Poly(L-lysine)-graft-dextran Copolymer Promotes Pyrimidine Motif Triplex DNA Formation at Physiological pH
THERMODYNAMIC AND KINETIC STUDIES*

Hidetaka TorigoeDagger §, Anwarul Ferdous, Hiromitsu Watanabe, Toshihiro Akaike, and Atsushi Maruyama§

From the Dagger  Gene Bank, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, and  Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuda, Midori, Yokohama 226-8501, Japan

    ABSTRACT
Top
Abstract
Introduction
References

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.

    INTRODUCTION
Top
Abstract
Introduction
References

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 (1-3). A triplex is usually formed through the sequence-specific interaction of a single-stranded homopurine or homopyrimidine triplex-forming oligonucleotide (TFO)1 with the major groove of the homopurine-homopyrimidine stretch in duplex DNA (1-5). 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 (1-5). 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 (1-5). 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 (6-10). The extreme instability of the pyrimidine motif triplex 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. Numerous efforts such as the replacement of cytosine bases in a homopyrimidine TFO with 5-methylcytosine (9, 11-13) or other chemically modified bases (14-18), the conjugation of different DNA intercalators to TFO (19, 20) and/or the use of polyamines such as spermine or spermidine as triplex stabilizers (21) 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 (2, 18).

We have previously reported that poly (L-lysine)-graft-dextran (PLL-g-Dex) copolymer (poly (L-lysine) with grafts of hydrophilic dextran chains; Fig. 1a) not only significantly increased the thermal stability of the triplex involving poly(dA)·2poly(dT) (22) but also stabilized the pyrimidine motif triplex DNA at neutral pH using a 30-bp homopurine-homopyrimidine target duplex from rat alpha 1(I) collagen gene promoter and an unmodified cytosine-rich TFO (23, 24). 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. 1b) and its specific 15-mer homopyrimidine TFO (Pyr15T) (Fig. 1b) have been analyzed by isothermal titration calorimetry (ITC) (25, 26), interaction analysis system (IAsys) (27-29), and electrophoretic mobility shift assay (EMSA) (23, 24). 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 (30). 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.

    MATERIALS AND METHODS

Preparation of Oligonucleotides-- We synthesized 23-mer complementary oligonucleotides for target duplex Pur23A and Pyr23T (Fig. 1b) and the 15-mer homopyrimidine TFO Pyr15T (Fig. 1b) 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. 1b), 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 a Mr 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 mM magnesium chloride) with or without the triplex stabilizer (0.84 mM spermine or 0.038 mM PLL-g-Dex copolymer).

Preparation of the PLL-g-Dex Copolymer-- The PLL-g-Dex copolymer (number average molecular weight = 7.9 × 104) (Fig. 1a) was prepared by a reductive amination reaction between poly (L-lysine) and dextran T-10, as described in detail previously (22, 31). The purified copolymer solution was dialyzed (molecular weight cutoff = 1000) extensively against Buffer A.

ITC-- Isothermal titration experiments were carried out on a MCS ITC system (Microcal Inc.) essentially as described previously (25, 26). 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.

IAsys-- Kinetic experiments were performed on an IAsys instrument (Affinity Sensors Cambridge Inc.), where a real-time biomolecular interaction was measured with a laser biosensor (27-29). 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 and N-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-- EMSA experiments were performed essentially as described previously, with slight modifications (23, 24). 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 mM magnesium 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 (23, 24). 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 (23, 24).

    RESULTS

Thermodynamic Effect of PLL-g-Dex Copolymer on the Pyrimidine Motif Triplex Formation at Neutral pH-- 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. 1b), at 25 °C and pH 6.8 by ITC under three different conditions: 1) Buffer A (10 mM sodium 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. 2a 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. 2b 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. 2c. The resulting titration plot was fitted to a sigmoidal curve by using a nonlinear least-squares method. The binding constant (Ka) and the enthalpy change (Delta H) were obtained from the fitted curve (25, 26). The Gibbs free energy change (Delta G) and the entropy change (Delta S) were calculated from the equation Delta G = -RTlnKa = Delta H - TDelta S. The titration plots with TFO alone or in the presence of spermine are also shown in Fig. 2c. The thermodynamic parameters under these conditions were obtained from these plots in the same way.


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Fig. 1.   a, structural formula of PLL-g-Dex copolymer. b, oligonucleotide sequences of the target duplex Pur23A·Pyr23T, the specific TFO Pyr15T, and the nonspecific TFOs Pyr15NS-1 and Pyr15NS-2.


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Fig. 2.   Thermodynamic analyses of pyrimidine motif triplex formation at neutral pH by ITC. a, the ITC profiles of the initial three injections for triplex formation between Pyr15T and Pur23A·Pyr23T at 25 °C and pH 6.8 in the absence or presence of the triplex stabilizer. The Pyr15T solution (120 µM in Buffer A; see "Materials and Methods") in the absence or presence of the triplex stabilizer (0.84 mM spermine or 0.038 mM PLL-g-Dex copolymer) was injected in 5-µl increments into 5 µM Pur23A·Pyr23T solution in the same buffer. Injections occurred over 10 s at 10-min intervals. The concentration of amino groups in 0.84 mM spermine is equivalent to that in 0.038 mM PLL-g-Dex copolymer. The profile in the presence of spermine is indistinguishable from that observed in the absence of the triplex stabilizer. b, a total ITC profile for triplex formation between Pyr15T and Pur23A·Pyr23T in the presence of the PLL-g-Dex copolymer. The Pyr15T solution was injected 20 times into the Pur23A·Pyr23T solution. Other experimental conditions were the same as those described in a. c, titration plots in the absence or presence of the triplex stabilizer against the molar ratio of [Pyr15T]/[Pur23A·Pyr23T]. The data were fitted using a nonlinear least-squares method.

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 Delta H and Delta S were negative under all reaction conditions. Because an observed negative Delta S was unfavorable for triplex formation, triplex formation was driven by a large negative Delta H under each condition. The magnitude of the Delta H 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. 2a. The Ka 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 mM PLL-g-Dex copolymer is equivalent, an increase in Ka by the addition of 0.038 mM PLL-g-Dex copolymer was much higher than that by the addition of 0.84 mM spermine.

                              
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Table I
Thermodynamic 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 measurements
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.

Kinetic Effect of PLL-g-Dex Copolymer on Pyrimidine Motif Triplex Formation at Neutral pH-- To understand the putative mechanism involved in the tremendous increase in Ka 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. 3a compares the IAsys sensorgrams representing the triplex formation and dissociation involving 2.0 µM of the specific (Pyr15T) or nonspecific (Pyr15NS-1) TFOs (Fig. 1b) 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 mM copolymer) 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. 3b. The on-rate constant (kon) was obtained from the analysis of each association curve. Fig. 3c shows a plot of kon against the concentrations of Pyr15T. The resultant plot was fitted to a straight line (r2 = 0.98) using a linear least-squares method. The association rate constant (kassoc) was determined from the slope of the fitted line (27-29). On the other hand, the off-rate constant (koff) was obtained from the analysis of each dissociation curve (Fig. 3a; data not shown). Because koff is usually independent of the concentration of the injected solution, the dissociation rate constant (kdissoc) was determined by averaging koff for several concentrations (27-29). Ka was calculated from the equation Ka = kassoc/kdissoc. The kinetic parameters with TFO alone and in the presence of spermine were obtained in the same way.


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Fig. 3.   Kinetic analyses of pyrimidine motif triplex formation at neutral pH by IAsys. Analysis of triplex formation by IAsys is described in detail under "Materials and Methods." a, typical IAsys sensorgrams for triplex formation at 25 °C and pH 6.8 after injecting 2.0 µM specific TFO alone (Pyr15T in Buffer A without the triplex stabilizer) or in the presence of spermine (Pyr15T in Buffer A with 0.84 mM spermine) or the PLL-g-Dex copolymer (Pyr15T in Buffer A with 0.038 mM PLL-g-Dex copolymer) into the Bt-Pyr23T·Pur23A-immobilized cuvette are shown. A solution containing 2.0 µM nonspecific TFO, Pyr15NS-1, and the copolymer (Pyr15NS-1 in Buffer A with 0.038 mM PLL-g-Dex copolymer) was also injected into the Bt-Pyr23T·Pur23A-immobilized cuvette as a control experiment. b, a series of IAsys sensorgrams for triplex formation between Pyr15T and Pur23A·Pyr23T at 25 °C and pH 6.8 in the presence of 0.038 mM PLL-g-Dex copolymer. The Pyr15T solution, which was diluted in Buffer A to achieve the indicated final concentrations, was injected with 0.038 mM PLL-g-Dex copolymer into the Bt-Pyr23T·Pur23A-immobilized cuvette. The binding of Pyr15T to Bt-Pyr23T·Pur23A was monitored as the response against time. c, measured kon values of triplex formation in b were plotted against the respective concentrations of Pyr15T. The plot was fitted to a straight line (r2 = 0.98) using a linear least-squares method.

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 Ka were consistent with those obtained from ITC under the three conditions (Table I), the magnitudes of Ka calculated from the ratio of kassoc/kdissoc in Table II were slightly smaller than those in Table I. The precise source of this discrepancy is not clearly understood. However, at least two possible reasons can interpret this. First, in IAsys, the target duplex Pur23A·Pyr23T is immobilized on the matrix surface. Therefore, the triplex formation in IAsys, rather than that in solution in ITC, may be accompanied by a larger negative change in entropy, which in turn leads to loss in Delta G and thereby Ka. Second, triplex formation on the matrix surface might be affected by the molecular exclusion effect due to the accumulation of oligonucleotides and the polymer on the matrix surface. Of course, further analysis is needed to provide a precise explanation for this observation.

                              
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Table II
Kinetic 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 IAsys measurements
Several different concentrations of Pyr15T were injected over the immobilized Bt-Pyr23T · Pur23A. The concentrations of the injected Pyr15T were between 2 and 40 µM for None, between 2 and 20 µM for 0.84 mM spermine, and between 0.5 and 2 µM for 0.038 mM PLL-g-Dex copolymer.

The kassoc of pyrimidine motif triplex formation increased 3.6 times or 45.6 times by the addition of 0.84 mM spermine or 0.038 mM PLL-g-Dex copolymer, respectively. Once again, we have found a more significant increase in kassoc by adding 0.038 mM PLL-g-Dex copolymer rather than 0.84 mM spermine, although the concentration of their amino groups is almost equal. In contrast, when the dissociation rate constant (kdissoc) of pyrimidine motif triplex formation was compared, a 1.1 times or 1.5 times lower kdissoc was obtained by the addition of 0.84 mM spermine or 0.038 mM PLL-g-Dex copolymer, respectively. Thus, the much larger Ka in the presence of the PLL-g-Dex copolymer resulted mainly from the increase in kassoc rather than the decrease in kdissoc.

Electrophoretic Mobility Shift Assay of Pyrimidine Motif Triplex Formation at Neutral pH-- Finally, we have investigated pyrimidine motif triplex formation between Pyr15T and Pur23A·Pyr23T in the absence or presence of the triplex stabilizer at pH 7.0 by EMSA. Fig. 4 shows that in the absence of the triplex stabilizer (None), Pyr15T had a very poor binding affinity for Pur23A·Pyr23T; therefore, a stable triplex was formed only at higher concentrations of Pyr15T. The addition of 5 µM nonspecific oligonucleotides (Pyr15NS-2 (see lane 1 for None) or Pyr15NS-1 (data not shown)) failed to form a triplex, suggesting a sequence-specific interaction of Pyr15T with Pur23A·Pyr23T to form a triplex. The addition of 1.0 mM spermine (Spermine) with Pyr15T did indeed slightly increase (~2 times) the binding affinity (see lane 5 for None and Spermine). It is important that the addition of 1.9 µM (1/20 concentration of that used in ITC and IAsys) PLL-g-Dex copolymer (Polymer) along with Pyr15T significantly increases triplex formation. Note that whereas no triplex was detected when 0.02 µM TFO was incubated in either the absence of the triplex stabilizer or the presence of 1.0 mM spermine (see lane 2 for None and Spermine), more than 60% of the triplex was formed in the presence of the PLL-g-Dex copolymer (see lane 2 for Polymer), although the charge ratio of (amino groups)stabilizer to (phosphate groups)DNA in the presence of the PLL-g-Dex copolymer was 25 times lower than that in the presence of spermine (see the figure legends). The Ka of pyrimidine motif triplex formation in the presence of the PLL-g-Dex copolymer was about 100 times higher than that in the absence of the triplex stabilizer or even in the presence of spermine. The tremendously higher triplex-promoting efficiency of the PLL-g-Dex copolymer observed by ITC (Fig. 2a; Table I) and IAsys (Fig. 3a; Table II) was further supported by EMSA.


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Fig. 4.   EMSA of pyrimidine motif triplex formation at neutral pH in either the absence or presence of the triplex stabilizer. Triplex formation was initiated by adding 32P-labeled Pur23A·Pyr23T duplex (~1 ng) with the indicated final concentrations of the specific TFO Pyr15T. Nonspecific oligonucleotide Pyr15NS-2 was added to adjust the equimolar concentrations of TFO (5.0 µM) in each lane. The reaction mixtures in the buffer (50 mM Tris acetate, pH 7.0, 100 mM sodium chloride, and 10 mM magnesium chloride) without the triplex stabilizer (None) or with 1.0 mM spermine (the charge ratio of (amino groups)spermine/(phosphate groups)DNA was 50) or with 1.9 µM PLL-g-Dex copolymer (the charge ratio of (amino groups)copolymer/(phosphate groups)DNA was 2) were incubated for 6 h at 37 °C and then electrophoretically separated on a 15% native polyacrylamide gel. The positions of the duplex (D) and triplex (T) are indicated.


    DISCUSSION

Using the ITC study, we have previously reported that the Ka, Delta H, and Delta S values of pyrimidine motif triplex formation between Pyr15T and Pur23A·Pyr23T at pH 4.8 were 9 × 107 M-1, -83.8 kcal mol-1, and -245 cal mol-1 K-1, respectively (26). The Ka observed for the same triplex formation at pH 6.8 without the triplex stabilizer (Table I) is more than 100 times lower than that at pH 4.8 (26), which is consistent with the previously reported results (6-10) showing that neutral pH is unfavorable for pyrimidine motif triplex formation involving C+·GC triads. In contrast, the Ka at pH 6.8 in the presence of the PLL-g-Dex copolymer (Table I) is similar in magnitude to that at pH 4.8 (26), indicating that the copolymer tremendously promotes pyrimidine motif triplex formation at pH 6.8 up to a similar level at pH 4.8.

The Delta H measured by ITC reflects a contribution from the protonation of cytosine bases and the deprotonation of the buffer (26) in addition to a contribution from the hydrogen bonding and base stacking involved in the triplex formation. Because the pKa value of cytosine in a homopyrimidine oligonucleotide is 4.4 (13) and Pyr15T contains five cytosines, approximately 1.4 and 0.02 cytosines of Pyr15T are protonated in solution at pH 4.8 and pH 6.8, respectively. Therefore, the residual 3.6 and 4.98 cytosines need to be protonated for effective pyrimidine motif triplex formation. After correcting for the deprotonation enthalpy change of the buffer (-0.47 kcal mol-1) and the protonation enthalpy change of cytosine (-2.8 kcal mol-1) (32), the corrected Delta H at pH 4.8 in the absence of the copolymer and at pH 6.8 in the presence of the copolymer was about -72.0 and -71.6 kcal mol-1, respectively. These values are consistent with each other, suggesting that pyrimidine motif triplex formed almost stoichiometrically even at pH 6.8 in the presence of the copolymer (Table I).

The Delta S measured by ITC includes a positive entropy change from a release of structured water upon triplex formation, an entropy change accompanying the counterion condensation effect described below (33, 34), and a major contribution of a negative entropy change from the conformational restraint of TFO upon triplex formation (26). The magnitudes of Delta S at pH 4.8 in the absence of the copolymer (-245 cal mol-1 K-1) (26) and at pH 6.8 in the presence of the copolymer (-262 cal mol-1 K-1) (Table I) are quite similar, which also supports effective pyrimidine motif triplex formation at pH 6.8 in the presence of the copolymer (Table I). In contrast to stoichiometric triplex formation in the presence of the copolymer, triplex formation without the triplex stabilizer (Table I) and in the presence of spermine (Table I) is less stoichiometric, because the magnitudes of Delta H and Delta S under these conditions are considerably lower than those at pH 4.8 (26).

Because the Ka of pyrimidine motif triplex formation at pH 6.8 increased in the presence of spermine or the PLL-g-Dex copolymer (Tables I and II; Fig. 4), both of them have the ability to stabilize the triplex, but to a different extent. The counterion condensation (CC) model developed by Manning (33) and elaborated by Record et al. (34) provides reasonable descriptions of the electrostatic interaction of counterions with DNA. The association of TFO with the duplex to form a triplex results in an increase in the linear charge density of DNA. Because the extent of CC per phosphate charges is a function of the linear charge density of DNA (33, 34), triplex formation facilitates the CC in the vicinity of the triplex from bulk solution (35, 36). However, the extent of the CC, which is entropically unfavorable for triplex formation, should be reduced in the presence of spermine or PLL-g-Dex copolymer because the association of these polycationic (or oligocationic) materials with DNA is a counterion release process (37).

Whereas the concentration of amino groups in 0.038 mM PLL-g-Dex copolymer is equivalent to that in 0.84 mM spermine, the Ka in the presence of the former was much larger than that seen in the presence of the latter (Tables I and II). Furthermore, as shown by EMSA (Fig. 4), triplex formation was still promoted while a 1/20 concentration (1.9 µM) of the copolymer was used. The difference in the promoting efficacy between spermine and the copolymer probably results from their association constant with polynucleotides. An association constant of a cationic substance with polynucleotides increases with the valency of the cation (37, 38). Compared with spermine, which bears only four cationic charges, the polycationic copolymer associates more stably with polynucleotides. Indeed, the electrostatically equivalent amount of the copolymer (the charge ratio of (amino groups)copolymer to (phosphate groups)DNA was 1) completely abrogated the electrophoretic migration of 20-mer oligonucleotides into polyacrylamide gels (31), confirming a stoichiometric and stable complex formation between the copolymer and the oligonucleotide. Furthermore, our previous melting curve analysis also supports the stable association of the copolymer with polynucleotides at physiological ionic strength (31). Because the association of a polycationic (or oligocationic) substance with polyanions, such as polynucleotides, is largely driven by entropic gain from counterion release (37, 38), the more stable association of the copolymer rather than spermine should be accompanied by a larger counterion release. Thus, the stable association of the copolymer would considerably reduce the extent of the CC and isolate triplex formation from the CC effect. Isolation from the CC effect causes a net increase in Delta S upon triplex formation, providing a favorable component to Delta G and Ka, although the apparent Delta S observed by ITC in the presence of the copolymer (Table I) is decreased compared with that observed in the absence of triplex stabilizers (Table I) or in the presence of spermine (Table I) due to the negative conformational entropy change from the conformational restraint of TFO upon the stoichiometric triplex formation discussed above (26).

Despite such a strong association of the copolymer, it is unique that the copolymer allows polynucleotides to recognize and associate with each other on the basis of the base sequences (22, 31). As shown in the present (Fig. 3a) and previous (23) studies, little association of nonspecific oligonucleotides with the target duplex was observed. This result further supports the idea that the copolymer does not hinder the sequence specificity of triplex formation. The abundant dextran moieties of the copolymer seem to play an important role in the maintenance of sequence-specific recognition of polynucleotides. Polycationic homopolymer, such as poly L-lysine and polyarginine, formed insoluble complexes with polynucleotides (39). The higher-order structure of the polynucleotides was considerably affected in these complexes (40, 41). The sequence-specific recognition and association between polynucleotides was disturbed by these polycations (31). In contrast, the copolymer containing hydrophilic dextran chains in addition to poly L-lysine formed soluble complexes with polynucleotides (22) and did not disorder the higher-order structure of polynucleotides, such as poly(dA)·poly(dT) duplex, poly(dA)·2poly(dT) triplex, and bovine thymus DNA (22, 31). The maintenance of the solubility and the higher-order structure of polynucleotides may be important for conserving the sequence-specific interaction between polynucleotides.

In addition to the influence of the copolymer upon the CC effect, other mechanisms might be involved in the enhancement of triplex formation by the copolymer. The copolymer is designed to consist of 90 weight % dextran and 10 weight % poly L-lysine. The polynucleotides associated with the copolymer may be forced to merge into the dextran-rich phase, which is low in dielectric constant. Such an environment under a low dielectric constant may enhance hydrogen bonding between TFO and the target duplex to promote triplex formation. Indeed, it has been reported that triplex stabilized by polyamines was further stabilized by the addition of an organic solvent to aqueous media (42).

Kinetic data have demonstrated that the copolymer considerably increases the kassoc of pyrimidine motif triplex formation (Table II). The increase in the kassoc is a plausible kinetic reason to explain the remarkable gain in the Ka at neutral pH by the copolymer. Both our group (26) and others (43) have previously proposed a model in which triplexes form along nucleation-elongation processes; in a nucleation step, only a few base contacts of the Hoogsteen hydrogen bonds may be formed between TFO and the target duplex, and this may be followed by an elongation step, in which Hoogsteen base pairings progress to complete triplex formation. Both groups (26, 43) have also suggested that the observed Ka, which is the ratio of kassoc/kdissoc, may largely reflect a rapid equilibrium of the nucleation step, which is probably the rate-limiting process of triplex formation. In this sense, the copolymer is considered to accelerate the nucleation step by reducing the extent of the CC to increase the Ka in pyrimidine motif triplex formation.

The present study has clearly demonstrated that the PLL-g-Dex copolymer promotes pyrimidine motif triplex formation at neutral pH. The triplex-promoting effect of the PLL-g-Dex copolymer does not affect the sequence specificity of triplex formation. We conclude that the design of polycationic copolymer with grafts of hydrophilic side chains (i.e. comb-type copolymer) is certainly a promising strategy for the promotion of triplex formation and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.

    ACKNOWLEDGEMENTS

We gratefully acknowledge M. Aoyagi and Y. Kanaya of Nissei Sangyo Co., Ltd. for generous help with the IAsys measurements.

    FOOTNOTES

* This work was supported in part by Grants-in-Aid 08249247 (to H. T.) and 09750967 (to A. M.) from the Ministry of Education, Science, Sports and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 81-298-36-9082; Fax: 81-298-36-9080; E-mail: torigoe{at}rtc.riken.go.jp (H. T.). Tel.: 81-45-924-5809; Fax: 81-45-924-5815; E-mail: amaruyam{at}bio.titech.ac.jp (A. M.).

    ABBREVIATIONS

The 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; kon, on-rate constant; kassoc, association rate constant; koff, off-rate constant; kdissoc, dissociation rate constant; CC, counterion condensation; bp, base pair.

    REFERENCES
Top
Abstract
Introduction
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