Selective and Robust Stabilization of Triplex DNA Structures Using

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Selective and Robust Stabilization of Triplex DNA Structures using Cationic Comb-type Copolymers Asako Yamayoshi, Daisuke Miyoshi, Yu-ki Zouzumi, Yohei Matsuyama, Jumpei Ariyoshi, Naohiko Shimada, Akira Murakami, Takehiko Wada, and Atsushi Maruyama J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b01926 • Publication Date (Web): 31 Mar 2017 Downloaded from http://pubs.acs.org on April 3, 2017

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The Journal of Physical Chemistry

Title Selective and Robust Stabilization of Triplex DNA Structures using Cationic Comb-type Copolymers

Authors’ Names Asako Yamayoshi,a,b* § Daisuke Miyoshi,c* § Yu-ki Zouzumi,c Yohei Matsuyama,d Jumpei Ariyoshib,d, Naohiko Shimada,e Akira Murakamid, Takehiko Wadaf and Atsushi Maruyamae*

Affiliations a

The Hakubi Center for Advanced Research, Kyoto University, Yoshida-ushinomiyacho, Sakyo-ku,

Kyoto 606-8501, Japan b

Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho,

Sakyo-ku, Kyoto 606-8502, Japan c

Faculty of Frontiers of Innovative Research in Science and Technology (FIRST), Konan University,

7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan d

Department of Biomolecular Engineering, Graduate School of Science and Technology, Kyoto

Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan e

Department of Life Science and Technology, Tokyo Institute of Technology, 4259 B-57 Nagatsuta,

Midori-ku, Yokohama, Kanagawa 226-8501, Japan f

Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1, Katahira,

Aoba-ku, Sendai 980-8577, Japan

ACS Paragon Plus Environment


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§

These authors equally contributed to this work.

*Corresponding authors & E-mail Asako Yamayoshi The Hakubi Center for Advanced Research, Kyoto University, E-mail: [email protected] Daisuke Miyoshi Faculty of Frontiers of Innovative Research in Science and Technology (FIRST), Konan University E-mail: [email protected] Atsushi Maruyama Department of Life Science and Technology, Tokyo Institute of Technology E-mail: [email protected]


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Abstract DNA sequences capable of forming triplexes induce DNA double-strand breaks that have attracted attention in genome editing technologies (e.g., CRISPR/Cas9 system, TALEN, and ZFN). Therefore, novel functional tools that stabilize triplex DNA structures must be further investigated to spark renewed interest. In this study, we investigated the unique character of cationic comb-type copolymers for the selective stabilization of triplex DNA. The melting temperature (Tm) of triplex DNA increased from 24.5 ºC to 73.0 ºC (∆Tm = 48.5 ºC) by the addition of poly(allylamine)-graft-dextran (PAA-g-Dex) under physiological conditions (at pH 7.0), while PAA-g-Dex did not stabilize but rather destabilized the DNA duplex. On the other hand, poly(L-lysine)-graft-dextran (PLL-g-Dex) stabilized both the duplex and triplex structures at pH 7.0. Thermodynamic parameters evaluated by isothermal titration calorimetry (ITC) revealed that the binding constant (Ka) for the intermolecular triplex formation in the presence of PAA-g-Dex was 1.1 × 109 M-1 at 25 ºC which is more than 10 times larger than that in the presence of PLL-g-Dex (8.6 × 107 M-1). Stabilizing activity and selectivity of cationic copolymers toward DNA assemblies were successfully controlled by selecting appropriate backbone structures of the copolymer. Various functional molecules that stabilize DNA duplexes have been developed and used in biological research. However, there are few cationic polymers that stabilize triplex DNA selectively. This study indicates that PAA-g-Dex has great potential to regulate the biological activities of triplex DNA.



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Introduction Dynamical structural changes in nucleic acids play important roles in human cells. Such changes in the constitutions of nucleic acid structures are involved in gene regulation. For example, cruciform DNA (Holiday junction), Z-DNA, and triplex DNA have several biological functions, including gene expression, replication pausing, and recombination.1-3 In particular, naturally occurring sequences capable of forming triplex DNA (e.g., triplet repeat symmetry and purine-pyrimidine tracts) are found in the human genome as frequently as 1 in every 50,000 base pairs (60,000 total locations). Some of them affect the progression of DNA replication forks, initiating a stalled replication fork and leading DNA double-strand breaks (DSBs).4,5

DSBs have

attracted attention in genome editing technologies (e.g., CRISPR/Cas9 system, TALEN, and ZFN) because gene repair pathway for DSBs stimulate short DNA insertions or deletions by homologous recombination or non-homologous end joining.2,6-8 Furthermore, triplex-forming oligonucleotides (TFOs) that can bind the double-stranded DNA and inhibit transcription have received considerable attention for their potential application in controlling a particular gene expression.

8

Therefore,

development of novel functional molecules that stabilize triplex DNA structures is of particular interest as candidates for gene editing tools. We have developed various kinds of cationic comb-type copolymers comprising a polycationic backbone with hydrophilic side chains and have discovered that the copolymers increase the stability of both duplex and triplex DNAs under physiological conditions.9-11 Furthermore, it was recently revealed that one of our cationic comb-type copolymers, poly(allylamine)-graft-dextran (PAA-g-Dex), induced a reversible B-A transition of DNA duplex, regulated by polymer binding that caused the dehydration of DNA duplex.12

Thus, we speculate

that PAA-g-Dex has potential to selectively stabilize triplex DNA stabilize through formation of


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Hoogsteen base-pairs that release water molecules upon the base pair formation.13 Herein, we report the unique effects of the cationic backbone structures on triplex DNA stabilization and interesting findings are disclosed.

MATERIALS AND METHODS All oligonucleotides described in this study (sequences are shown in Figure 1a) were purchased from Gene Design, Inc. (Osaka, Japan). Other reagents were purchased from Wako Pure Chemicals Inc. (Osaka, Japan) and used without further purification. Preparation of the comb-type copolymers, poly(L-lysine)-graft-dextran (PLL-g-Dex) and poly(allylamine)-graft-dextran (PAA-g-Dex) have been previously described in detail.14 Briefly, Dex was covalently coupled with pendant amino groups of PLL or PAA by a reductive animation reaction in borate buffer (pH 8.5) using sodium cyanoborohydride as a reductant. The copolymers were isolated by ultrafiltration and dialysis from unreacted Dex and the reductant followed by lyophilizzation. The grafting ratio of the copolymers was evaluated by 1H-NMR (JNM-EX-270, JEOL, Tokyo, Japan). The resulting copolymers were characterized by size-exclusion chromatography (Model 800, JASCO, Japan) equipped with a multiangle light scattering detector (DAWN EOS, Wyatt Technology, Santa Barbara). UV-melting profiles of the duplexes and triplexes were obtained using a UV-spectrophotometer equipped with a programmed thermal controller at an increase rate of 0.5 ºC /min. All measurements were performed with Robinson buffer containing 100 mM NaCl or 10 mM sodium phosphate (pH6.5) containing 0.1 mM ETDA and 200 mM NaCl. Before measurements, all the samples were



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heated at 90 ºC for 2 min and gently cooled from 90 ºC to 25 ºC at 0.5 ºC min-1 for annealing. Circular dichroism (CD) spectra were obtained on a CD spectrophotometer (J-720, JASCO) equipped with a thermo controller (RET-100, Neslab). Oligonucleotides solutions used for the measurement of CD spectra were prepared in Robinson buffer containing 100 mM NaCl or 10 mM sodium phosphate (pH6.5) containing 0.1 mM ETDA and 200 mM NaCl. The isothermal titration calorimetry (ITC) experiments were performed using a MicroCal ITC200 instrument (Malvern Instruments, Piscataway, NJ). Samples were degassed prior to use for 20 min with a MicroCal ThermoVac unit (Malvern Instruments, Piscataway, NJ). Cell samples containing of 1 µM Triplex-stem were prepared in a Robinson buffer (pH 6.0) with 100 mM NaCl and 207 µM cationic polymer (PAA-g-Dex or PLL-g-Dex). Syringe sample containing of 16.5 µM Triplex H-15 was prepared in a Robinson buffer (pH 6.0) with 100 mM NaCl and 207 µM cationic polymer. All experiments were performed at 25°C and corrected for the heat of dilution of the cell sample. During the experiment, 1 µL of syringe sample was added in successive injections to the cell samples with 180 sec intervals, and the first injection was 0.5 µL. Post-measurement concentration of the polymers was N/P = 3 [NP: (number of amino groups in the polymer) / (number of phosphate groups in the DNA strand)]. Heat released with injection was recorded. The resulting thermograms were fit to a one-set-of-sites binding model within Origin software (Malvern Instruments, Piscataway, NJ).


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Results and Discussion Two cationic comb-type copolymers, PLL-g-Dex and PAA-g-Dex (Figure 1b), were prepared to explore their effects of the cationic comb-type copolymers on the stabilization of duplex/triplex DNA.14 The graft copolymers composed of more than 80 wt.% hydrophilic graft chains and less than 20 wt.% positively charged main chain form water-soluble inter-polyelectrolyte complexes (wsIPECs) with DNA. The wsIPECs enabled us to spectroscopically evaluate the effect of the copolymers on the hybridization properties of DNA.14,15

Two parallel-stranded triplex forming

oligonucleotides were prepared: Triplex15 and Triplex11. Triplex15 contains a 46-mer strand forming hairpin loop structure with 21 Watson-Crick base pairs as stem, and a 15-mer third strand forming 15 Hoogsteen base pairs with the hairpin loop stem. Triplex11 contains the same 46-mer strand and a 11-mer third strand (Figure 1a). Figure 2a shows CD spectra of 10 µM Triplex15 at pH 6.0 in the presence of various concentrations of PAA-g-Dex from N/P = 0 to N/P = 3. The negative peak around 212 nm, which is the signature of the triplex formation17, was induced by the addition of PAA-g-Dex. These results indicate that the parallel triplex structure of Triplex15 was stabilized by PAA-g-Dex. CD intensity at 212 nm of Triplex15 was plotted at pH 6.0 and pH 7.0 against the concentrations of PAA-g-Dex or PLL-g-Dex (Figure 2b). Original CD spectra are shown in Figures S1a, S2a, and S2b. Triplex structure of Triplex15 was induced by PAA-g-Dex even at pH 7.0. CD intensity at 212 nm of Triplex15 with PLL-g-Dex revealed that the triplex structure was also stabilized by this polymer (original CD spectra are shown in Figure S1a and S2b), though the increment of the CD intensity was less significant compared with that of PAA-g-Dex. These results indicate that PAA-g-Dex is a better inducer of the triplex than PLL-g-Dex. Figure 2c shows CD spectra of 10 µM Triplex11 at pH



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6.0 in the presence of various concentrations of PAA-g-Dex, indicating that the polymer induces the triplex structure for Triplex11. Figure 2d shows CD intensity traced at 212 nm in the presence of PAA-g-Dex or PLL-g-Dex at pH 6.0 and pH 7.0 (original CD spectra are shown in Figure S1b, S2c and S2d). The 11-mer third strand was not able to form a stable triplex structure at pH 6.0 in the presence of PLL-g-Dex whereas it was possible at pH 7.0 in the presence of PAA-g-Dex. This supports that PAA-g-Dex is a better inducer of the triplex than PLL-g-Dex, as was shown in the case of Triplex 15. Moreover, in the presence or absence of the copolymer, we evaluated the pH

dependence of the triplex structures by measurement of CD signals at 212 nm (25 oC). The CD measurements were performed using intramolecular triplex DNA at pH values between 4 and 8 (data not shown). In the absence of copolymers, the pKa value was determined as around 6.5, which is consistent with pKa value of a cytosine base in a DNA triplex structure.18 On the other hand, the values of pKa in the presence of both copolymers were determined to be more than 8.0. These results suggest that specific stabilization effects on triplex DNA by PAA-g-Dex are kept in wide pH range, and we speculate that PAA-g-Dex would be act as a good triplex-selective stabilizer in various cellular conditions. Effects of the copolymers on the thermal stability of the triplexes were further investigated by taking UV-melting curves. Figure 3a shows UV-melting curves at 260 nm of 2.5 µM Triplex15 in the presence of PAA-g-Dex (from N/P = 0 to N/P = 3) at pH 6.0. At N/P = 0, two melting transitions at lower and higher temperatures were observed. The melting transitions at the lower and higher temperatures correspond to Hoogsteen base pair dissociation and the Watson-Crick base pair dissociation, respectively. Hyperchromicity of melting at the lower temperature was smaller than that at the higher temperature, because of the smaller number of Hoogsteen base pairs than that of


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Watson-Crick base pairs. This assignment was confirmed by a UV melting curve traced at 295 nm that shows thermal denaturation of the Hoogsteen base pairs (Figure 3b).13 In the presence of PAA-g-Dex (N/P = 3), the two melting transitions were merged and a single transition was observed. This single transition shows that both the triplex and the duplex denatured around the same temperature. These results indicate that the triplex with Hoogsteen base pairs is as stable as the duplex with Watson-Crick base pairs, even though the length of the triplex is shorter than the duplex. Surprisingly, the melting temperature (Tm) of triplex increased from 24.5 ºC to 73.0 ºC by the addition of PAA-g-Dex (N/P = 3) as listed in Table 1 (melting curves at 260 nm and 295 nm are shown in Figures S3, S4, S5 and S6). To the best of our knowledge, among additives previously reported, PAA-g-Dex has the largest stabilization effect on DNA triplex structures (∆Tm = 48.5 ºC). Tm of Triplex15 at pH 7.0 is 69.5 ºC, which is significantly higher than 37 ºC. Tm values obtained in the presence of PLL-g-Dex showed that PLL-g-Dex also stabilized the triplex structure. The stabilization effect of PLL-g-Dex is smaller than that of PAA-g-Dex and Tm at pH 7.0 is lower than 37 ºC. These results are consistent with the CD results shown in Figure 2 and confirmed that PAA-g-Dex allowed Triplex15 to form a stable parallel triplex structure under the physiological conditions (pH 7.0 and 37ºC). Notably, PAA-g-Dex did not stabilize but rather destabilized the DNA duplex at both pH 6.0 and pH 7.0. Similar effects of PAA-g-Dex were also observed for Triplex 11 (Table 1). These results strongly imply that PAA-g-Dex selectively stabilizes the triplex but not the duplex structure, while PLL-g-Dex stabilizes both the DNA duplex and triplex structures. In our

previous study,12 it was reported that PAA-g-Dex significantly stabilized GC-rich DNA duplexes. The stabilization effect was observed when the DNA strands folded into A-form duplex. In the same study, we also reported that PAA-g-Dex leaded to dehydration of the DNA



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duplexes, which was favorable for the A-form structure, resulting in the B-form to A-form structural transition. On the contrary, in this study, we designed AT-rich oligonucleotides to form an antiparallel B-form duplex as the Watson and Crick strands. It was also reported that DNA oligonucleotides take up water molecules upon formation of the B-form duplex,13 resulting in destabilization of the B-form duplex in dehydrated conditions. In this study, we found that PAA-g-Dex did not stabilize but rather destabilized the B-form duplex. This effect of PAA-g-Dex is consistent with the previous reports as described above. We further investigated the mechanism of the stabilization of the triplex structure by the copolymers using ITC. Figures 4 shows ITC results of Triplex15 in the absence of the polymer, in the presence of PLL-g-Dex (N/P = 3), and in the presence of PAA-g-Dex (N/P = 3) at pH 6.0 and 25 ºC. As shown in Figure 4a, no heat was observed in the binding between the hairpin loop stem strand and the third strand in the absence of the polymer. These results demonstrate no triplex formation of Triplex15 in this experimental condition, confirming structural and thermal analysis by use of CD spectroscopy and UV-melting experiments, respectively. On the other hand, heat was observed in the presence of the PLL-g-Dex (Figure 4b) and PAA-g-Dex (Figure 4c). Thermodynamic parameters evaluated from these ITC results are listed in Table 2. In the presence of PAA-g-Dex, the binding constant (Ka) was evaluated to be 1.1 × 109 M-1 at 25 ºC which is more than 10 times larger than that in the presence of PLL-g-Dex (8.6 × 107 M-1). The free energy change (∆Gº at 25 ºC) in the presence of PAA-g-Dex (-12.3 kcal mol-1) is smaller than that in the presence of PLL-g-Dex (-10.8 kcal mol-1). These results quantitatively show that PAA-g-Dex stabilizes the triplex structure more significantly than PLL-g-Dex, which is consistent with the CD spectra and UV-melting curves. Moreover, the values of enthalpy change in the presence of PLL-g-Dex (∆Hº = -56.2 kcal/mol) and PAA-g-Dex


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(∆Hº = -77.2 kcal/mol) showed that the stabilization of the triplex structure by PAA-g-Dex was mainly originated from enthalpic contribution. It was previously reported that the enthalpic stabilization of DNA structures by molecular crowding was attributable to the hydration state of the DNA structure because co-solutes that induce molecular crowding conditions decrease the activity of water molecules.13 Moreover, we recently reported that PAA-g-Dex-induced B-A transition of GC-rich DNA duplexes is accompanied by dehydration.12 PAA-g-Dex also entropically stabilized triplex structure by promoting dehydration of Triplex15. From these observations, in the case of PLL-g-Dex, the predominant stabilization mechanism toward both duplex and triplex structures involves electrostatic interactions between the anionic DNA structures and the cationic polymers rather than the dehydration effects.19 On the other hand, it is suggested that PAA-g-Dex stabilizes the triplex structure by two mechanisms: (I) the stabilization effect as a cationic polymer toward the anionic DNA triplex, and (II) the dehydration effect derived from the backbone structure of PAA. For the DNA duplex, which takes up water molecules upon formation,13 the enthalpically stabilization effect by PAA-g-Dex as a cationic polymer is canceled by the dehydration effect of the PAA backbone of entropic destabilization. Finally, we intended to examine the effect of PAA-g-Dex on the stabilization of triplex DNA structure that is composed of three independent DNA strands. A target duplex (py21 and pu21) and a triplex-forming oligonucleotide (TFO) that can bind to pu21with Hoogsteen base-pairs were designed to form an intermolecular parallel triplex (Figure 1a).16

CD measurements were then

performed to investigate the secondary structures of DNA hybrids in the absence or presence of copolymers. The results are shown in Figure 5. In the absence of copolymers, the py21/pu21 mixture showed a typical B-type duplex DNA conformation, which provides a conservative CD spectrum



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with a positive band around 280 nm and a negative band around 245 nm (Figure 5a, gray line). In the case of the TFO/py21/pu21 mixture at 20 oC, it was determined that the TFO/py21/pu21 mixture formed a typical triplex conformation from an appearance of a negative peak at 212 nm (Figure 5a, black line).17 At 60 oC, the negative short wavelength peak of the TFO/py21/pu21 mixture was weakened and the shape of the CD spectrum showed a typical B-type duplex DNA conformation (Figure 5a, dashed line). These data suggest that TFO was dissociated from the py21/pu21 duplex at 60 oC in the absence of copolymers. Similar trends were observed in the presence of PLL-g-Dex. CD spectra of the py21/pu21 mixture and TFO/py21/pu21 mixture showed a typical B-type DNA conformation and triplex conformation at 20 oC, respectively (Figure 5b). At 60 oC, the negative peak around 210 mm of the TFO/py21/pu21 mixture was weakened, suggesting that TFO also dissociated from the py21/pu21 duplex in the presence of PLL-g-Dex. In contrast, a significant difference was observed in CD profiles when using PAA-g-Dex (Figure 5c). The negative peak around 210 mm of the TFO/py21/pu21 mixture still remained at 60 oC, suggesting that TFO did not completely dissociate from the py21/pu21 duplex and that TFO/py21/pu21 kept the triplex structure. The effects of copolymers on the thermal stabilization of DNA hybrids (Figure 6) were also assessed. The Tm values of DNA hybrids in the absence or presence of copolymers were determined from the UV-melting profiles (Table 3). In the absence of copolymers, the TFO/py21/pu21 triplex showed a biphasic melting profile (Figure 6a). The first and second transitions at 29.5 oC and 69.1 oC corresponded to the melting of the TFO/py21/pu21 triplex and py21/pu21 duplex, respectively. This assignment was confirmed from a UV-melting curve traced at 295 nm (Figure 7) derived from thermal denaturation of Hoogsteen base pairs.13 In the presence of PLL-g-Dex, these two transitions shifted to higher temperatures of 45.1 oC and 80.0 oC, respectively (Figure 6b). These data suggest


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that PLL-g-Dex increased the thermal stability of both duplex and triplex formation as reported previously14. On the other hand, in the presence of PAA-g-Dex, the two transitions observed in Figure 6a merged into one broad transition. From the 260nm UV transition of py21/pu21 duplex with PAA-g-Dex (Figure 6c, dashed line) and 295nm UV transition of TFO/py21/pu21 triplex with PAA-g-Dex (Figure 7 dashed line), this broad transition was determined to be a merged one with thermal denaturation of triplex and duplex DNA. The first thermal transition in Figure 6c (58.2 oC) corresponds to the conformational change of the TFO/py21/pu21 (61.7 oC) from the temperature dependence of the CD spectra of DNA mixtures (Figure S7c). Our findings clearly show that PAA-g-Dex selectively increases thermal stability of triplex formation even though in the case of a triplex DNA structure that is composed of three independent DNA strands. Moreover, we found an interesting behavior in the CD spectra of TFO/pu21/py21 mixture in the presence of PAA-g-Dex at 60 ºC (Figure 5c). The negative peak around 210 nm at 60 ºC strengthened compared with that at 20 ºC. Some research groups reported that the B-A transition

of DNA duplex is accompanied by the appearance of a deep negative band at 210 nm.20, 21 Moreover, we also found that the positive peak around 280 nm was strengthened and was blue-shifted in Fugure 5c. These features of the CD spectrum are also useful to follow the B-A transition of DNA duplex. We previously reported that PAA-g-Dex induces the B-A transition of GC-rich oligonucleotides.12 A higher temperature is also favorable for the A-form duplex. Therefore a biphasic transition trough a thermal denaturation from the B-form duplex to the A-form duplex, and then random coil is one of the typical behaviors of a DNA oligonucleotide, which is able to form both the B-form and A-form duplexes in a experimental condition. From these previous results, it is possible to consider that the B-A transition of the pu21/py21 DNA



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was induced by rising temperature from 20 ºC to 60 ºC in the presence of PAA-g-Dex, because about one-half of the TFOs dissociated from DNA duplex at that temperature (Tm value of TFO/pu21/py21 in the presence of PAA-g-Dex was 61.7 oC). In fact, similar result was observed for the pu21/py21 DNA duplex in the presence of PAA-g-Dex (Figure S8c). These results suggest that a structural transition from the parallel triplex to a random coil via the B-A duplex transition of the TFO/pu21/py21 is induced by addition of PAA-g-Dex, although further studies are required to confirm this complex structural transition.

Conclusions In this study, we identified and confirmed the unique character of PAA-g-Dex for the selective and robust stabilization of the DNA conformation. Many research groups have demonstrated the development of functional molecules to stabilize DNA duplexes, however, there are few reports for cationic polymers that selectively stabilize triplex DNA in biological research. Triplex DNAs are implicated to have biological activities as genetic effectors or scaffold roles to control gene expression. We believe that PAA-g-Dex has great potential to regulate the biological activities of triplex DNA.

Acknowledgments This paper was supported by JSPS KAKENHI (Grant Number 15K05564 for AY, 15H03840 and 16K14042 for DM, 15H01807 for AM) and was also supported by Grant-in-Aid for a Network Joint Research Center for Materials and Devices of Japan (20163039 for AY and 20164023 for AM). We also acknowledge financial support from Grants-in-Aid for Scientific Research on Innovative Areas


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"Molecular Robotics" (No. 15H00804 for NS) and the Center of Innovation (COI) Program, Japan Science and Technology Agency (JST).

Supporting Information Figure S1. CD spectra of 10 µM Triplex15 (a) or Triplex11 (b) in the presence of various concentrations of PLL-g-Dex in 100 mM NaCl Robinson Buffer (pH 6.0) at 25 ℃. N/P = 0, 0.1, 0.3, 1.0, and 3.0.

Figure S2. CD spectra of Triplex15 in the presence of various concentrations of PAA-g-Dex (a) or PLL-g-Dex (b) and of Triplex11 in the presence of various concentrations of PAA-g-Dex (c) or PLL-g-Dex (d). All spectra were obtained with 10 µM DNA strand in 100 mM NaCl Robinson Buffer (pH 7.0) at 25 oC. N/P = 0, 0.1, 0.3, 1.0, and 3.0.

Figure S3. Normalized UV melting curves at 260 nm of Triplex15 in the presence of various concentrations of PLL-g-Dex (a), and Triplex11 in the presence of PAA-g-Dex (b) or PLL-g-Dex (c). All measurements were carried with out 10 µM DNA strand concentration in 100 mM NaCl Robinson Buffer (pH 6.0) at 25 oC. N/P = 0, 0.1, 0.3, 1.0, and 3.0.

Figure S4. Normalized UV melting curves at 295 nm of Triplex15 in the presence of various concentrations of PLL-g-Dex (a), and Triplex11 in the presence of PAA-g-Dex (b) or PLL-g-Dex (c). All measurements were performed with 10 µM DNA strand concentration in 100 mM NaCl



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Robinson Buffer (pH 6.0) at 25 oC. N/P = 0, 0.1, 0.3, 1.0, and 3.0.

Figure S5. Normalized UV melting curves at 260 nm of Triplex15 in the presence of various concentrations of PAA-g-Dex (a) or PLL-g-Dex (b), and Triplex11 in the presence of PAA-g-Dex (c) or PLL-g-Dex (d). All measurements were carried out with 10 µM DNA strand in 100 mM NaCl Robinson Buffer (pH 7.0) at 25 oC. N/P = 0, 0.1, 0.3, 1.0, and 3.0.

Figure S6. Normalized UV melting curves at 295 nm of Triplex15 in the presence of various concentrations of PAA-g-Dex (a) or PLL-g-Dex (b), and Triplex11 in the presence of PAA-g-Dex (c) or PLL-g-Dex (d). All measurements were carried out with 10 µM DNA strand in 100 mM NaCl Robinson Buffer (pH 7.0) at 25 oC. N/P = 0 , 0.1, 0.3, 1.0, and 3.0.

Figure S7. Temperature dependence of CD spectra of triplex-forming oligonucleotides in the absence or presence of graft copolymers (N/P = 2). Without copolymers (a), with PLL-g-Dex (b), with PAA-g-Dex (c). Concentration of oligonucleotides: 1 µM each, 10 mM phosphate buffer (pH 6.5), 0.1 mM EDTA, 200 mM NaCl.

Figure S8. CD spectra of py21/pu21 mixtures in the absence (a) or presence of PLL-g-DEX, (b) or PAA-g-DEX (c) (N/P = 2). Black line: 20

o

C, and pink line: 60

o

C. Concentration of

oligonucleotides: 1 µM each in 10 mM phosphate buffer (pH 6.5), 0.1 mM EDTA, and 200 mM NaCl.


Page 17 of 32

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References 1.

Lilley, D.M. Structures of helical junctions in nucleic acids. Q. Rev. Biophys. 2000, 33,

109-159. 2.

Wang, G.; Vasquez, K.M. Naturally occurring H-DNA-forming sequences are mutagenic in

mammalian cells. Proc Natl Acad Sci U S A., 2004, 101, 13448-13453. 3.

Bacolla, A.; Wells, R.D., Non-B DNA conformations, genomic rearrangements, and human

disease, Journal of biological chemistry, 2004, 279, 47411-47414. DOI: 10.1074/jbc.R400028200. 4.

Schroth, G.P.; Ho, P.S. Occurrence of potential cruciform and H-DNA forming sequences in

genomic DNA. Nucleic Acids Res., 1995, 23, 1977-1983. 5.

Wang, G.; Seidman, M.M.; Glazer, P.M., Mutagenesis in mammalian cells induced by triple

helix formation and transcription-coupled repair. Science, 1996, 271, 802-805. 6.

Saglio,G.; Borrello, M.G.; Guerrasio A.; Sozzi G.; Serra, Di; Celle A.P.F.; Foa, R.; Ferrarini,

M.; Roncella S.; Pignatti, C.. et al., Preferential clustering of chromosomal breakpoints in Burkitt's lymphomas and L3 type acute lymphoblastic leukemias with a t(8;14) translocation, Genes Chromosomes Cancer, 1993, 8, 1-7. 7.

Tiwari, M.K.; Rogers, F.A. XPD-dependent activation of apoptosis in response to triplex-

induced DNA damage. Nucleic Acids Res., 2013, 41, 8979-8994. 8.

Tiwari, M.K.; Adaku, N.; Natoya P., Rogers, F.A.; Triplex structures induce DNA double strand

breaks via replication fork collapse in NER deficient cells, Nucleic Acids Res., 2016, 44, 7742-7754. 9.

Maruyama, A.; Ishihara, T.; Kim, J.-S.; Kim, S.W.; Akaike, T., Nanoparticle DNA Carrier with

Poly(L-lysine) Grafted Polysaccharide Copolymer and Poly(D,L-lactic acid), Bioconjugate Chem., 1997, 8, 735-742.



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10. Maruyama, A.; Watanabe, H.; Ferdous, A.; Katoh, M.; Ishihara, T.; Akaike, A., Characterization of Interpolyelectrolyte Complexes between Double-Stranded DNA and Polylysine Comb-Type Copolymers Having Hydrophilic Side Chains, Bioconjugate Chem., 1998, 9, 292-299. 11. Moriyama, R.; Shimada, N.; Kano, A.; Maruyama, A. The role of cationic comb-type copolymers in chaperoning DNA annealing. Biomaterials, 2011, 32, 7671-7676. 12. Yamaguchi, N., Zouzumi, Y.; Shimada, N.; Nakano, S.; Sugimoto, N.; Maruyama, A.; Miyoshi, D., A reversible B–A transition of DNA duplexes induced by synthetic cationic copolymers, Chem. Commun., 2016, 52, 7446-7449. 13. Miyoshi, D.; Nakamura, K.; Tateishi-Karimata, H.; Ohmich, T.; Sugimoto, N., Hydration of Watson − Crick Base Pairs and Dehydration of Hoogsteen Base Pairs Inducing Structural Polymorphism under Molecular Crowding Conditions, J. Am. Chem. Soc., 2009, 131, 3522-3531. 14. Sato, Y.; Kobayashi, Y.; Kamiya, T.; Watanabe, H.; Akaike, T.; Yoshikawa, K.; Maruyama, A. The effect of backbone structure on polycation comb-type copolymer/DNA interactions and the molecular assembly of DNA. Biomaterials, 2005, 26, 703-711. 15. Miyoshi, D.; Ueda, Y.M.; Shimada, N.; Nakano, S.; Sugimoto, N.; Maruyama, A., Drastic stabilization of parallel DNA hybridizations by a polylysine comb-type copolymer with hydrophilic graft chain, Chem. Med. Chem., 2014, 9, 2156-2163. 16. Moser, H.E.; Dervan. P.B. Sequence-specific cleavage of double helical DNA by triple helix formation. Science, 1987, 238, 645-650. 17. Manzini, G.; Xodo, L.E.; Gasparotto, D., Triple helix formation by oligopurineoligopyrimidine DNA fragments. Electrophoretic and thermodynamic behavior, J. Mol. Biol., 1990, 213, 833-843.


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18. Asensio, J.L.; Lane, A.N.; Dhesi, J.; Bergqvist, S.; Brown, T., The contribution of cytosine

protonation to the stability of parallel DNA triple helices, J. Mol. Biol., 1998, 275, 811–822. 19. Maruyama, A.; Ohnishi, Y.; Watanabe, H.; Torigoe, H.; Ferdous, A.; Akaike, T., Polycation comb-type copolymer reduces counterion condensation effect to stabilize DNA duplex and triplex formation, Colloids Surf. B, 1999, 16, 273-280.

20. Yang, J.T.; Samejima, T., Effect of base tilting on the optical activity of nucleic acids: A hypothesis, Biochem. Biophys. Res. Commun., 1968, 33, 739-745. 21. Vorlícková, M.; Subirana, J.A., Chládková, J.; Tejralová, I.; Huynh-Dinh, T; Arnold, L.; Kypr, J., Comparison of the solution and crystal conformations of (G + C)-rich fragments of DNA, Biophys. J., 1996, 71, 1530-1538.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 32

Figure Legends Figure 1. Sequences of oligonucleotides (a) and structural formulas of graft copolymers (b) used in this study.

Figure 2. CD spectra of the Triplex15 (a) and Triplex11 (c) with PAA-g-Dex at pH 6.0. CD intensities at 212 nm for the Triplex15 (b) and Triplex11 (d) with PLL-g-Dex at pH 6.0 (○), PLL-g-Dex at pH 7.0 (□), PAA-g-Dex at pH 6.0 (●), and PAA-g-Dex at pH 7.0 (■). All measurements were performed with 10 µM DNA in 100 mM NaCl Robinson buffer at 25℃. N/P = 0, 0.1, 0.3, 1.0, and 3.0.

Figure 3. Normalized UV-melting curves of 2.5 µM Triplex15 in the presence of various concentrations of PAA-g-Dex at pH 6.0. Melting curves are traced at 260 nm (a) and 295 nm (b) in 100 mM NaCl Robinson buffer. N/P = 0, 0.1, 0.3, 1.0, and 3.0.

Figure 4. ITC measurement for the complex formation of Triplex H15 in the absence of cationic polymer (N/P = 0) (a), in the presence of PLL-g-Dex (N/P = 3.0) (b), and in the presence of PAA-g-Dex (N/P = 3.0) (c). All measurements were carried out in 100 mM NaCl Robinson buffer (pH 6.0) at 25 ℃.

Figure 5. CD spectra of oligonucleotides in the absence (a) or presence of PLL-g-DEX, (b) or PAA-g-DEX (c) (N/P = 2). Black line: TFO/py21/pu21 mixture (20

o

C), dashed line:

TFO/py21/pu21 mixture (60 oC) and gray line: py21/pu21 mixture (20 oC). Concentration of


Page 21 of 32

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oligonucleotides: 1 µM each in 10 mM phosphate buffer (pH 6.5), 0.1 mM EDTA, and 200 mM NaCl.

Figure 6. Normalized UV-melting curves of 1.0 µM of oligonucleotides of triplex (TFO/pu21/py21) in the absence (a) or presence of copolymers PLL-g-DEX (b), or PAA-g-DEX (c) (N/P = 2). Melting curves are traced at 260 nm in 10 mM phosphate buffer (pH 6.5), 0.1 mM EDTA, and 200 mM NaCl. Dased line: pu21/py21 mixture, solid line: TFO/pu21/py21 mixture.

Figure 7. Normalized UV melting curves of 1.0 µM of TFO/pu21/py21 mixture with PAA-g-DEX (N/P = 2). Melting curves are traced at 295 nm in 10 mM phosphate buffer (pH 6.5), 0.1 mM EDTA, and 200 mM NaCl.

Table 1. Tm values of the Triplex15 and Triplex11 obtained from UV-melting curves traced at 260 nm and 295 nm.

Table 2. A summary of thermodynamic parameters, Hill coefficient (n), association constants (Ka), free energy change (∆Gº), enthalpy changes (∆Hº), and entropy changes (∆Sº), for the intermolecular triplex formation of Triplex15 in the absence of the polymer, in the presence of PLL-g-Dex or in the presence of PAA-g-Dex in 100 mM NaCl Robinson buffer (pH 6.0) at 25 ºC.

Table 3. Tm values of TFO/pu21/py21 mixture evaluated by UV melting curves at 260 nm and 295 nm.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(a)

(b) PLL-g-Dex

Backbone: Poly(L-lysine) （Mw 30,000) Side chain: Dextran （Mw 10,000) Dextran content 90 wt%

PAA-g-Dex

Backbone: Polyallylamine （Mw 15,000) Side chain: Dextran （Mw 10,000) Dextran content 92 wt%

Figure 1. Yamayoshi et al.


Page 22 of 32

Page 23 of 32

10

(a)

(b) 10-5 [θ] at 212 nm / deg cm2 dmol-1

10-5 [θ] / deg cm2 dmol-1

N/P = 0

N/P = 3

5

0

-5

-10 200

250 300 Wavelength / nm

2 0 -2 -4 -6 -8 -10

350

0

(c) N/P = 0

N/P = 3

5

0

-5

-10 200

0.5

1

1.5 N/P

2

2.5

3

0.5

1

1.5 N/P

2

2.5

3

(d) 10-5 [θ] at 212 nm / deg cm2 dmol-1

10


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2 0 -2 -4 -6 -8 -10

250

300

350

0

Wavelength / nm




1.1

(a)

1.2

(b)

N/P = 0

1.0

Normalized Abs. at 295 nm


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 32

N/P = 3

0.9

0.8

1.1

1.0

0.9 N/P = 0

0.8 N/P = 3

0.7

0.7 0

20

40 60 Temperature / ºC

80

0

20

40 60 Temperature / ºC



80

Page 25 of 32

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Table 1. Yamayoshi et al.



(a)

Kcal mol-1 of injectant

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(b)

(c)

0

0

0

-20

-20

-20

-40

-40

-40

-60

-60

-60

-80

-80

-80

0

0.5

1

1.5

Molar Ratio

2

0

Page 26 of 32

0.5

1

1.5

2

Molar Ratio



0

0.5

1

1.5

Molar Ratio

2

Page 27 of 32

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(a) 10-5 [θ] / deg cm2 dmol-1

15 10 5 0 -5 -10 -15 -20 200

220

240

260

280

300

320

340

300

320

340

300

320

340

Wavelength / nm


(b)

15 10 5 0 -5 -10 -15 -20 200

220

240

260

280

Wavelength / nm

(c)

15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 32

10 5 0 -5 -10 -15 -20 200

220

240

260

280

Wavelength / nm



Page 29 of 32

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



with PAA-g-Dex with PLL-g-Dex

w/o polymers

Temperature / ℃



Page 30 of 32

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Page 32 of 32

PAA-g-Dex CH2 CH

CH2 CH

CH2

CH2

+NH3 1-y

Duplex

CH2OH O OH O OH OH

CH2 OH OH

+NH2 y

CH2 O O OH n

OH OH

OH CH2

Triplex

OH

SELECTIVE STABILIZATION

O H N CH C

H N

CH2

CH2

CH2

CH2

CH2

CH2

CH2 +NH3

CH2OH O OH O OH OH

CH2 OH OH

O CH C

CH2 1-x

+NH2

CH2 O O OH n

OH OH

x

OH CH2 OH

PLL-g-Dex


Selective and Robust Stabilization of Triplex DNA Structures Using

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