 and GQ 4 with the trans form of 8PVG may derive from differences in the difficulty of unwinding Gquadruplexes during transcription. One mechanism for stabilizing G-quadruplexes by installing PCN involves stacking PCNs between two aromatic compounds such as fluorene and pyrene, in this case, when PCN is in the trans form.26 Therefore, it is difficult to unwind the Gquadruplex in GQ 4 when the stacking position is near the base of the G-quadruplex, thus effectively inhibiting RNA polymerase progression. Tandem G-quadruplexes enhance Inhibition of RNA polymerase progression. Based on the above results, GQ 4 was used as a photo-controllable Gquadruplex in all subsequent experiments. We designed templates in which two G-quadruplexes were introduced downstream of the T7 promoter to test whether inhibition was enhanced (Figure 3a). For transcription of the template containing two GQ 4 with 8PVG in the trans form, production of the full-length 105-nt RNA was dramatically reduced compared with that of single GQ 4; a new band of approximately 64-nt appeared, corresponding to a transcript produced when RNA polymerase progression was stopped by the second Gquadruplex (Figure 3b, Figure S8). A 23-fold difference in production of full-length RNA was observed between the trans and cis forms. Reversible photoregulation of 8PVG,

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transcription was subsequently performed using a template with two GQ 4. In advance, it was confirmed that illumination with 370 nm and 460 nm light does not affect the transcription system (Figure S7). Transcription of full-length RNA was inhibited until the reaction mixture was illuminated with 460 nm light at the 20 min time point, when 8PVG was in the trans form due to illumination with 370 nm light at the start of the experiment (Figure 3c, Figure S9). Production of fulllength transcripts rapidly increased after illumination with 460 nm light. Subsequent illumination with 370 nm light at 40 min resulted in cessation of full-length transcript production, suggesting that RNA polymerase progression was inhibited by the GQ 4 quadruplex. Production of full-length transcripts increased again after second illumination with 460 nm light at the 60 min time point. These results indicate that the stability of G-quadruplexes can be reversibly controlled by cis-trans photoisomerization of 8PVG in the transcription system. Next, luciferase DNA installed two GQ 4 in downstream of the T7 promoter was constructed and tested for its ability to modulate the expression in rabbit reticulocyte lysate. As we expected, GQ 4 with 8PVG in the cis form yielded larger amount of protein than in the trans form (Figure 3d). Photoregulation of Gene Expression in Zebrafish Embryo. To demonstrate the applicability of my method to photo-control of gene expression, a photo-controllable Venus plasmid (pCS2-Venus) in which two GQ 4 were introduced into downstream of the cytomegalovirus (CMV) promoter was constructed and applied to zebrafish embryos (Figure 4a). Plasmid (50 pg) was injected into one-cell stage zebrafish embryos by microinjection. For isomerization of 8PVG, the embryos were illuminated with 370 nm or 460 nm light for 30 sec or 2 min, respectively, with a 100-W metal halide lamp under a confocal microscope. It was found in a previous study that illumination with 370 nm UV light does not adversely affect development of zebrafish embryos.30 After 24 h of incubation, fluorescence associated with Venus expression was observed. The fluorescence intensity of embryos injected with GQ 4–modified plasmid and illuminated with 460 nm light recovered to 72% of that of embryos injected with non-modified plasmid, whereas the fluorescence intensity of embryos injected with GQ 4–modified plasmid and illuminated with 370 nm light was reduced to near background levels (Figure 4b, c). A 68-fold difference in Venus expression levels was observed between the trans and cis forms of 8PVG. The spatial control achievable using this system was tested by inducing local Venus expression in an embryo by illumination with a 460 nm spotlight created by a patterned photostimulator. Strong fluorescence was observed only in the illuminated area (Figure 4d). Moreover, reversible photo-activation of the GQ 4–modified plasmid was performed in embryo by


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alternate illumination with 460 nm and 370 nm light (Figure 4e). The rate of increase in fluorescence intensity increased or decreased after illumination with 460 nm or 370 nm light, respectively, suggesting that Venus was expressed or suppressed by destabilization or stabilization, respectively, of the GQ 4 quadruplex via cis-trans photoisomerization of 8PVG. CONCLUSION In summary, we developed a method for reversible photoregulation of transcription via photo-control of Gquadruplex stability using cis-trans isomerization of 8PVG. 8PVG can be reversibly photoisomerized by alternate illumination with 460 nm and 370 nm light without any side reactions in G-quadruplexes and switching between a non-structured and hyper-stable Gquadruplex state in a reversible manner based on isomerization. By introducing two photo-controllable Gquadruplexes, QG 4, downstream of the T7 promoter, transcription could be controlled by light illumination in an in vitro transcription system with a very high on/off ratio. Moreover, the applicability of this technique to complex biological system was demonstrated by regulating gene expression in zebrafish embryos using a photo-controllable plasmid containing two QG 4 downstream of the CMV promoter; spatial and temporal activation and inactivation of gene expression were also demonstrated. Since the injected photo-controllable plasmid is diluted due to cell division, our method need to be used in conjunction with drug delivery system that can persistently supply a nucleic acid for a week31 in order to control gene expression for long periods of time in vivo. This strategy controlling inhibition of RNA polymerase progression using photo-controllable Gquadruplexes could be applied to any promoter. Additionally, the photo-control of hyper-stable Gquadruplexes could be applied to other nucleic acids such as aptamers,32, 33 DNAzymes,34 mRNAs,35-37 and telomeres,38, 39 as well as to nanotechnologies such as DNA computing,40, 41 DNA machines,42 and construction of photo-controllable DNA structures.43, 44 METHODS Photoisomerization. Cis-trans photoisomerization of 8PVG incorporated into G-quadruplexes was performed in an aqueous solution containing 5 M oligonucleotide, 10 mM potassium phosphate (pH 7.2), and 30 mM KCl at room temperature using a 300 W xenon lamp (MAX-302, ASAHI SPECTRA), which can extract a specific wavelength with a 10 nm peak width at half height by employing an adequate bandpass filter (MX0370, MX0460, ASAHI SPECTRA). Light (370 or 460 nm) from the xenon lamp (70% intensity) was applied to a solution from a 3 cm distance for 30 sec for cis to trans or 2 min for trans to cis isomerization, respectively.

CD spectroscopy and thermal denaturation experiments. CD spectra were acquired on a JASCO J-720 spectropolarimeter over the wavelength range 200-320 nm at 20°C in a 1 cm path length cuvette. Each trace was the average of 3 scans at 50 nm/min, with a 1 s time constant, 0.1 nm step resolution, and 1 nm bandwidth. The Tm curves were measured by monitoring the absorbance at  = 295 nm on a JASCO model V-730 BIO spectrophotometer equipped with a programmable temperature controller using a 1 cm path length cuvette. The absorbance of the samples was monitored from 20 to 100°C at a heating rate of 0.5°C/min. Polymerase stop assay. The reaction solution (20 L total volume) containing 1× reaction buffer, 1.5 M template DNA, 1 mM dNTPs, 5 mM DTT, 30 mM KCl, and 50 units of T7 polymerase (TAKARA) was incubated at 37°C for 90 min. After the reaction,  L of DNase was added to the mixture and incubate at 37°C for 30 min to hydrolyze the template DNA. Electrophoresis analysis was performed on a 15% denatured polyacrylamide gel in Tris-glycine buffer and run for 60 min with a field of 100 V at room temperature. Subsequently, the gel was stained with SYBR gold (Thermo Fisher Scientific). Luciferase reporter assay. NheI restriction site were cloned into luciferase DNA using PCR with the lucforward primer and the luc-reverse primer. PCR amplification was carried out using KOD-plus-DNA polymerase (TOYOBO). The reaction solution (300 L total volume) containing 1 x reaction buffer, 300 ng plasmid, 0.2 M dNTPs, 1 mM MgSO4, 6 l of KOD-pluspolymerase and 0.3 M primers was subjected to 35 cycles of amplification. The temperatures and incubation times were as follows: the sample was denatured at 96°C for 2 min (15 sec in subsequent cycles), cooled to 58°C for 30 sec to anneal the primers, and heated to 68°C for 90 sec for primer extension. After reaction, product was purified using Wizard SV Gel and PCR Clean-Up System (Promega). The purified product was digested with NheI (New England Biolads). GQ DNAs with complement strand were digested with NheI. The digested luciferase DNA and GQ fragment were mixed and annealed with 95°C for 5 min and slowly cooled to room temperature. The reaction mixture was ligated using ligase (New England Biolads). The ligated product was purified using E-Gel EX 1% Agarose (Thermo Fisher Scientific). In vitro transcription and translation were performed using TNT Quick Coupled Transcription/Translation System (Promega). Modified luciferase DNA (0.1 g) was added in reaction mixture (25 L total volume) containing 20 l of TNT Quick Master Mix, 0.5 l of 1mM methionine and 0.5 l of TNT PCR Enhancer and incubated at 30°C for 90 min. After reaction, the mixture was added in Luciferase Assay System (Promega) and measured with luminometer (AB-2270 Luminescencer Octa, ATTO Inc.) Construction of the GQ-modified plasmids. NheI and BamHI restriction site were cloned into pCS2-Venus using PCR with the forward primer and the reverse primer. PCR condition was the same as above except for primer extension time, 5 min. The purified product and GQ DNAs



were digested with NheI and BamHI (New England Biolads). The digested plasmid and GQ fragment were mixed and annealed with 95°C for 5 min and slowly cooled to room temperature. The reaction mixture was ligated using ligase (New England Biolads). The ligated product was purified using E-Gel EX 1% Agarose (Thermo Fisher Scientific). Photo-activation and inactivation of plasmid in zebrafish embryos. The 50 pg plasmids (100 ng/L) were injected into the yolk of one cell stage embryo using FemtoJet (eppendorf Inc.). After microinjection, embryos were cultured at 28.5 oC in Ringer’s solution containing 0.01 % kanamycin and penicillin/streptomycin. The plasmid-injected embryos were transferred to an incubation chamber on the stage of a confocal laser scanning microscope (LSM 710, Carl Zeiss) equipped with a 100-W metal halide lamp (X-Cite 120PC). Embryos were illuminated at 28.5°C with 370 nm or 460 nm light from the metal halide lamp (25% intensity) for 30 sec or 2 min, respectively. Local illumination of embryos with 460 nm light was carried out using a MAX-303 (70% intensity) equipped with a patterned photostimulator (Mightex Polygon400 Dynamic Spatial Illuminator, Mightex System) mounted on the side port of the LSM 710.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. CD spectra and Tm curves of 8PVG-modified Gquadruplexes, DNA sequences and supporting figures (PDF)

AUTHOR INFORMATION Corresponding Author

[email protected] Notes

The author declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by JST PRESTO Grant Number JPMJPR15P3, and JSPS KAKENHI Grant Number 16K14733.

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