The role of polyamines, Na⁺ and K⁺ in the formation of triple helices between purine oligonucleotides and the promoter region of the human c-src proto-oncogene

Abstract

Binding constants for triplex formation between purine-rich oligonucleotides and a pyrimidine·purine tract of the human c-src proto-oncogene were measured by fluorescence polarization in the presence of polyamines, Na⁺ and K⁺. In both the hexamine and tetramine series, the longer polyamines had the larger binding constants for triplex formation at low concentrations of polyamine. At higher concentrations all values tended to plateau in the 10⁹/M range. In contrast to previous reports, K⁺ did not inhibit triplex formation and at 150 mM the binding constants were again in the 10⁹/M range for both an 11mer and 22mer oligonucleotide. At 150 mM K⁺ the addition of polyamines did not lead to any significant increase in the binding constants. It was determined that the lack of inhibition by K⁺ was due to the low concentration (1 nM) of purine oligonucleotide required for the fluorescence polarization technique. At higher concentrations (1 µM) self-association of the oligonucleotide was observed. These results suggest that in vivo, at least for the c-src promoter, the inhibition of triplex formation by K⁺ may not be detrimental. However, it may be difficult to achieve binding constants above ~10⁹/M even in the presence of polycations.

INTRODUCTION

Many eukaryotic genes contain pyrimidine·purine (pyr·pur) tracts in the promoter regions, which are potential targets for antigene therapy with triplex-forming oligonucleotides (1–4). Upon forming a triplex, transcription is blocked. Because of the inherent sequence specificity of triplex formation it may be possible to target a single gene. There are two major types of triplexes. pyr·pur·pyr triplexes can be formed by the addition of a pyrimidine oligonucleotide to the pyr·pur tract by Hoogsteen pairing (5). This requires a protonated cytosine so that, in general, the higher the pH the lower the triplex stability (6). In contrast, the pur·pyr·pur type of triplex is formed by addition of a purine oligonucleotide to the pyr·pur tract and is stable at neutral pH (7). However, it requires high concentrations of divalent metal ions for stability and there have been some reports that formation is severely inhibited by K⁺ (8,9).

Polyamines are naturally occurring multivalent cations which are known to have a profound effect on nucleic acid structure and function. For example, polyamines stabilize RNA/DNA duplexes and enhance the cellular uptake of oligonucleotides (10,11), both of which are important considerations for gene therapy. Polyamines also stabilize pyr·pur·pyr triplexes such that formation is favored at neutral pH (6,12). In the case of pur·pyr·pur triplexes, polyamines enhance the rate of triplex formation and can partially overcome the inhibition by K⁺ (13). Therefore, polyamines, which are present at high concentrations in the eukaryotic nucleus, may play an essential role in the success of antigene therapy.

In this report we have studied the effect of polyamines, Na⁺ and K⁺ on oligonucleotide-directed pur·pyr·pur triplex formation in the c-src promoter region. The c-src gene encodes a non-receptor tyrosine kinase which is activated in a number of human cancers and one of the pyr·pur tracts (TC1) is a potential target for antigene therapy (14–16). Previously we demonstrated that Mg²⁺ promoted the formation of triplexes within the TC1 tract (17). However, because of enthalpy–entropy compensation, there was an apparent upper limit to the binding constant of ~10⁸–10⁹/M which was broadly independent of both length and sequence of the oligonucleotide. This may limit the effectiveness of oligonucleotide-directed gene therapy. Therefore, it is important to understand to what extent polyamines might increase the affinity of triplex formation.

MATERIALS AND METHODS

The oligonucleotides were puchased from the Calgary Regional DNA Synthesis Laboratory. The measurement of binding constants by fluorescence polarization has been described in detail (17). Briefly, increasing concentrations of the TC1 duplex were added to a fluorescein-labeled oligonucleotide (1 nM) in 1 ml of buffer (20 mM HEPES, pH 7.0, 10 mM NaCl) with added polyamines or KCl or NaCl at 20°C. The millipolarization value (mP) was measured at equilibrium after incubation for 30 min. As shown previously (17), under these conditions triplex formation is complete within a few minutes. Upon triplex formation the mP value increases and the binding constant (K) was determined by fitting the data to a single site binding isotherm.

The hexamines and tetramines were a gift of Dr A. Shirahata (18).

The self-association of the fluorescein-labeled 22 nt long Aap22 purine oligonucleotide was measured as follows. Aliquots of Aap22 from a stock solution were added to a solution of 20 mM HEPES containing 10 mM NaCl and 150 mM KCl. Polarization values of fluorescein-labeled Aap22 were measured after incubating for 30 min at 20°C.

RESULTS

The structures of the polyamines, either hexamines or tetramines, are shown in Figure 1a. For example, spermine is a tetramine represented as 343, where the digits denote the number of methylene (-CH₂-) groups present between the two amine groups. Binding parameters for two 5′-end fluorescein-labeled purine oligonucleotides (AapL and Aap22) were measured by fluorescence polarization (17). AapL is an 11mer which binds to the left end of the TC1 tract of the c-src promoter (Fig. 1b) whereas Aap22 is a 22mer which covers the whole TC1 tract (17). As shown in Figure 2, upon addition of the TC1 duplex the millipolarization value (mP) of Aap22 increases until, at high concentrations, a plateau is reached. In all cases the hexamines gave larger initial and final mP values than the tetramines (see Discussion). Binding constants were calculated as described previously (17) and are listed in Table 1 for these and the other polyamines. In the hexamine series the binding constants are in the 10⁹/M range with a 2-fold increase for the longest polyamine, 34343, compared to the shortest, 33333. Increasing the polyamine concentration had no effect. Suprisingly, the 11mer oligonucleotide had a similar binding constant as the 22mer in the presence of 1 µM 33333. For the tetramine series at 1 µM there is again a trend towards higher binding constants with increasing polyamine length. Thermodynamic parameters were also estimated by measuring binding constants over a range of temperatures (data not shown). As was found previously for Mg²⁺ (17), polyamine-mediated triplex formation is entropy driven.

Figure 1.

(a) Structures of the polyamines. (b) Sequences of the oligonucleotides and target duplex.

Figure 2.

Triplex formation between Aap22 and the TC1 duplex in the presence of polyamines measured by fluorescence polarization. Upon addition of the duplex, the polarization of the fluorescein-labeled oligonucleotide increases. Squares, 1 µM 333; diamonds, 5 µM 333; circles, 1 µM 33333; triangles, 5 µM 33333.

Table 1. Values of binding constants (K) at 1 µM concentrations of polyamines.

Polyamines	TFO used	K (per M × 109)
33333	Aap22	0.74 (0.74)a
33433	Aap22	1.1
34343	Aap22	1.6
323	Aap22	0.06
333	Aap22	0.21 (0.2)
343 (spermine)	Aap22	0.19
353	Aap22	0.32
363	Aap22	0.43
383	Aap22	0.72
33333	AapL	0.89
333	AapL	0.036

The standard buffer was 20 mM HEPES, pH 7.0, 10 mM NaCl.

^aNumbers in parentheses indicate the values of K at 5 µM polyamines.

Initial experiments showed that once a triplex was formed in the presence of polyamines, it remained stable upon addition of high concentrations of K⁺, in contrast to previous reports which suggested that K⁺ inhibited triplex formation (8,9). Therefore, the interplay between polyamines and monovalent cations was studied directly. Figure 3a shows binding curves for AapL to the TC1 tract in the presence of increasing concentrations of Na⁺. As expected, Na⁺ promotes triplex formation and much higher concentrations are required compared to Mg²⁺ and polyamines. The calculated binding constants are listed in Table 2. A similar experiment with K⁺ is shown in Figure 3b. Suprisingly, K⁺ promotes triplex formation; the binding constants are lower than with Na⁺, but still within the 10⁸/M range under physiological conditions (150 mM K⁺). As shown in Table 2, the longer purine oligonucleotide, Aap22, also binds well in the presence of K⁺, with a binding constant of 1.8 × 10⁹/M at the highest concentrations.

Figure 3.

Triplex formation between Aap22 and the TC1 duplex in the presence of (a) NaCl and (b) KCl measured by fluorescence polarization. Squares, 50 mM; circles, 100 mM; triangles, 150 mM; diamonds, 200 mM.

Table 2. Effect of Na⁺ or K⁺ on the values of the binding constant (K).

[Na+] or [K+] (M)	K (per M × 109)
	AapL + TC1 duplex with NaCl	AapL + TC1 duplex with KCl	Aap22 + TC1 duplex with KCl	AapL + TC1 duplex with 1 µM 33333 and NaCl	AapL + Tc1 duplex with 1 µM 33333 and KCl	Aap22 + Tc1 duplex with 1 µM 33333 and KCl	Aap22 + Tc1 duplex with 1 µM 333 and KCl
0a				0.81	0.89	0.74	0.21
0.025				0.48	0.31
0.05	0.085	0.0089	0.32	0.29	0.21	1.8	1.0
0.1	0.42	0.11	1.7	0.21	0.23	1.5	1.3
0.15	0.59	0.32	1.8	0.41	0.22	1.8	1.4
0.2	0.85	0.36	1.8	0.61	0.3	1.5	1.3

^aThe standard buffer was 20 mM HEPES, pH 7.0, 10 mM NaCl.

Can the presence of polyamines increase the binding constants still further? As shown in Table 2, the answer is clearly no. Although polyamines are required for triplex formation at low K⁺ concentrations (e.g. 25 mM), at high concentrations of K⁺ there is no significant difference in binding constants in the presence of 1 µM 33333.

The polarization technique used in the present work requires only nanomolar concentrations of purine oligonucleotide, whereas previous studies of triplex formation used micromolar concentrations (8,9). Therefore, we investigated the effect of concentration on the structure of Aap22 in the presence of 150 mM KCl with or without 1 µM spermine. As shown in Figure 4, the mP value for the oligonucleotide alone is concentration dependent and the sigmoidal curve suggests a very cooperative process; this is attributed to tetraplex or G-quartet formation (19,20). Up to 10 nM there is little effect, whereas at 1 µM self-association is complete. Kinetic studies confirmed that at 1 nM oligonucleotide the mP value is stable for several days, whereas at 1 µM tetraplex formation occurs within seconds (data not shown).

Figure 4.

Self-association of oligonucleotide Aap22 measured by fluorescence polarization. Squares, 150 mM KCl; circles, 150 mM KCl + 1 µM spermine.

DISCUSSION

The fluorescence polarization method for evaluating triplex formation is applicable under a wide variety of conditions (17). In the presence of polyamines the initial mP value of the fluorescein-labeled oligonucleotide is higher than with mono- or divalent cations (17) and the final mP value representing complete conversion to triplex is also increased. However, the data still fit well to a single binding isotherm and reliable binding constants can be calculated. The increase in mP values in the presence of polyamines is most likely due to an increase in their molecular mass and an increased stiffness when large cations are bound. Both of these factors will tend to increase the mP values.

In general, the hexamines were better at promoting triplex formation than the tetramines at 1 µM, a result which is consistent with a requirement for charge neutralization in the stabilization of purine-rich triplexes. Binding constants for triplex formation between Aap22 and the c-src duplex did not increase upon increasing the concentrations of 33333 and 333 to 5 µM (Table 1). Under these conditions higher concentrations (10 µM) of the polyamines could not be evaluated because the polymers tended to precipitate. There was also a trend to lower binding constants for the shorter polyamines. For example, at 1 µM tetramine 323 the binding constant was only 0.06 × 10⁹/M. However, the central amino groups have the lowest pK_a values due to charge repulsion and this effect is exacerbated when the methylene bridge is only two in length. Therefore, the shorter polyamines may not be fully charged at pH 8, reducing their effectiveness.

On the other hand, in the presence of physiological concentrations of K⁺ the addition of polyamines had no effect on the binding constant for either the 11mer or 22mer. Presumably the cations are competing for binding to the phosphodiester backbone of the DNA and once charge neutralization is complete no further increase in the binding constant for triplex formation can occur. Polyelectrolyte theory would tend to support this view, since above a moderate concentration of cation no further decrease in the electrostatic charge of DNA is observed (21,22). Previously it had been thought that K⁺ inhibited triplex formation by sequestering the purine oligonucleotide as a tetraplex (8,9). Our results clearly show that at low concentrations of oligonucleotide, triplex formation is much faster than tetraplex formation. The kinetics of triplex formation are probably first order with respect to the oligonucleotide (17), whereas tetraplex formation is expected to be at least second order. Therefore, K⁺ promotes triplex formation at low oligonucleotide concentrations.

In summary, it would appear that with conventional purine oligonucleotides with physiological concentrations of K⁺, the highest binding constant that can be achieved is ~10⁹/M. Our previous results suggest that this limit is largely independent of sequence and length, since both variables show entropy/enthalpy compensation (17). Also, the highest oligonucleotide concentration which will not rapidly associate to a tetraplex is in the nanomolar range. With these values the following calculation is instructive. For a cell of radius 20 µm the volume is ~2 × 10^–14 m³, so that a single target sequence has a concentration of ~10^–13 M, i.e much lower than the maximum oligonucleotide concentration of 1 nM. Therefore, for a binding constant of 10⁹/M with 1 nM oligonucleotide the target will be ~50% bound. In other words, the apparent effectiveness of antigene oligonucleotides in vivo even in the presence of K⁺ can be rationalized. It is also clear that improvements in antigene therapy must come about either by reducing the ability of the purine oligonucleotide to form a tetraplex or by increasing the binding constant above 10⁹/M. Our results would suggest that increasing the binding constant by the addition of polycations may be very difficult to achieve.