| Nucleic Acids Research | Pages |
Nucleic acid duplex stability: influence of base composition on cation effects
Introduction
Materials And Methods
Materials
UV measurements
Determination of thermodynamics for duplex formations
Prediction of thermodynamics by nearest-neighbor parameters
Circular dichroism (CD) measurements
Results And Discussion
Thermodynamics of nucleic acid duplex in 100 mM NaCl-phosphate buffer
Correlation among thermodynamics for nucleic acid duplex formations
Sodium ion concentration sensitivity of duplex stabilities
Effects of monovalent and divalent cations on nucleic acid duplex stability
Insights of cation binding to nucleic acid duplexes
Acknowledgements
References
Nucleic acid duplex stability: influence of base composition on cation effects
Received February 21, 1999; Revised and Accepted June 3, 1999
ABSTRACT The effects of counter ion on a nucleic acid duplex stability were investigated. Since a linear free energy relationship for the thermostability of oligonucleotide duplexes between those in 1 M and in 100 mM NaCl-phosphate buffer were observed regardless of whether they are DNA-DNA, RNA-RNA or RNA-DNA duplexes, simple prediction systems for [Delta]G°37 as well as Tm values in 100 mM NaCl-phosphate buffer were established. These predictions were successful with an average error of only 2.4°C for Tm and 5.7% for G°37 values. The number of Na+ newly bound to a duplex when the duplex forms (-[Delta]n) was significantly influenced by the base composition, and -[Delta]n for d(GCCAGTTAA)/d(TTAACTGGC) was different for MgCl2, CaCl2, BaCl2 and MnCl2 (from 0.70 to 0.76 with the same order of the duplex stability). Almost no additive effects on the duplex stability was observed for NaCl and MgCl2, suggesting a competitive binding for these cations. The sequence-dependent manner of [Delta]n suggests the presence of preferential base pairs or nearest-neighbor base pairs for the cation binding, which would affect nearest-neighbor parameters.
INTRODUCTION
The stability of many kinds of nucleic acid duplexes were measured to investigate their stable folding structures, potential as recognition sites by a protein, as candidate target sites for an antisense nucleotide, etc. (1-3). Now, it is possible to estimate the thermodynamics for oligonucleotide duplexes by nearest-neighbor parameters, and we can easily evaluate any duplex stability with good agreement with experimental results (4-9). However, since all of these prediction parameters are determined in 1 M NaCl-phosphate buffer, they are ambiguous for the prediction of duplex stabilities under physiological salt conditions, e.g., 100 mM NaCl and NaCl with MgCl2. Despite the importance of the salt concentration and species on the duplex thermostability, detailed experimental data are lacking, though some information for polynucleotides of different base composition were reported described as a melting temperature (Tm) (10). Since the Tm for a polynucleotide is the temperature at which 50% of the base pairs in a duplex have been dissociated (11), it is difficult to determine the number of dissociated base pairs and to estimate the effects of loops and single-stranded nucleotides on duplex stability. They also had less possibilities for forming secondary structures such as hairpin loops, thereby lowering the observed duplex stability (12). There are publications about salt effect on oligonucleotide duplex stability of d(AT) oligomers (13), r(A7U7p)2 (14), dumb-bell shaped DNAs (15), d(GCATGC)2 (16), r(AUGCAU)2 with 3[prime] dangling end (17), parallel-stranded DNA (18) etc. However, systematic information about nucleic acid hybridization and stability data also exist. Lesnik and Freier reported Tm and [Delta]G° data in 100 mM NaCl-phosphate buffer (19,20) though they used relatively longer oligonucleotides (up to 21 bp) and values were estimated from only a single melting curve. To determine quantitative salt effects on a nucleic acid duplex, reliable thermodynamic data on short oligonucleotides are needed. Here, we used short (14 bp) oligonucleotide duplexes that dissociate completely to become separated strands at temperatures higher than the Tm value and that can be applied to the nearest-neighbor model for the duplex stability (4-9). We measured thermodynamics in 100 mM NaCl-phosphate buffer for 30 kinds of oligonucleotide duplexes including those DNA-DNA, RNA-RNA and RNA-DNA composition, and developed a prediction system for their stabilities. Furthermore, several monovalent and divalent cations were also investigated. With these results, we described physical roles for cations on a duplex stability in comparison to reported information about cations with nucleotides arising from ab initio, semi-empirical and empirical results.
MATERIALS AND METHODS
Materials
All oligodeoxyribonucleotides and oligoribonucleotides were synthesized chemically on a solid support using phosphoramidite procedures and purified with reversed-phase high performance liquid chromatography (HPLC) after deblocking operations. These nucleotides were desalted with a C-18 Sep-Pak cartridge. All of these oligonucleotide duplexes were designed to be from 6 to 14 nt long and to form Watson-Crick base pairs without producing unpaired nucleotides nor a hairpin-looped structure. Oligonucleotide concentration was determined from the absorbance at 260 nm with single strand extinction coefficients calculated from mononucleotide and dinucleotide data of a nearest-neighbor approximation (21). All duplexes, DNA duplex (DNA-DNA), RNA duplex (RNA-RNA) and RNA-DNA hybrid duplex (RNA-DNA), were prepared by mixing an equimolar concentration of DNA and RNA strands.
UV measurements
Absorbance measurements were made on Hitachi U-3200, U-3210 and Beckman DU 640 spectrophotometers. Melting curves (absorbance versus temperature curves) were measured at 260 nm with a connected temperature controller. The water condensation on the cuvette exterior at a low-temperature range was avoided by flushing with a constant stream of dry N2 gas. The heating rate was 0.5 or 1.0°C/min. UV melting curves were collected in NaCl-phosphate buffer, which contains NaCl, 10 mM Na2HPO4 and 1 mM Na2EDTA (pH 7.0). Cation chloride-cacodylate buffers were prepared by dissolving each cation chloride in 10 mM sodium cacodylate solution to the desired cation chloride concentration; thereafter the pH value was adjusted to 7.0.
Determination of thermodynamics for duplex formations
Melting curves were fitted with a procedure to obtain thermodynamic parameters ([Delta]H°, [Delta]S° and [Delta]G°37) for a double helix formation as described elsewhere (22). This method makes an estimation of the thermodynamic values from the shape of each melting curve. To increase the accuracy of these determinations, we also evaluated them from plots of Tm-1 versus ln(Ct/s). These thermodynamic values were also analyzed by the following equations (5,6):
| Tm-1 = R·ln(Ct/s)/[Delta]H° + [Delta]S°/[Delta]H° | 1 |
| [Delta]G°37 = [Delta]H° - 310.15·[Delta]S° | 2 |
where Ct is the total strand concentration, and s reflects a sequence symmetry of the self (s = 1) or non-self complementary strands (s = 4).
Estimated errors for thermodynamic values ([sigma][Delta]H°, [sigma][Delta]S° and [sigma][Delta]G°37) derived from a curve fitting procedure were the standard deviations among data points of each melting curves measured at different Cts. Those for [Delta]H° and [Delta]S° ([sigma][Delta]H°, [sigma][Delta]S°) from Tm-1 versus ln(Ct/s) plots were estimated from the linearity of the plots and those for [Delta]G°37 ([sigma][Delta]G°37) were calculated by the following equation:
| ([sigma][Delta]G°37)2 = ([sigma][Delta]H°)2 + 310.152([sigma][Delta]S°)2 - 2·310.15·(R[Delta]H°, [Delta]S°)·[sigma][Delta]H°·[sigma][Delta]S°, | 3 |
where R[Delta]H°, [Delta]S° is the correlation coefficient between [Delta]H° and [Delta]S° (8,9,23). The final thermodynamic parameters were evaluated from the average values obtained from the curve fitting and Tm-1 versus ln (Ct/s) plots.
Prediction of thermodynamics by nearest-neighbor parameters
Nearest-neighbor parameters for the nucleic acid duplexes were reported by Sugimoto et al. (6) and SantaLucia Jr et al. (7,8) for DNA-DNA (DNA duplex), Freier et al. (4) and Xia et al. (9) for RNA-RNA (RNA duplex) and Sugimoto et al. (5) for RNA-DNA hybrid duplexes. According to the nearest-neighbor model, thermodynamic values ([Delta]H°, [Delta]S° and [Delta]G°37) for a duplex formation consists of the following three terms: (i) a free energy change for a helix propagation as a sum of each subsequent base pair, 10 kinds of data sets for both DNA-DNA and RNA-DNA but 16 sets for RNA-DNA (5); (ii) a free energy change for the helix initiation; and (iii) a free energy change of a mixing entropy term when a duplex is composed of a self-complementary strand. The sequences of oligonucleotide duplexes were divided into nearest-neighbor base pairs and these three terms were summed up according to the nearest-neighbor parameters (4-9). Melting temperatures (Tm values) at 8 µM total nucleotide strand concentration were calculated with predicted [Delta]H° and [Delta]S° from the equation 1.
Circular dichroism (CD) measurements
CD spectra were obtained on a JASCO J-600 spectropolarimeter equipped with a temperature controller. The experimental temperature was 5°C. The cuvette-holding chamber was flushed with a constant stream of dry N2 gas to avoid water condensation on the cuvette exterior. All CD spectra were measured from 320 to 200 nm in 0.1 cm path-length cuvettes. The concentration of the samples was 70 µM in 100 mM NaCl/10 mM phosphate/1 mM Na2EDTA (pH 7.0) or 1 mM MgCl2/10 mM sodium cacodylate buffer (pH 7.0).
RESULTS AND DISCUSSION
Thermodynamics of nucleic acid duplex in 100 mM NaCl-phosphate buffer
Predictions of the stability of nucleic acid duplexes (DNA-DNA, RNA-RNA and RNA-DNA) in 1 M NaCl concentration are successful using nearest-neighbor parameters (4-9). To estimate the duplex stabilities in biological salt conditions, an extended usage of these nearest-neighbor parameters for the stability predictions in 100 mM NaCl was examined. Prediction of the duplex stability in 100 mM NaCl buffer is badly needed in view of the usage of antisense oligonucleotides in biological systems. Thermodynamics ([Delta]H°, [Delta]S° and [Delta]G°37) for 30 kinds of oligonucleotide duplexes (21 DNA-DNA, 6 RNA-RNA and 3 RNA-DNA duplexes) in 100 mM NaCl-phosphate buffer were measured and their parameters are listed in Table 1 including Tm values at 8 µM. All of these duplexes showed a two-state transition in 1 M NaCl-phosphate buffer and their thermodynamics in 1 M NaCl were predicted by nearest-neighbor parameters with a high accuracy. Here, all of these duplexes also showed a two-state transition in 100 mM NaCl with <10% differences among the parameters determined by curve fittings and Tm-1 versus ln(Ct/s) plots. As expected, these duplexes decreased their stability with respect to those measured in 1 M NaCl (5,6) because of the anionic character of the nucleotide. Three DNA duplexes of d(GACTAGTC)2, d(GAGTACTC)2 and d(GTCTAGAC)2 having the same nearest-neighbor base pairs presented similar thermodynamic parameters, indicating the nearest-neighbor model can also be applied in 100 mM NaCl. We employed a relationship between thermodynamics in 100 mM NaCl and those predicted for 1 M NaCl (4-6). Plots between Tm in 100 mM NaCl and those predicted for 1 M NaCl are given in Figure 1A, including those determined by Lesnik and Freier, although they used relatively longer (more stable) oligonucleotides and estimated Tm and [Delta]G°37 values from the shape of a single melting curve (19,20). They showed good correlations of Tm values obtained in 100 mM with their predicted values. For [Delta]G°37, linear plots were also found as shown in Figure 1B. These linear plots were fitted to a straight line, giving the following equations:
| Tm (100 mM) = 0.876 Tm (1 M) - 5.148 | 4 |
|
[Delta]G°37 (100 mM) = 0.630 [Delta]G°37 (1 M) - 1.667 |
5 |
These equations indicate the possibility of estimating Tm and [Delta]G°37 values in 100 mM NaCl by using nearest-neighbor parameters determined in 1 M NaCl. Equation 5 indicates a linear free energy relationship (LFER) between [Delta]G°37 values in 1 M and in 100 mM NaCl, and when the other prediction parameters reported by Allawi and SantaLucia for DNA duplex (8) and Xia et al. for RNA duplex (9) were applied, almost the same equation of [Delta]G°37 (100 mM) = 0.632 [Delta]G°37 (1 M) - 1.360, was obtained. Conveniently, Tm and [Delta]G°37 values in Table 1 can be predicted by these equations with an average error of 2.4°C and 5.7%. These are improved estimates with regard to previously reported methods (those of 3.7°C and 8.6%, respectively) which emphasize the number of phosphate groups of a duplex on the salt effect (7,24). These equations are also used to predict another 86 data sets that have been measured by several groups (19,20,25,26). The resulting average errors of the predictions for Tm and [Delta]G°37 are 3.8°C and 10.2%, respectively. The equations 4 and 5 agree well with the relatively shorter (unstable) nucleotides, but less well with the longer (stable) ones. This might be because of a non-two-state transition or a limitation of the nearest-neighbor model for some longer nucleotides examined by Lesnik and Freier (19,20). Lesnik and Freier also reported linear relationships of Tm and [Delta]G°37 in 100 mM NaCl versus predicted Tm and [Delta]G°37 in 1 M NaCl for RNA duplex and for RNA-DNA hybrid duplex separately (20). Here, we suggest the equations 4 and 5 regardless of DNA-DNA, RNA-RNA or RNA-DNA duplex.
Figure 1. Relationship beween measured (A) Tm and (B) [Delta]G°37 in 100 mM NaCl and those in 1 M NaCl predicted by nearest-neighbor parameters. Data with open and closed symbols are data presented by Lesnik and Freier (19,20) and those shown in Table 1, respectively. These straight lines are drawn as least-square fits and their correlation coefficients are presented as r2.
 |
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Table 1. Thermodynamic parameters of helix formation in NaCl-phosphate buffer determined with Tm-1 versus lnCt and curve fitting procedurea
aAll experiments were done in buffer containing 100 mM NaCl/10 mM Na2HPO4/1 mM Na2EDTA, pH 7.0.
bMelting temperatures were calculated at the total oligomer strand concentration of 8 µM.
Quantitatively, effects of the sodium ion concentration on the thermostability of nucleic acid duplex are considered with the following equilibrium relations (27,28):
| -[alpha]·[Delta]n = d logKa/d log[Na+] | 6 |
| (d Tm/d log[Na+])/2.303 = [alpha]·[Delta]n·R·Tm2/H[prime]° | 7 |
where [alpha] correlates with an activity coefficient of the sodium ion ([gamma]), equal to (1 + d ln[gamma]/d ln[Na+]) and which is regarded as a constant of 0.92 over the NaCl concentration range considered here (17,28,29). [Delta]n is the number of sodium ions released from a duplex in the duplex formation, resulting in a negative n value for a duplex formation (28). The association constant of Ka, [Delta]H[prime]° (enthalpy change per mole of nucleotide) and Tm values can be obtained from the measurements of duplex stability. The linear correlation observed for [Delta]G°37 values between 1 M and 100 mM NaCl in spite of nucleotide sequence, chain length and duplex species (Fig. 1B) means any two duplexes with the same stability show the same d logKa/d log[Na+] value suggested from equation 5, resulting in the same number of Na+ required for duplex formation given in equation 6. Thus, [Delta]n for Na+ would be significantly affected by the base composition as well as the number of phosphate groups.
Correlation among thermodynamics for nucleic acid duplex formations
Figure 2A shows a relationship between T·[Delta]S° and [Delta]H° for 204 duplex formations, 87 DNA-DNA (6,7), 49 RNA-RNA (4,11,30) and 68 RNA-DNA (5) measured in 1 M NaCl-phosphate buffer. Although a rectangular hyperbola relationship between them has been suggested (31), a good straight line (T·[Delta]S° = 0.868·[Delta]H° - 0.304) with a correlation coefficient of r2 = 0.99 was revealed due to a compensation between [Delta]H° and [Delta]S°. It is considered that the slope of the plot is attributable to the structural demands necessary for the nucleotide association, and that of the intercept is an entropy contribution owing to the release of solvent when a complex forms (32). For T·[Delta]S° and [Delta]H° plots for porphyrin derivatives associated with quinoline analogs showed 0.62 and 0.11 kcal mol-1 for the slope and its intercept, respectively (32). A larger slope for oligonucleotides than that for porphyrin with quinoline is reasonable in view of the flexible structure of nucleotides; however it is unlikely to explain the negative intercept for oligonucleotides because water molecules dissociated from nucleotides result in a positive entropy change upon a duplex formation. Thus, the negative intercept of Figure 2A indicates that an association such as cation bindings to a duplex should be incorporated for the nucleotide duplex formation. This suggests a role of cation in the duplex initiation, which also reflects to a helix initiation factor in the nearest-neighbor parameter (6).
Figure 2. Plots between (A) T·[Delta]S° and [Delta]H°, and (B) Tm-1 and -[Delta]G°37-1 for DNA-DNA, RNA-RNA and RNA-DNA duplexes measured in 1 M NaCl-phosphate buffer. Data are presented by Freier et al. (4), Sugimoto et al. (5,6), SantaLucia Jr et al. (7), Longfellow et al. (18) and Peritz et al. (30). The open and closed symbols in (B) indicate whether a nucleotide sequence is a non-self or self-complementary strand, respectively. The gray symbols are those measured in 100 mM NaCl-phosphate buffer (Table 1). Thermodynamics described here were estimated at the total strand concentration of 8 µM.
Tm and [Delta]G°37 values measured in both 1 M and 100 mM NaCl buffer also revealed a linear correlation between reciprocals of Tm (K) and those of -[Delta]G°37. The plots in Figure 2B were grouped into two straight lines depending on whether a self-complementary sequence of the duplex was involved. With these two linear equations of Tm-1 = -3.36 × 10-3·[Delta]G°37-1 + 2.81 × 10-3 for self complementary strands and Tm-1 = -3.27 × 10-3·[Delta]G°37-1 + 2.77 × 10-3 for non-self complementary strands, it is possible to estimate [Delta]G°37 from Tm values-the most easily available experimental parameter. Also, this equation agrees well with thermodynamic data measured in 100 mM NaCl reported by Lesnik and Freier (19,20) (data not shown). Theoretically, approximation of the straight line between T·[Delta]S° and [Delta]H°, ignoring its intercept, can yield a linear equation between Tm-1 and [Delta]G°37-1; however, the duplexes with a lower stability deviate from linear plots due to the limitation of the approximation. As a result, the equations give good theoretical agreement with the two linear plots in Figure 2B (Tm-1 = -3.43 × 10-3·[Delta]G°37-1 + 2.80 × 10-3 for self complementary strands and Tm-1 = -3.07 × 10-3·[Delta]G°37-1 + 2.80 × 10-3 for non-self complementary strands). Moreover, the fact that the relationship between Tm-1 and [Delta]G°37-1 is derived from thermodynamic equations means the reciprocal correlation of Tm and [Delta]G°37 could be applied for any oligonucleotide concentration and to duplexes including non-Watson-Crick pairs such as mismatches. Note that both plots in Figure 2A and B were the same regardless of DNA-DNA, RNA-RNA or RNA-DNA duplexes.
Sodium ion concentration sensitivity of duplex stabilities
Effects of sodium ion concentration on Tm for five kinds of DNA-DNA, RNA-RNA and RNA-DNA duplexes with scrambled sequences were measured. Figure 3 shows Tm values for d(ACCGCA)/d(TGCGGT), d(GCCAGTTAA)/d(TTAAC-TGGC), r(GCCAGUUAA)/d(TTAACTGGC), r(AUUGGAU-ACAAA)/r(UUUGUAUCCAAU) and r(AUUGGAUACAAA)/-d(TTTGTATCCAAT) measured at several NaCl concentrations. All of these duplex stabilities decreased with decreasing NaCl concentration and indicated a linear dependency of their Tm values on the logarithm of the sodium ion concentration. A good correlation for Tm versus log[Na+] plots in the range of 1 M to 10 mM NaCl for each duplex was observed; however the slopes of these lines were different. Although d(GCCAGTT-AA)/d(TTAACTGGC) and r(GCCAGUUAA)/d(TTAACTGGC), and r(AUUGGAUACAAA)/r(UUUGUAUCCAAU) and r(AU-UGGAUACAAA)/d(TTTGTATCCAAT) have the same number of phosphate groups, effects of the sodium ion concentration on their Tm values were different. A more stable duplex was enhanced more by increments of sodium ion concentration; d Tm/d log[Na+] values for d(ACCGCA)/d(TGCGGT), r(GC-CAGUUAA)/d(TTAACTGGC), d(GCCAGTTAA)/d(TTAA-CTGGC), r(AUUGGAUACAAA)/d(TTTGTATCCAAT) and r(AUUGGAUACAAA)/r(UUUGUAUCCAAU) were 5.2, 7.5, 8.6, 10.7 and 11.5°C, respectively, which is the same order of their duplex stability (27.9, 37.6, 38.0, 45.4 and 57.3°C for Tm measured at 1 M NaCl, respectively). This result suggests that d Tm/d log[Na+] values are related to Tm values measured at 1 M NaCl, that is, the sodium ion concentration effect on a duplex stability is affected by the base composition as well as the number of phosphate groups in the duplex.
Figure 3. Tm values measured at the total nucleotide strand concentration of 8 µM in a phosphate buffer containing various NaCl concentrations. Oligonucleotides with sequence are as follows: d(ACCGCA)/d(TGCGGT) (closed circles) and d(GCCAGTTAA)/d(TTAACTGGC) (open circles) for a DNA duplex, r(AUUGGAUACAAA)/r(UUUGUAUCCAAU) (triangles) for an RNA duplex, and r(GCCAGUUAA)/d(TTAACTGGC) (open squares) and r(AUUGGAUACAAA)/d(TTTGTATCCAAT) (gray squares) for an RNA-DNA hybrid duplex.
For polynucleotides, R·Tm2/[Delta]H[prime]° (= [beta]) is constant independent of temperature, counter cation concentration, nucleotide sequence and chain length (28). Reported [beta] values for T4 DNA and poly(rA)·poly(rU) were 50 2 and 55°C, respectively (28). We estimated [beta] for oligonucleotide duplexes from data in Table 1. Because [beta] is presented per mole of nucleotides, we introduced the number of nucleotides (m) into the equation 7, proving the slope of the (d Tm/d log[Na+])·m/2.303 versus [Delta]n plots equal to [alpha]·[beta]. Though [Delta]n was roughly estimated from two Ka values measured in 1 M and 100 mM NaCl by the equation 6, the plots in Figure 4 fit a good straight line giving 58.0 1.6°C for the [beta] value at 8 µM of the total strand concentration when [alpha] values for oligonucleotides is considered 0.92 (17,28,29). Further-more, almost the same [beta] (58.3 1.6°C) was revealed for 100 µM of the total strand concentration (data not shown), indicating less influence of Ct on [beta]. The [beta] value determined for oligonucleotides is similar to that observed for polynucleotides. Thus, -[Delta]n values for d(ACCGCA)/d(TGCGGT), r(GCCAGUUAA)/d(TTAACTGGC), d(GCCAGTTAA)/d(TT-AACTGGC), r(AUUGGAUACAAA)/d(TTTGTATCCAAT) and r(AUUGGAUACAAA)/r(UUUGUAUCCAAU) can be calculated as 0.42 0.02, 0.98 0.03, 1.12 0.05, 1.91 0.14 and 2.05 0.05, respectively. These values are proportional to the duplex stabilities. Moreover, the linear plots in Figure 4 indicate the close relationship between n/m for sodium ion and the Tm dependency on the Na+ concentration, because d Tm/d log[Na+] is proportional to n/m when [alpha]·[beta] is constant suggested from equation 7. Therefore, d Tm/d log[Na+] values for these oligonucleotides (in the range of 11.5-5.2°C) less than those reported for polynucleotide duplexes, 17.5°C for poly(dA)·poly(dT), 19.6°C for poly(rA)·poly(rU) (10,28) might be the contribution of the n term.
Figure 4. Plot of (d Tm/d log[Na+])·m/2.303 versus -[Delta]n for DNA duplexes listed in Table 1. d Tm/d log[Na+] and [Delta]n values were estimated from data previously measured in 1 M NaCl (6) and those in 100 mM NaCl-phosphate buffer (Table 1) as described in the text. These points were fitted to a straight line through the (0,0) point with a least-squares calculation.
Effects of monovalent and divalent cations on nucleic acid duplex stability
Considering the above results, we thought it advisable to investigate cation effects on nucleotides using DNA duplexes. Seventeen kinds of cation chlorides, MCln (LiCl, NaCl, KCl, CsCl, MgCl2, CaCl2, BaCl2, MnCl2, CoCl2, NiCl2, CuCl2, ZnCl2, SnCl2, PbCl2, YCl3, LaCl3 and GdCl3) were examined for their ability to the stabilize the d(GCCAGTTAA)/d(TTAACTGGC) duplex. Cation chlorides of CoCl2, NiCl2, ZnCl2, YCl3, LaCl3 and GdCl3 did not yield a melting curve for the duplex, but obscured its absorbance band around 260 nm. In contrast, CuCl2, PbCl2 and SnCl2 precipitated under the experimental conditions used. Figure 5A shows melting curves of the duplex in 10 mM LiCl, NaCl, KCl, CsCl, MgCl2, CaCl2, MnCl2 and BaCl2-cacodylate buffer. As expected, monovalent cations enhanced Tm of the duplex but divalent cations stabilized the duplex more effectively. Linear correlations of the Tm value with the cation concentration are indicated in Figure 5B, although Tm values measured in 100 mM divalent cation chlorides deviated from the linear plots. Monovalent cations (Na+, K+ and Cs+) stabilized the duplex to the same extent over the salt concentration examined here. Li+ was slightly more effective on the duplex stabilization than Na+, K+ and Cs+ by ~1.5°C. This curious Li+ effect has also been reported for a polynucleotide duplex (33). One possible reason is due to a higher ionic character (Li+ ion has only s-orbitals) compared with the other monovalent cations (34). For alkaline earth metals, smaller atomic numbers seemed to correlate better with the duplex stabilization. MgCl2 was the most effective for duplex stabilization. When the same salt concentration was examined, Tm values of the duplex were in the following order; MgCl2 > CaCl2 > MnCl2 > BaCl2 >> LiCl > NaCl ~ KCl ~ CsCl. To achieve the same Tm values with divalent cation chlorides as that in 100 mM NaCl (29.9°C) requires only 0.7 mM for MgCl2, 1.6 mM for CaCl2, 2.8 mM for MnCl2 and 4.3 mM for BaCl2. Thus, from 140 to 20 times lower concentrations of these divalent cation chlorides can provide the same duplex stability as 100 mM NaCl. The stabilization effect of magnesium ion on the DNA duplex is about 140 times larger than that of sodium ion, which is similar to the ratio of association constants for a B-DNA duplex with hydrated Na+ (150 M-1) and with hydrated Mg2+ (12 800 M-1) determined by NMR (35). Moreover, sensitivity of Tm value to the cation concentration (d Tm/d log[Mn+]) was similar (8.8 ~ 8.9°C) for Na+, K+ and Cs+ monovalent cations, while those for divalent cations were slightly different, 5.6, 5.4, 5.3 and 5.2°C for Mg2+, Ca2+, Mn2+ and Ba2+, respectively. The difference of d Tm/d log[Mn+] between Na+ and Mg2+ was not expected owing to Tm2/[Delta]H° (= [beta] value), because their Tm2/[Delta]H° values are not so different according to the thermodynamic parameters in Table 2. Thus, the [Delta]n contributions are dominant for the difference of d Tm/d log[Mn+] between Na+ and Mg2+ as suggested from equation 7. Assuming [beta] values for divalent cation chlorides are the same as that for NaCl, [Delta]n values were estimated from equation 7 as well as those from equation 6 shown in Table 3. Li+, Na+, K+ and Cs+ showed the same -[Delta]n values within the experimental error (1.33 ~ 1.35), but divalent cations were remarkably different from each other (0.70 ~ 0.76), and the order is Mg2+ > Ca2+ > Mn2+ > Ba2+, which is the same as their ability to stabilize the duplex. Also, [Delta]n values for Na+ and Mg2+ determined by Tm versus log [Mn+] are similar to those obtained from log Ka versus log[Mn+] plots. More than half of [Delta]n anticipated for divalent cations and the different [Delta]n requirements among divalent cations disagree with their valence, meaning not only the electronegativity of nucleotides decides the number of these cations bound to the duplex. Thus, the affinity of cations with duplexes would affect the [Delta]n values and duplex stability.
Figure 5. (A) Normalized melting curves of d(GCCAGTTAA)/d(TTAACTGGC) in 10 mM cation chloride/10 mM sodium cacodylate buffer (pH 7.0). (B) Tm plots of d(GCCAGTTAA)d(TTAACTGGC) against a cation chloride concentration for LiCl (open squares), NaCl (open circles), KCl (open triangles), CsCl (open diamonds), MgCl2 (closed circles), CaCl2 (closed triangles), BaCl2 (closed squares) and MnCl2 (closed diamonds). All melting curves were measured at 8 µM total strand concentration.
Table 2. Thermodynamic parameters of d(GCCAGTTAA)/d(TTAACTGGC) formation in NaCl and/or MgCl2 containing buffera
aAll experiments were conducted in buffer containing NaCl/10 mM Na2HPO4/1 mM Na2EDTA or MgCl2/10 mM sodium cacodylate (pH 7.0).
bMelting temperatures were calculated at the total oligomer strand concentration of 8 µM.
Table 3. The number of released cations when a duplex forms determined by two methodsa
| Cation (Mn+)b | -[Delta]n |
| Tm versus log[Mn+] plots | |
| Li+ | 1.35 0.01 |
| Na+ | 1.33 0.02 |
| K+ | 1.33 0.01 |
| Cs+ | 1.35 0.04 |
| Mg2+ | 0.76 0.02 |
| Ca2+ | 0.74 0.01 |
| Ba2+ | 0.70 0.02 |
| Mn2+ | 0.72 0.02 |
| log Ka versus log[Mn+] plots | |
| Na+ | 1.32 0.18 |
| Mg2+ | 0.78 0.05 |
b[alpha] and [beta] values for monovalent cations are used 0.92 and 58.1, respectively. Those for divalent cations are 0.88 and 58.1, respectively.
CD spectra of d(GCCAGTTAA)/d(TTAACTGGC) in NaCl and in MgCl2 show lesser cation effects on the global conformation of its duplex (data not shown) but did influence the duplex stability as shown in Table 2. A buffer including both NaCl and MgCl2, since it is often used in order to stabilize the secondary structure of nucleotides (36) and to allow ribozyme activity (37-40), was also employed. These cations are considered to have different binding sites on nucleotides; Na+ binds more with the phosphate group and Mg2+ prefers the N7 of purine bases (41). Figure 6 indicates Tm values for d(GCCAGTTAA)/d(TT-AACTGGC) measured in various MgCl2 concentrations in the presence or absence of 100 mM NaCl. From the view point of an ionic strength, MgCl2 with 100 mM NaCl is more effective for the duplex stabilization than MgCl2 alone. However, Tm values measured in >1 mM MgCl2 concentration with 100 mM NaCl were similar to those in MgCl2 alone, and Tm values in <1 mM MgCl2 concentration with 100 mM NaCl were similar to these in 100 mM NaCl alone (29.9°C). This was also observed from the thermodynamic values in Table 2. Williams et al. (15) reported Tm values measured in several Mg2+ concentrations in the presence of Na+ and showed similar Tm dependencies on Mg2+ concentration as shown in Figure 6. Thus, establishment of the prediction system for duplex stability under MgCl2 with NaCl might be easily possible. Furthermore, these results suggest that Na+ and Mg2+ compete with each other in their effects on the duplex stability, and the binding sites for Na+ and Mg2+ to the duplex are the same, possibly with nucleobases as well as phosphate groups. Accordingly, we propose that the stabilization mechanisms for a nucleic acid duplex by these monovalent and divalent cations are the same, but they differ in their efficiencies.
Figure 6. Tm values of 8 µM d(GCCAGTTAA)/d(TTAACTGGC) as a function of the magnesium ion concentration in 100 mM NaCl/10 mM sodium cacodylate buffer (triangles) or in 10 mM sodium cacodylate buffer (circles). The dotted line at 29.9°C indicates Tm values obtained in 100 mM NaCl/10 mM sodium cacodylate buffer.
Insights of cation binding to nucleic acid duplexes
Both stacking interactions and hydrogen bonds are widely understood to stabilize nucleic acid duplex structure (41,42). Here, we have revealed the importance of [Delta]n values on the duplex stability. The importance of cation binding for duplex structure are also reported. For instance, magnesium and potassium ions are needed for the folding of the Tetrahymena group I intron P4-P6 domain (37,38). Also, for d(CGCGAATTCGCG)2, Na+ and K+ ions partially occupy the primary water spine located at the minor groove of the duplex, and the bent structure at the AT-GC junction is due to the bound sodium ion bridging both nucleotide strands (43,44). Considering these facts, it is probable that cations bound to a double helix passively affect nucleic acid duplex stability (39,45).
It has been considered that cations, especially alkali metal ions, bind only to phosphate groups of nucleic acids (41). However, recent studies concerning cations and nucleotides arising from ab initio, semi-empirical and empirical results reveal other possibilities. Computational ab initio calculations often indicate N7 and O6 of guanine as the preferential cation binding sites. N7 of adenine is also a probable binding site (33,46). Longtime computer simulations to a nanosecond with AMBER 4.1 revealed the number of hits of sodium ions with A-RNA and B-DNA most frequent for GpG step at N7 and O6 of guanine bases (47). Moreover, hydrated magnesium ions are found in the major groove located between G/C base pairs of d(CGCGAATTCGCG)2 by high resolution structural analysis (43). These results support preferential cation binding sites of N7 and O6 of guanine bases as well as phosphate groups, which are components of one of the most stable nearest-neighbor base pairs of CG/CG (6-9,24). Here, we have demonstrated that the number of Na+ required for a duplex formation was related to the duplex stability. The duplex stability is accounted for by nearest-neighbor interactions (4-9). Accordingly, it is possible that newly bound Na+ when a duplex forms binds with nucleobases as well as phosphate groups, though its affinity toward nucleobases, perhaps N7 and O6 of a guanine base, is much lower than that of Mg2+. It is probable that naked or hydrated cations required for duplex formation are preferentially coordinated by both phosphate groups and nucleobases. The influence of nucleobases on the salt effect suggests that there are preferential base pairs or nearest-neighbor base pairs for the counter cation binding. Such sequence-dependent bindings might affect all nearest-neighbor parameters. As mentioned, it was impossible to distinguish among duplex species on the basis of linear plots of the thermodynamic values for duplex formation. This finding means that 2[prime]-OH of RNA strand, sugar puckering and the methyl group of thymine may affect sequence-dependent cation bindings. Theoretical studies of cation distribution around a DNA duplex were performed based on counterion condensation (CC), Monte Carlo (MC) or Poisson-Boltzmann (PB) methods (46). These calculations take into account electronegative points located only at the phosphates. However, if cations interact with nucleobases as well as the phosphates, it will be necessary to reconsider this model by including the possible cation binding sites in both grooves of the duplex surface. Such a consideration might reduce the end-effects on cation distributions of a duplex (48).
Johnson et al. (49) reported that differences in the stacking energies under high ionic conditions are conserved even at a lower salt condition. This concept means the base pair geometry is not significantly affected by the salt condition. Our own report would be consistent with the results obtained here in that duplex stabilities in 100 mM NaCl are linearly correlated with those in 1 M NaCl. It is said that cation binding to nucleobases influences the strength of hydrogen bonding between base pairs as determined by ab initio calculations (46,50), thus the salt effect on the strength of hydrogen bonds needs to be examined experimentally. Furthermore, the relationship between [Delta]G°37 and [Delta]n would provide novel equations for the prediction of a duplex stability that would apply over a large range of salt concentrations. We are presently determining the number of the cations bound to nucleic acid duplexes to reveal quantitatively their effects on nucleic acid duplex stabilities (51,52), and have found the [Delta]G°37 salt dependence is not explained by simple counterion condensation theories, which explain the salt effect as an oligomer length dependence.
ACKNOWLEDGEMENTS
We thank Dr R. I. Gumport for his comments on the manuscript. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture, Japan, and Grants from `Research for the Future' Program of the Japan Society for the Promotion of Science.
REFERENCES
*To whom correspondence should be addressed at: Department of Chemistry, Faculty of Science, Konan University, 8-9-1 Okamoto, Higashinada-ku, Kobe 658-8501, Japan. Tel: +81 78 435 2497; Fax: +81 78 435 2539; Email: sugimoto{at}konan-u.ac.jp
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