Comb-Type Polycations Effectively Stabilize DNA Triplex

Isolation of the resulting copolymers from unreacted Dex was confirmed by gel ... which meant that Dex chains were grafted onto an average of one of e...
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Bioconjugate Chem. 1997, 8, 3−6

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COMMUNICATIONS Comb-Type Polycations Effectively Stabilize DNA Triplex Atsushi Maruyama,* Maiko Katoh, Tsutomu Ishihara, and Toshihiro Akaike Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori, Yokohama 226, Japan. Received May 6, 1996X

DNA triplex formation has been studied as a potential strategy for regulation of gene expression. The triplex is, however, unstable under physiological conditions, so that an effective stabilizer for the triplex formation is needed. Here is shown a novel strategy to stabilize the triplex based on the molecular design of a comb-type polycation. Linear polycations, such as poly(L-lysine) and poly(Larginine), thermally stabilize DNA duplexes (and triplexes). The complexes between DNA and the polycation are irreversible and are liable to precipitate out of aqueous media. The irreversibility and phase separating properties of the complex impede association of single-stranded (ss) DNAs in the complex to form duplexes and triplexes. A comb-type polycation consisting of a poly(L-lysine) backbone and grafted chains of hydrophilic polymers was prepared. The comb-type copolymers increased solubility of their complex with DNA and suppressed conformational changes of DNA. Thermal melting curve analyses revealed that the comb-type copolymer markedly stabilized DNA triplexes and did not disturb ssDNAs in forming duplexes and triplexes. Reversible and one-step melting/reassociation transitions of poly(dA)‚2poly(dT) triplex were shown in the presence of the copolymers. The stabilizing effect of the copolymer was larger than that of spermine, a polyamine considered effective in stabilizing triplexes. These results indicated that molecular design of polycations with a comb-type structure is a novel strategy to create efficient triplex stabilizers. Such comb-type copolymer consisting of various types of polycation backbones and hydrophilic graft chains may have many applications in which specific and precise interactions of polynucleotides are involved.

Deactivation of a target gene with oligonucleotides which form triplexes with that gene has been proven to be a potential strategy for regulation of gene translation (1, 2). Triplex formation is, however, unstable in physiological conditions, which limits the utility of the triplex strategy. To extend the usefulness of the triplex strategy, several compounds that stabilize triplexes have been investigated (3-7). Polyamines, such as spermine, spermidine, and putrescine, stabilized duplexes and triplexes (8-10). The stabilizing effect of polyamines was largely due to neutralization of electrostatic repulsion among phosphate anions in DNA molecules associating in the triplex and duplex. Their effect was, however, reduced considerably under physiological conditions, because interaction of polyamines with DNA was hampered by coexisting cations (9-11). For neutralizing the electrostatic repulsion among DNA strands associated in triplexes or duplexes, macromolecular polycations, such as poly(L-lysine) (PLL) and poly(L-arginine), are more effective than oligomeric cations, leading to a considerable rise in the melting temperature (Tm) of the duplex (12, 13). Polycations, however, interact strongly with polyanions to form irreversible polyion complexes (or inter-polyelectrolyte complexes) (13, 14). Polycations severely compacted the DNA conformation (15, 16). Coacervation or precipitation * Author to whom correspondence should be addressed (telephone +81-45-924-5809; fax +81-45-924-5815; e-mail [email protected]). X Abstract published in Advance ACS Abstracts, December 15, 1996.

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of the complex occurred (12, 14). Single-stranded (ss) DNAs rarely form duplexes and triplexes in the presence of polycations, resulting in irreversibility of duplex and triplex transitions (melting and reassociation). The reversible transition of DNA might be, therefore, attainable by increasing the solubility of the complex and reducing the conformational changes of DNA. For this approach, the interactions of polycations with DNA have to be regulated. The interactions of polycations with DNA could be regulated by modifying polycations with DNA-immiscible chains such as electrostatically neutral polymer chains that interfere with these interactions. Modification of polycations with hydrophilic chains like polysaccharides may improve the solubility of a DNA/ polycation complex, leading to prevention of phase separation of the complex from aqueous medium. On the basis of the hypothesis mentioned above, we prepared comb-type copolymers of poly(L-lysine) with polysaccharide side chains and evaluated their ability to stabilize DNA triplexes. The comb-type copolymers, PLL-graft-dextran, were prepared by a reductive amination reaction of PLL‚HBr (100 mg, Mw ) 4.5 × 104 from Peptide Institute, Inc., Osaka, Japan) with dextran (Dex, 600 mg, Mn ) 5900, Dextran T-10 from Pharmacia Biotech, Uppsala, Sweden) using sodium cyanoborohydride (30 mg, from Wako Pure Chemical Industries, Ltd., Osaka, Japan) as a reductant in dimethyl sulfoxide. After the mixture was incubated for 48 h at 40 °C, the copolymers were isolated by dialysis against water for 7 days using Spectra/Por membrane (MWCO ) 12 000-14 000, Spectrum, Los Angeles, CA). Isolation of the resulting copolymers from unreacted Dex © 1997 American Chemical Society

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Maruyama et al.

Figure 1. Structural formula of PLL-graft-Dex comb-type copolymer. The copolymer with m ) 36.5 (calculated from the number-average MW of Dextran T-10) and n ) 0.2 (determined by 1H-NMR spectrum) was used in this study.

Figure 3. CD of calf thymus DNA mixed with PLL or PLLgraft-Dex comb-type copolymer in 1/100 diluted PBS at room temperature (ca. 20 °C). The samples were created using the same procedure described in Figure 2, except PBS concentration. The DNA/comb-type copolymer mixture in PBS showed almost the same CD signal as that in this figure, while CD of the DNA/ PLL mixture was not obtained because of turbidity and precipitation of the mixture.

Figure 2. Turbidity of DNA solution to which the indicated amounts of PLL (O) or PLL-graft-Dex comb-type copolymer (b) were added. To 1 mL of 100 µg/mL calf thymus DNA in Dulbecco’s PBS was added the appropriate amount of polymers from 10 mg/mL stock solution at room temperature. The mixtures were diluted to 2.5 mL, followed by optical density measurement at 500 nm.

was confirmed by gel permeation chromatography on Waters Ultrahydrogel 250 and 500 columns connected with a multiangle light scattering detector and a differential refractive index detector. The molecular weight of the resulting copolymers was determined to be Mn ) 2.5 × 105 (as free salt). Composition of the copolymer was determined by 1H-NMR spectrum in D2O to be 90 wt % Dex and 10 wt % PLL, which meant that Dex chains were grafted onto an average of one of every five lysine units. Further, it was estimated that the coupling efficacy of Dex to PLL was >90%. The structural formula of the copolymer is given in Figure 1. The Dex-grafted chains on PLL increased the solubility of the complex with DNA. Figure 2 shows the turbidity change of calf thymus DNA in phosphate-buffered saline (PBS) by an addition of either PLL homopolymer or the comb-type graft copolymer. While significant turbidity appeared in PLL/DNA mixture, no turbidity was seen for corresponding comb-type copolymer mixtures. Thus, PLL-graft-Dex comb-type copolymer seems to form a soluble complex with DNA. We then examined the comb-type copolymer for induced structural changes of DNA. Structural change of DNA is detectable by circular dichroism (CD). Figure 3 shows CD spectra of calf thymus DNA mixed with PLL homopolymer or the comb-type copolymer in 1/100 diluted PBS, where both mixtures are transparent. CD signals due to DNA base groups were considerably altered by the addition of PLL homopolymer. Complex formation between DNA and PLL is known to show cooperative association and irreversibility (13, 14). PLL condenses DNA into rod and toroid-like structures (15, 16), which accompanies a change in CD signals (14). In contrast to the PLL homopolymer, addition of the comb-type copolymers only shows a slight change. The CD signals did

Figure 4. UV-Tm profile of poly(dA)‚2poly(dT) triplex in the presence or absence of PLL-graft-Dex comb-type copolymer. Poly(dA) and poly(dT) were dissolved in 150 mM NaCl containing 10 mM sodium phosphate (pH 7.2) and 0.1 mM EDTA. Concentrations of the solutions were calculated using molar extinction coefficients of 8900 at 257 nm for poly(dA) and 9000 at 265 nm for poly(dT) (10). To a 1:2 mixture of poly(dA) and poly(dT) was added the comb-type copolymer at a polymer/DNA ([amino group]polymer/[phosphate group]DNA) ratio ) 2. After dilution to a final DNA concentration of 14.5 (bp) µmol/L with the same buffer, the mixtures were heated at 90 °C for 30 min, cooled to room temperature, and allowed to stand for 16 h. The UV-Tm curves were recorded at 0.2 K/min with a DU-640 spectrometer (Beckman) equipped with a micro-Tm apparatus.

not vary even when electrostatically excessive amounts of the copolymer were added (data not shown). Further, CD spectra of a comb-type copolymer/DNA mixture in PBS showed almost the same signals as that shown in Figure 3, while that of a PLL/DNA mixture was not obtained because of turbidity and precipitation of the mixture. Thus, the Dex graft was effective in preventing serious structural changes to DNA. It was predictable from the results described above that the DNAs associated with the comb-type copolymer preserve an ability to form duplexes or triplexes. The stabilizing effect of the comb-type copolymers on DNA triplexes was then examined by recording thermal melting profiles with a UV spectrometer. Figure 4 shows UV-Tm curves of a poly(dA)‚2poly(dT) triplex in PBS. At physiological ionic strength, the triplex showed two-step melting. The first transition at lower temperature (37

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Figure 5. CDs of poly(dA)‚poly(dT) duplex (dotted line), poly(dA)‚2poly(dT) triplex (broken line), and the 2:1 mixture (solid line) of the comb-type copolymer and the triplex. The samples were prepared according to the same procedure as that described in Figure 4, except a 1:1 molar ratio of poly(dA)/poly(dT) was used for the duplex. CD measurements were done after Tm measurements. The spectra were recorded on a Jasco J-600 using a 0.5 cm length quartz cell at 20 nm/min as a scanning speed, 0.1 nm/point as a wavelength step, and 1 s/point as a time step. Each spectrum shown was the average of three scans and has been smoothed by a computer. The CD signal attributed to the comb-type copolymer was negligible under the condition.

°C) was the melting of the triplex to a duplex and a ssDNA, and the second transition at higher temperature (72 °C) was that of the duplex. In the presence of excess comb-type copolymers over DNAs, only one transition was observed at higher temperature (89 °C). As the magnitude in UV absorbance change (∆ABS) at Tm in the presence of the copolymer was equal to the sum of those at Tm1 and Tm2 in the absence of the copolymer, the transition is indicated to be a direct melting of the triplex to its constituting ssDNAs. The direct melting of the triplex was further indicated by ∆ABS measurement with changing poly(dT)/poly(dA) molar ratio [while the concentration of poly(dA) was kept constant]. The ∆ABS values reached a plateau at the changing poly(dT)/poly(dA) molar ratio over 2 (data not shown). It should be noted that the UV-Tm profile in the cooling process demonstrated reversibility of the transition even in the presence of the comb-type copolymer. It was indicated that the comb-type copolymer thermally stabilized the triplex but did not disturb triplex formation from its constituting ssDNAs. Formation of the poly(dA)‚2poly(dT) triplex was further evaluated by CD measurements. As shown in Figure 5, whereas the CD spectrum of the poly(dA)‚poly(dT) duplex has a strong positive band near 220 nm, the poly(dA)‚2poly(dT) triplex has no positive band near this wavelength (17). The mixture of poly(dA)‚2poly(dT) and the comb-type copolymers showed almost the same signals as the triplex alone even after Tm measurement. We conclude that the combtype copolymers do not disturb inter-polynucleotide associations of ssDNAs to form the triplex. The efficacy of the comb-type copolymer for stabilizing the triplex was then compared to that of spermine, a polyamine effective in triplex stabilization (10). Figure 6 shows the effects of comb-type copolymer and spermine on melting temperatures of the triplex at various stabilizer/DNA ratios. An electrostatically equivalent amount ([amino group]polymer /[phosphate group]DNA) of the combtype copolymer increased the melting temperature of the

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Figure 6. Stabilizing effect of spermine and the comb-type copolymer. All experiments were conducted under the conditions described in Figure 4. Tm was determined by generating the first derivative, dA/dT (where A and T are absorbance and temperature, respectively), of the UV-Tm curves.

triplex by 50 °C, while a large excess of spermine increased it by 20 °C. Moreover, one-step melting of the triplex was observed in the presence of the comb-type copolymer at a stabilizer/DNA ratio over 1, whereas twostep melting was still seen even in the presence of a large excess of spermine. Poly(dA)‚2poly(dT) triplex stabilized with 7.5 µM spermine was reported to melt in a one-step manner in low ionic strength medium (10). The stabilizing effect was, however, significantly reduced in the presence of 0.15 M NaCl, resulting in the two-step melting of the triplex. Spermine is believed to be competitively displaced with Na cations. In contrast to spermine, the comb-type copolymer considerably stabilized the triplex of poly(dA)‚2poly(dT). The one-step melting and considerable rise in Tm observed with an electrostatically equivalent amount of the comb-type copolymer implied its stable association with DNA. The strong and stable stabilizing effect of the combtype copolymer was considered to be directed by its high multivalency of cations. The copolymer, however, allows ssDNAs to form duplexes and triplexes. The Dex graft chains on the copolymer interfere with interaction between the PLL backbones and DNAs. Those graft chains might inhibit the close contact of DNAs to the PLL backbones and thereby dehydration and compaction, which are presumably involved in the irreversible complex formation between DNAs and homopolycations. Regardless of the weakened interaction, the comb-type copolymer could suppress the repulsive forces among DNA strands enough to stabilize the triplex. In addition to the shielding effect upon the repulsive forces, Dex chains may play a role in stabilizing hydrogen bonding between base pairs. As the comb-type copolymer consists of 90 wt % Dex and 10 wt % PLL, the PLL backbone in the copolymer is likely surrounded by a phase concentrated with Dex segments. The DNAs that are attracted to the PLL backbone by electrostatic interactions are forced to merge with the Dex-enriched phases, which are low in dielectric constant. Such low dielectric environments might enhance hydrogen bonding between base pairs, leading to DNA triplex stabilization. It was reported that the triplex in a medium containing spermine was further stabilized by moderate concentrations of organic solvents (18). If the electrostatic repulsion was neutralized by polyamines or polycations,

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medium with a low dielectric constant may further stabilize triplexes. Furthermore, specific interaction between Dex chains and DNA may affect the stabilization behavior (19). These results indicate that the molecular design of polycations with a comb-type structure is a successful approach to create efficient triplex stabilizers. Combtype copolymers consisting of various types of polycation backbones and hydrophilic graft chains may have many applications in which specific and precise interactions of polynucleotides are involved. Conjugation of triplexforming oligonucleotides (TFOs) with the comb-type copolymer may allow specific and stable triplex formation at target genes. Modification of the comb-type copolymers with a cellular specific ligand and other functional groups will provide novel types of delivery systems for TFOs, which is not available with low molecular weight stabilizers. ACKNOWLEDGMENT

We are grateful to Professor Mizuo Maeda of Kyushu University for helpful discussion. LITERATURE CITED (1) Felsenfeld, G., Davies, D. R., and Rich, A. (1957) Formation of a three-stranded polynucleotide molecule. J. Am. Chem. Soc. 79, 223-224. (2) For review: He´le`ne, C., and Toulme, J. J. (1990) Specific regulation of gene expression by antisense, sense and antigene nucleic acids. Biochim. Biophys. Acta 1049, 99-125. (3) Le Doan, T., Perrouault, L., Praseuth, D., Habhoub, N., Decout, J. L., Thuong, N. T., Lhomme, J., and He´le`ne, C. (1987) Sequence-specific recognition, photocrosslinking and cleavage of the DNA double helix by an oligo-[R]-thymidylate covalently linked to an azidoproflavine derivative. Nucleic Acids Res. 15, 7749-7760. (4) Tung, C-H., Breslauer, J. B., and Stein, S. (1993) Polyaminelinked oligonucleotides for DNA triplex formation. Nucleic Acids Res. 23, 5489-5494. (5) Nara, H., Ono, A., and Matsuda, A. (1995) Nucleosides and nucleotides. 135. DNA duplex and triplex formation and resistance to nucleolytic degradation of oligodeoxynucleotides containing syn-norspermidine at the 5-position of 2′-deoxyuridine. Bioconjugate Chem. 6, 54-61. (6) Mergny, J. L., Duval-Valentin, G., Nguyen, C. H., Perrouault, L., Faucon, B., Rouge´e, M., Montenay-Garestier, T.,

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