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• W1983157047 abstract "Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use for artificial control of gene expression in vivo. Stabilization of the pyrimidine motif triplex at physiological pH is therefore of great importance in improving its therapeutic potential. To this end, isothermal titration calorimetry interaction analysis system and electrophoretic mobility shift assay have been used to explore the thermodynamic and kinetic effects of our previously reported triplex stabilizer, poly (l-lysine)-graft-dextran (PLL-g-Dex) copolymer, on pyrimidine motif triplex formation at physiological pH. Both the thermodynamic and kinetic analyses have clearly indicated that in the presence of the PLL-g-Dex copolymer, the binding constant of the pyrimidine motif triplex formation at physiological pH was about 100 times higher than that observed without any triplex stabilizer. Of importance, the triplex-promoting efficiency of the copolymer was more than 20 times higher than that of physiological concentrations of spermine, a putative intracellular triplex stabilizer. Kinetic data have also demonstrated that the observed copolymer-mediated promotion of the triplex formation at physiological pH resulted from the considerable increase in the association rate constant rather than the decrease in the dissociation rate constant. Our results certainly support the idea that the PLL-g-Dex copolymer could be a key material and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo. Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use for artificial control of gene expression in vivo. Stabilization of the pyrimidine motif triplex at physiological pH is therefore of great importance in improving its therapeutic potential. To this end, isothermal titration calorimetry interaction analysis system and electrophoretic mobility shift assay have been used to explore the thermodynamic and kinetic effects of our previously reported triplex stabilizer, poly (l-lysine)-graft-dextran (PLL-g-Dex) copolymer, on pyrimidine motif triplex formation at physiological pH. Both the thermodynamic and kinetic analyses have clearly indicated that in the presence of the PLL-g-Dex copolymer, the binding constant of the pyrimidine motif triplex formation at physiological pH was about 100 times higher than that observed without any triplex stabilizer. Of importance, the triplex-promoting efficiency of the copolymer was more than 20 times higher than that of physiological concentrations of spermine, a putative intracellular triplex stabilizer. Kinetic data have also demonstrated that the observed copolymer-mediated promotion of the triplex formation at physiological pH resulted from the considerable increase in the association rate constant rather than the decrease in the dissociation rate constant. Our results certainly support the idea that the PLL-g-Dex copolymer could be a key material and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo. In recent years, triplex DNA has attracted considerable interest because of its possible biological functions in vivo and its wide variety of potential applications, such as regulation of gene expression, site-specific cleavage of duplex DNA, mapping of genomic DNA, and gene-targeted mutagenesis (1Mirkin S.M. Frank-Kamenetskii M.D. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 541-576Crossref PubMed Scopus (227) Google Scholar, 2Frank-Kamenetskii M.D. Mirkin S.M. Annu. Rev. Biochem. 1995; 64: 65-95Crossref PubMed Scopus (658) Google Scholar, 3Soyfer V.N. Potaman V.N. Triple-Helical Nucleic Acids. Springer-Verlag New York, Inc., New York1996Crossref Google Scholar). A triplex is usually formed through the sequence-specific interaction of a single-stranded homopurine or homopyrimidine triplex-forming oligonucleotide (TFO) 1The abbreviations used are: TFO, triplex-forming oligonucleotide; PLL-g-Dex, poly (l-lysine)-graft-dextran; ITC, isothermal titration calorimetry; IAsys, interaction analysis system; EMSA, electrophoretic mobility shift assay; k on, on-rate constant; k assoc, association rate constant; k off, off-rate constant; k dissoc, dissociation rate constant; CC, counterion condensation; bp, base pair. with the major groove of the homopurine-homopyrimidine stretch in duplex DNA (1Mirkin S.M. Frank-Kamenetskii M.D. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 541-576Crossref PubMed Scopus (227) Google Scholar, 2Frank-Kamenetskii M.D. Mirkin S.M. Annu. Rev. Biochem. 1995; 64: 65-95Crossref PubMed Scopus (658) Google Scholar, 3Soyfer V.N. Potaman V.N. Triple-Helical Nucleic Acids. Springer-Verlag New York, Inc., New York1996Crossref Google Scholar, 4Sun J.-S. Helene C. Curr. Opin. Struct. Biol. 1993; 3: 345-356Crossref Scopus (170) Google Scholar, 5Sun J.-S. Garestier T. Helene C. Curr. Opin. Struct. Biol. 1996; 6: 327-333Crossref PubMed Scopus (116) Google Scholar). In the purine motif triplex, a homopurine TFO binds antiparallel to the homopurine strand of the target duplex by reverse Hoogsteen hydrogen bonding to form A·A:T (or T·A:T) and G·G:C triplets (1Mirkin S.M. Frank-Kamenetskii M.D. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 541-576Crossref PubMed Scopus (227) Google Scholar, 2Frank-Kamenetskii M.D. Mirkin S.M. Annu. Rev. Biochem. 1995; 64: 65-95Crossref PubMed Scopus (658) Google Scholar, 3Soyfer V.N. Potaman V.N. Triple-Helical Nucleic Acids. Springer-Verlag New York, Inc., New York1996Crossref Google Scholar, 4Sun J.-S. Helene C. Curr. Opin. Struct. Biol. 1993; 3: 345-356Crossref Scopus (170) Google Scholar, 5Sun J.-S. Garestier T. Helene C. Curr. Opin. Struct. Biol. 1996; 6: 327-333Crossref PubMed Scopus (116) Google Scholar). On the other hand, in the pyrimidine motif triplex, a homopyrimidine TFO binds parallel to the homopurine strand of the target duplex by Hoogsteen hydrogen bonding to form T·A:T and C+·G:C triplets (1Mirkin S.M. Frank-Kamenetskii M.D. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 541-576Crossref PubMed Scopus (227) Google Scholar, 2Frank-Kamenetskii M.D. Mirkin S.M. Annu. Rev. Biochem. 1995; 64: 65-95Crossref PubMed Scopus (658) Google Scholar, 3Soyfer V.N. Potaman V.N. Triple-Helical Nucleic Acids. Springer-Verlag New York, Inc., New York1996Crossref Google Scholar, 4Sun J.-S. Helene C. Curr. Opin. Struct. Biol. 1993; 3: 345-356Crossref Scopus (170) Google Scholar, 5Sun J.-S. Garestier T. Helene C. Curr. Opin. Struct. Biol. 1996; 6: 327-333Crossref PubMed Scopus (116) Google Scholar). Because the cytosine bases in a homopyrimidine TFO are to be protonated to bind with the guanine bases of the G:C duplex, the formation of the pyrimidine motif triplex needs an acidic pH condition and is thus extremely unstable at physiological pH (6Lee J.S. Johnson D.A. Morgan A.R. Nucleic Acids Res. 1979; 6: 3073-3091Crossref PubMed Scopus (200) Google Scholar, 7Lyamichev V.I. Mirkin S.M. Frank-Kamenetskii M.D. J. Biomol. Struct. Dyn. 1985; 3: 327-338Crossref PubMed Scopus (118) Google Scholar, 8Frank-Kamenetskii M.D. Methods Enzymol. 1992; 211: 180-191Crossref PubMed Scopus (39) Google Scholar, 9Singleton S.F. Dervan P.B. Biochemistry. 1992; 31: 10995-11003Crossref PubMed Scopus (164) Google Scholar, 10Shindo H. Torigoe H. Sarai A. Biochemistry. 1993; 32: 8963-8969Crossref PubMed Scopus (65) Google Scholar). The extreme instability of the pyrimidine motif triplex at physiological pH severely limits its use for artificial control of gene expressionin vivo. Stabilization of the pyrimidine motif triplex at physiological pH is therefore of great importance in improving its therapeutic potential. Numerous efforts such as the replacement of cytosine bases in a homopyrimidine TFO with 5-methylcytosine (9Singleton S.F. Dervan P.B. Biochemistry. 1992; 31: 10995-11003Crossref PubMed Scopus (164) Google Scholar,11Lee J.S. Woodsworth M.L. Latimer L.J.P. Morgan A.R. Nucleic Acids Res. 1984; 12: 6603-6614Crossref PubMed Scopus (276) Google Scholar, 12Povsic T.J. Dervan P.B. J. Am. Chem. Soc. 1989; 111: 3059-3061Crossref Scopus (281) Google Scholar, 13Xodo L.E. Manzini G. Quadrifoglio F. van der Marel G.A. van Boom J.H. Nucleic Acids Res. 1991; 19: 5625-5631Crossref PubMed Scopus (205) Google Scholar) or other chemically modified bases (14Ono A. Ts'o P.O.P. Kan L.-S. J. Am. Chem. Soc. 1991; 113: 4032-4033Crossref Scopus (171) Google Scholar, 15Krawczyk S.H. Milligan J.F. Wadwani S. Moulds C. Froehler B.C. Matteucci M.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3761-3764Crossref PubMed Scopus (102) Google Scholar, 16Koh J.S. Dervan P.B. J. Am. Chem. Soc. 1992; 114: 1470-1478Crossref Scopus (141) Google Scholar, 17Jetter M.C. Hobbs F.W. Biochemistry. 1993; 32: 3249-3254Crossref PubMed Scopus (68) Google Scholar, 18Ueno Y. Mikawa M. Matsuda A. Bioconjugate Chem. 1998; 9: 33-39Crossref PubMed Scopus (44) Google Scholar), the conjugation of different DNA intercalators to TFO (19Sun J.S. Giovannangeli C. Francois J.C. Kurfurst R. MontenayGarestier T. Asseline U. Saison-Behmoaras T. Thuong N.T. Helene C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6023-6027Crossref PubMed Scopus (107) Google Scholar, 20Mouscadet J.-F. Ketterle C. Goulaouic H. Carteau S. Subra F. Le Bret M. Auclair C. Biochemistry. 1994; 33: 4187-4196Crossref PubMed Scopus (63) Google Scholar) and/or the use of polyamines such as spermine or spermidine as triplex stabilizers (21Hampel K.J. Crosson P. Lee J.S. Biochemistry. 1991; 30: 4455-4459Crossref PubMed Scopus (135) Google Scholar) have been made to improve the stability of the pyrimidine motif triplex at physiological pH. However, in some cases, modification strategies lessened the overall binding affinity of the TFO or increased its nonspecific interaction with DNA (2Frank-Kamenetskii M.D. Mirkin S.M. Annu. Rev. Biochem. 1995; 64: 65-95Crossref PubMed Scopus (658) Google Scholar, 18Ueno Y. Mikawa M. Matsuda A. Bioconjugate Chem. 1998; 9: 33-39Crossref PubMed Scopus (44) Google Scholar). We have previously reported that poly (l-lysine)-graft-dextran (PLL-g-Dex) copolymer (poly (l-lysine) with grafts of hydrophilic dextran chains; Fig. 1 a) not only significantly increased the thermal stability of the triplex involving poly(dA)·2poly(dT) (22Maruyama A. Katoh M. Ishihara T. Akaike T. Bioconjugate Chem. 1997; 8: 3-6Crossref PubMed Scopus (140) Google Scholar) but also stabilized the pyrimidine motif triplex DNA at neutral pH using a 30-bp homopurine-homopyrimidine target duplex from rat α1(I) collagen gene promoter and an unmodified cytosine-rich TFO (23Ferdous A. Watanabe H. Akaike T. Maruyama A. Nucleic Acids Res. 1998; 26: 3949-3954Crossref PubMed Scopus (53) Google Scholar, 24Ferdous A. Watanabe H. Akaike T. Maruyama A. J. Pharm. Sci. 1998; 87: 1400-1405Abstract Full Text PDF PubMed Scopus (23) Google Scholar). However, the mechanistic explanation for the PLL-g-Dex copolymer-mediated triplex stabilization was not clearly understood. Therefore, we have further extended our previous study to address this issue in the context of the thermodynamic and kinetic effects of the PLL-g-Dex copolymer on pyrimidine motif triplex formation at neutral pH. The thermodynamic and kinetic effects of the copolymer on pyrimidine motif triplex formation between a 23-bp homopurine-homopyrimidine target duplex (Pur23A·Pyr23T) (Fig.1 b) and its specific 15-mer homopyrimidine TFO (Pyr15T) (Fig. 1 b) have been analyzed by isothermal titration calorimetry (ITC) (25Wiseman T. Williston S. Brandts J.F. Lin L.-N. Anal. Biochem. 1989; 179: 131-137Crossref PubMed Scopus (2447) Google Scholar, 26Kamiya M. Torigoe H. Shindo H. Sarai A. J. Am. Chem. Soc. 1996; 118: 4532-4538Crossref Scopus (59) Google Scholar), interaction analysis system (IAsys) (27Cush R. Cronin J.M. Stewart W.J. Maule C.H. Molloy J. Goddard N.J. Biosens. Bioelectron. 1993; 8: 347-353Crossref Scopus (386) Google Scholar, 28Edwards P.R. Gill A. Pollard-Knight D.V. Hoare M. Buckle P.E. Lowe P.A. Leatherbarrow R.J. Anal. Biochem. 1995; 231: 210-217Crossref PubMed Scopus (160) Google Scholar, 29Bates P.J. Dosanjh H.S. Kumar S. Jenkins T.C. Laughton C.A. Neidle S. Nucleic Acids Res. 1995; 23: 3627-3632Crossref PubMed Scopus (72) Google Scholar), and electrophoretic mobility shift assay (EMSA) (23Ferdous A. Watanabe H. Akaike T. Maruyama A. Nucleic Acids Res. 1998; 26: 3949-3954Crossref PubMed Scopus (53) Google Scholar, 24Ferdous A. Watanabe H. Akaike T. Maruyama A. J. Pharm. Sci. 1998; 87: 1400-1405Abstract Full Text PDF PubMed Scopus (23) Google Scholar). Results from the three independent lines of experiments have clearly indicated the effect of the copolymer in promoting pyrimidine motif triplex formation at neutral pH. In the presence of the copolymer, the binding constant for pyrimidine motif triplex formation at neutral pH was about 100 times higher than that observed with TFO alone. Moreover, the triplex-promoting efficiency of the copolymer was more than 20 times higher than that of the physiological concentration (about 1 mm) of spermine, a putative intracellular triplex stabilizer (30Thomas T. Thomas T.J. Biochemistry. 1993; 32: 14068-14074Crossref PubMed Scopus (130) Google Scholar). Kinetic data have also demonstrated that the major contribution for the copolymer-mediated promotion of triplex formation resulted from the considerable increase in the association rate constant rather than the decrease in the dissociation rate constant. The ability of the PLL-g-Dex copolymer to promote pyrimidine motif triplex formation at physiological pH would support further progress in therapeutic applications of the antigene strategy in vivo. We synthesized 23-mer complementary oligonucleotides for target duplex Pur23A and Pyr23T (Fig. 1 b) and the 15-mer homopyrimidine TFO Pyr15T (Fig.1 b) on an ABI DNA synthesizer using the solid-phase cyanoethyl phosphoramidite method and purified them with reverse-phase high performance liquid chromatography on a Wakosil DNA column. 5′-Biotinylated Pyr23T (denoted as Bt-Pyr23T) was prepared from biotin phosphoramidite. Two different nonspecific TFOs, Pyr15NS-1 and Pyr15NS-2 (Fig. 1 b), were synthesized as mentioned above and purchased from Grainers Japan Co. (Tokyo, Japan), respectively. The concentration of all oligonucleotides was determined by UV absorbance. Complementary strands Pur23A and Pyr23T were annealed by heating at up to 90 °C, followed by a gradual cooling to room temperature. The annealed sample was applied on a hydroxyapatite column (KOKEN Inc.) to remove unpaired single strands. The concentration of the duplex DNA (Pur23A·Pyr23T) was determined by UV absorption considering the DNA concentration ratio of 1 OD = 50 μg/ml, with aM r of 15180. The purified oligonucleotide solutions were dialyzed (molecular weight cutoff = 500) extensively against Buffer A (10 mm sodium cacodylate-cacodylic acid (pH 6.8), 200 mm sodium chloride, and 20 mmmagnesium chloride) with or without the triplex stabilizer (0.84 mm spermine or 0.038 mm PLL-g-Dex copolymer). The PLL-g-Dex copolymer (number average molecular weight = 7.9 × 104) (Fig. 1 a) was prepared by a reductive amination reaction between poly (l-lysine) and dextran T-10, as described in detail previously (22Maruyama A. Katoh M. Ishihara T. Akaike T. Bioconjugate Chem. 1997; 8: 3-6Crossref PubMed Scopus (140) Google Scholar, 31Maruyama A. Watanabe H. Ferdous A. Katoh M. Ishihara T. Akaike T. Bioconjugate Chem. 1998; 9: 292-299Crossref PubMed Scopus (122) Google Scholar). The purified copolymer solution was dialyzed (molecular weight cutoff = 1000) extensively against Buffer A. Isothermal titration experiments were carried out on a MCS ITC system (Microcal Inc.) essentially as described previously (25Wiseman T. Williston S. Brandts J.F. Lin L.-N. Anal. Biochem. 1989; 179: 131-137Crossref PubMed Scopus (2447) Google Scholar,26Kamiya M. Torigoe H. Shindo H. Sarai A. J. Am. Chem. Soc. 1996; 118: 4532-4538Crossref Scopus (59) Google Scholar). Briefly, the TFO and Pur23A·Pyr23T duplex DNA solutions were prepared by extensive dialysis against Buffer A with or without the triplex stabilizer. The TFO solution in Buffer A with or without the triplex stabilizer was injected 20 times in 5-μl increments at 10-min intervals into the Pur23A·Pyr23T duplex solution without changing the reaction conditions. The heat for each injection was subtracted by the heat of dilution of the injectant, which was measured by injecting the TFO into Buffer A with or without the triplex stabilizer. Each corrected heat was divided by the number of moles of TFO injected and analyzed with Microcal Origin software supplied by the manufacturer. Kinetic experiments were performed on an IAsys instrument (Affinity Sensors Cambridge Inc.), where a real-time biomolecular interaction was measured with a laser biosensor (27Cush R. Cronin J.M. Stewart W.J. Maule C.H. Molloy J. Goddard N.J. Biosens. Bioelectron. 1993; 8: 347-353Crossref Scopus (386) Google Scholar, 28Edwards P.R. Gill A. Pollard-Knight D.V. Hoare M. Buckle P.E. Lowe P.A. Leatherbarrow R.J. Anal. Biochem. 1995; 231: 210-217Crossref PubMed Scopus (160) Google Scholar, 29Bates P.J. Dosanjh H.S. Kumar S. Jenkins T.C. Laughton C.A. Neidle S. Nucleic Acids Res. 1995; 23: 3627-3632Crossref PubMed Scopus (72) Google Scholar). The resonant layer of a cuvette was washed with 200 μl of 10 mm acetate buffer (pH 4.6) and then activated with 200 μl of a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide andN-hydroxysuccinimide solution. The activated surface was washed again with 10 mm acetate buffer (pH 4.6), and streptavidin in 10 mm acetate buffer (pH 4.6) was immobilized on the surface. After blocking the remaining reactive groups with 1 m ethanolamine (pH 8.5), the cuvette was washed extensively with 10 mm acetate buffer (pH 4.6) and then with 20 mm hydrochloric acid to remove the loosely associated protein. The cuvette was washed with Buffer A, and Bt-Pyr23T (1.2 μm in Buffer A) was added to bind with the streptavidin on the surface. After washing the cuvette with the same buffer, complementary oligonucleotide Pur23A (1.2 μm in Buffer A) was added to hybridize with Bt-Pyr23T. After extensive washing and equilibrating of the Bt-Pyr23T·Pur23A-immobilized surface with Buffer A with or without the triplex stabilizer for more than 30 min, the TFO in 200 μl of Buffer A with or without the triplex stabilizer was injected over the immobilized Bt-Pyr23T·Pur23A duplex, and then triplex formation was monitored for 30 min. This was followed by washing the cuvette with Buffer A with or without the triplex stabilizer, and the dissociation of the preformed triplex was monitored for an additional 20 min. Finally, 100 mm Tris-HCl (pH 8.0) was injected for 3 min for a complete break of the Hoogsteen hydrogen bonding between the TFO and Pur23A, during which the Bt-Pyr23T·Pur23A duplex may be partially denatured. The Bt-Pyr23T·Pur23A duplex was regenerated by injecting 1.2 μm Pur23A. The resulting sensorgrams were analyzed with Fastfit software supplied by the manufacturer to calculate the kinetic parameters. EMSA experiments were performed essentially as described previously, with slight modifications (23Ferdous A. Watanabe H. Akaike T. Maruyama A. Nucleic Acids Res. 1998; 26: 3949-3954Crossref PubMed Scopus (53) Google Scholar, 24Ferdous A. Watanabe H. Akaike T. Maruyama A. J. Pharm. Sci. 1998; 87: 1400-1405Abstract Full Text PDF PubMed Scopus (23) Google Scholar). In 9 μl of reaction mixture, 32P-labeled duplex (∼10,000 cpm; ∼1 ng) was mixed with increasing concentrations of Pyr15T and the nonspecific oligonucleotide Pyr15NS-2 in either the absence or presence of the triplex stabilizer, (spermine or the PLL-g-Dex copolymer) in a buffer containing 50 mm Tris acetate (pH 7.0), 100 mm sodium chloride, and 10 mmmagnesium chloride. Pyr15NS-2 was added to achieve equimolar concentrations of TFO in each lane as well as to minimize the adhesion of the DNA (target duplex and Pyr15T) to plastic surfaces during incubation and subsequent losses during processing. After a 6-h incubation at 37 °C, 2 μl of 50% glycerol solution containing bromphenol blue and 1 μl of thymus DNA (6 μg) were added without changing the pH and salt concentrations of the reaction mixtures. Thymus DNA was added to chase the ionic interaction between DNA (target duplex and TFO) and the copolymer as described previously (23Ferdous A. Watanabe H. Akaike T. Maruyama A. Nucleic Acids Res. 1998; 26: 3949-3954Crossref PubMed Scopus (53) Google Scholar, 24Ferdous A. Watanabe H. Akaike T. Maruyama A. J. Pharm. Sci. 1998; 87: 1400-1405Abstract Full Text PDF PubMed Scopus (23) Google Scholar). Samples were then directly loaded onto a 15% native polyacrylamide gel prepared in buffer (50 mm Tris acetate, pH 7.0, and 10 mm magnesium chloride), and electrophoresis was performed at 8 V/cm for 16 h at 4 °C. The percentage of the formed triplex was estimated as described previously (23Ferdous A. Watanabe H. Akaike T. Maruyama A. Nucleic Acids Res. 1998; 26: 3949-3954Crossref PubMed Scopus (53) Google Scholar, 24Ferdous A. Watanabe H. Akaike T. Maruyama A. J. Pharm. Sci. 1998; 87: 1400-1405Abstract Full Text PDF PubMed Scopus (23) Google Scholar). We have examined the thermodynamics of pyrimidine motif triplex formation between the 23-bp target duplex Pur23A·Pyr23T and its specific 15-mer TFO, Pyr15T (Fig.1 b), at 25 °C and pH 6.8 by ITC under three different conditions: 1) Buffer A (10 mmsodium cacodylate-cacodylic acid at pH 6.8 containing 200 mm sodium chloride and 20 mm magnesium chloride), 2) Buffer A + 0.84 mm spermine, and 3) Buffer A + 0.038 mm PLL-g-Dex copolymer. The concentration of amino groups in 0.84 mm spermine is equivalent to that in 0.038 mm PLL-g-Dex copolymer. Fig. 2 a compares the ITC profiles of the initial three injections for triplex formation between Pyr15T and Pur23A·Pyr23T at 25 °C and pH 6.8 with TFO alone or in the presence of spermine or the PLL-g-Dex copolymer. The magnitudes of the exothermic peaks in the presence of 0.038 mm PLL-g-Dex copolymer were much larger than those observed with TFO alone. Triplex formation in the presence of 0.038 mm PLL-g-Dex copolymer reached to equilibrium within 10 min after each injection of Pyr15T. On the other hand, the magnitudes of the exothermic peaks in the presence of 0.84 mm spermine were indistinguishable from those observed with TFO alone. Fig. 2 b shows a 200-min ITC profile for triplex formation in the presence of the PLL-g-Dex copolymer. An exothermic heat pulse was observed after each injection of Pyr15T into Pur23A·Pyr23T. The magnitude of each peak decreased gradually with each new injection, and a small peak was still observed at a molar ratio of [Pyr15T]/[Pur23A·Pyr23T] = 2. The area of the small peak was equal to the heat of dilution measured in a separate experiment by injecting Pyr15T into the same buffer (data not shown). The area under each peak was integrated, and the heat of dilution of Pyr15T was subtracted from the integrated values. The corrected heat was divided by the number of moles injected, and the resulting values were plotted as a function of the molar ratio of [Pyr15T]/[Pur23A·Pyr23T], as shown in Fig. 2 c. The resulting titration plot was fitted to a sigmoidal curve by using a nonlinear least-squares method. The binding constant (K a) and the enthalpy change (ΔH) were obtained from the fitted curve (25Wiseman T. Williston S. Brandts J.F. Lin L.-N. Anal. Biochem. 1989; 179: 131-137Crossref PubMed Scopus (2447) Google Scholar, 26Kamiya M. Torigoe H. Shindo H. Sarai A. J. Am. Chem. Soc. 1996; 118: 4532-4538Crossref Scopus (59) Google Scholar). The Gibbs free energy change (ΔG) and the entropy change (ΔS) were calculated from the equation ΔG = −RTlnK a = ΔH − TΔS. The titration plots with TFO alone or in the presence of spermine are also shown in Fig. 2 c. The thermodynamic parameters under these conditions were obtained from these plots in the same way. Table I summarizes the thermodynamic parameters of triplex formation at 25 °C and pH 6.8 obtained from ITC under the three different conditions. The signs of both ΔH and ΔS were negative under all reaction conditions. Because an observed negative ΔS was unfavorable for triplex formation, triplex formation was driven by a large negative ΔH under each condition. The magnitude of the ΔH of pyrimidine motif triplex formation in the presence of 0.038 mm PLL-g-Dex copolymer was 2.5 times larger than those observed with TFO alone or in the presence of 0.84 mm spermine, consistent with the ITC profiles in Fig.2 a. The K a of pyrimidine motif triplex formation was increased 2.6 times or 95.9 times by the addition of 0.84 mm spermine or 0.038 mm PLL-g-Dex copolymer, respectively. Although the concentration of amino groups in 0.84 mm spermine and 0.038 mmPLL-g-Dex copolymer is equivalent, an increase inK a by the addition of 0.038 mmPLL-g-Dex copolymer was much higher than that by the addition of 0.84 mm spermine.Table IThermodynamic parameters for triplex formation between the 15-mer TFO Pyr15T and the 23-bp duplex Pur23A · Pyr23T at 25 °C and pH 6.8 in 10 mm sodium cacodylate-cacodylic acid, 200 mm sodium chloride, and 20 mm magnesium chloride with or without the triplex stabilizer obtained from ITC measurementsTriplex stabilizerK aK a(relative)ΔGΔHΔSm −1kcal mol−1cal mol−1K−1None(1.97 ± 0.43) × 1051.0−7.22 ± 0.15−34.9 ± 2.2−92.7 ± 8.00.84 mm spermine(5.15 ± 0.29) × 1052.6−7.79 ± 0.03−34.1 ± 3.2−88.2 ± 10.80.038 mmPLL-g-Dex copolymer(1.89 ± 0.32) × 10795.9−9.93 ± 0.11−87.9 ± 2.3−262 ± 8.1The concentration of Pyr15T is 120 μm in the syringe. Pyr15T is injected 20-times in 5-μl increments into Pur23A · Pyr23T. The concentration of Pur23A · Pyr23T is 5 μm in the cell. The obtained values are the average of at least three ITC experiments. Open table in a new tab The concentration of Pyr15T is 120 μm in the syringe. Pyr15T is injected 20-times in 5-μl increments into Pur23A · Pyr23T. The concentration of Pur23A · Pyr23T is 5 μm in the cell. The obtained values are the average of at least three ITC experiments. To understand the putative mechanism involved in the tremendous increase in K a of pyrimidine motif triplex formation in the presence of the PLL-g-Dex copolymer (Table I), we have assessed the kinetic parameters for the association and dissociation of Pyr15T with Pur23A·Pyr23T at 25 °C and pH 6.8 by IAsys. Fig.3 a compares the IAsys sensorgrams representing the triplex formation and dissociation involving 2.0 μm of the specific (Pyr15T) or nonspecific (Pyr15NS-1) TFOs (Fig. 1 b) and the immobilized Bt-Pyr23T·Pur23A at 25 °C and pH 6.8 in either the absence or presence of the triplex stabilizer. The injection of Pyr15T alone (Pyr15T in no stabilizer) over the immobilized Bt-Pyr23T·Pur23A caused an increased response, and the injection of Pyr15T and spermine (Pyr15T in 0.84 mm spermine) slightly increased the response. However, the response was substantially changed when Pyr15T and the PLL-g-Dex copolymer were injected (Pyr15T in 0.038 mm copolymer). It is important to note that the negligible response observed when Pyr15NS-1 was injected with the PLL-g-Dex copolymer (Pyr15NS-1 in 0.038 mmcopolymer) indicates that the specificity of triplex formation was preserved in the presence of the copolymer. Taken together, it unambiguously indicates that the PLL-g-Dex copolymer significantly increased the association rate constant of triplex formation, and its triplex-promoting efficacy was remarkably higher than that of spermine. We have measured a series of association and dissociation curves at the various concentrations of Pyr15T in the presence of the PLL-g-Dex copolymer to obtain the kinetic parameters. An increase in the concentration of Pyr15T led to a gradual change in the response of the association curves as shown in Fig.3 b. The on-rate constant (k on) was obtained from the analysis of each association curve. Fig.3 c shows a plot of k on against the concentrations of Pyr15T. The resultant plot was fitted to a straight line (r 2 = 0.98) using a linear least-squares method. The association rate constant (k assoc) was determined from the slope of the fitted line (27Cush R. Cronin J.M. Stewart W.J. Maule C.H. Molloy J. Goddard N.J. Biosens. Bioelectron. 1993; 8: 347-353Crossref Scopus (386) Google Scholar, 28Edwards P.R. Gill A. Pollard-Knight D.V. Hoare M. Buckle P.E. Lowe P.A. Leatherbarrow R.J. Anal. Biochem. 1995; 231: 210-217Crossref PubMed Scopus (160) Google Scholar, 29Bates P.J. Dosanjh H.S. Kumar S. Jenkins T.C. Laughton C.A. Neidle S. Nucleic Acids Res. 1995; 23: 3627-3632Crossref PubMed Scopus (72) Google Scholar). On the other hand, the off-rate constant (k off) was obtained from the analysis of each dissociation curve (Fig. 3 a; data not shown). Because k off is usually independent of the concentration of the injected solution, the dissociation rate constant (k dissoc) was determined by averagingk off for several concentrations (27Cush R. Cronin J.M. Stewart W.J. Maule C.H. Molloy J. Goddard N.J. Biosens. Bioelectron. 1993; 8: 347-353Crossref Scopus (386) Google Scholar, 28Edwards P.R. Gill A. Pollard-Knight D.V. Hoare M. Buckle P.E. Lowe P.A. Leatherbarrow R.J. Anal. Biochem. 1995; 231: 210-217Crossref PubMed Scopus (160) Google Scholar, 29Bates P.J. Dosanjh H.S. Kumar S. Jenkins T.C. Laughton C.A. Neidle S. Nucleic Acids Res. 1995; 23: 3627-3632Crossref PubMed Scopus (72) Google Scholar).K a was calculated from the equationK a =k assoc/k dissoc. The kinetic parameters with TFO alone and in the presence of spermine were obtained in the same way. Table II summarizes the kinetic parameters of triplex formation at 25 °C and pH 6.8 obtained from IAsys under the three different conditions. Although the relative values of K a were consistent with those obtained from IT" @default.
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• W1983157047 title "Poly(l-lysine)-graft-dextran Copolymer Promotes Pyrimidine Motif Triplex DNA Formation at Physiological pH" @default.
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