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. 2016 Oct 4;111(7):1350–1360. doi: 10.1016/j.bpj.2016.08.031

Thermal Stability of RNA Structures with Bulky Cations in Mixed Aqueous Solutions

Shu-ichi Nakano 1,∗, Yuichi Tanino 1, Hidenobu Hirayama 1, Naoki Sugimoto 1,2
PMCID: PMC5052467  PMID: 27705759

Abstract

Bulky cations are used to develop nucleic-acid-based technologies for medical and technological applications in which nucleic acids function under nonaqueous conditions. In this study, the thermal stability of RNA structures was measured in the presence of various bulky cations in aqueous mixtures with organic solvents or polymer additives. The stability of oligonucleotide, transfer RNA, and polynucleotide structures was decreased in the presence of salts of tetrabutylammonium and tetrapentylammonium ions, and the stability and salt concentration dependences were dependent on cation sizes. The degree to which stability was dependent on salt concentration was correlated with reciprocals of the dielectric constants of mixed solutions, regardless of interactions between the cosolutes and RNA. Our results show that organic solvents affect the strength of electrostatic interactions between RNA and cations. Analysis of ion binding to RNA indicated greater enhancement of cation binding to RNA single strands than to duplexes in media with low dielectric constants. Furthermore, background bulky ions changed the dependence of RNA duplex stability on the concentration of metal ion salts. These unique properties of large tetraalkylammonium ions are useful for controlling the stability of RNA structures and its sensitivity to metal ion salts.

Introduction

Mixtures of water and organic solvents have several applications in studies of nucleic acids, including expanding the use of nucleic acids in apolar media, improving hydrophobic ligand solubility, modifying the functions of nucleic acids, and constructing DNA scaffolds for organic synthesis (1). Aqueous solutions containing high concentrations of organic polymer compounds, such as poly(ethylene glycol) (PEG) and dextran, can also be used to evaluate the effects of molecular crowding inside living cells that contain high concentrations of macromolecules, small molecule metabolites, and osmolytes (2, 3). These solution conditions affect the thermal stability of DNA and RNA structures (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). Some compounds added to a solution specifically interact with nucleotide bases and sugar-phosphate backbones, and in particular, urea, formamide (FA), dimethylsulfoxide (DMSO), and diethylsulfoxide greatly decrease the stability of DNA basepairs (15, 16, 17, 18). In addition, N,N,N-trimethylglycine (glycine betaine (GB)) and trimethylamine N-oxide (TMAO), which have methylammonium groups with a net zero charge and accumulate at high concentrations in osmotically stressed cells, affect to some extent the secondary and tertiary structures of DNA and RNA (16, 19, 20).

Negative charges of nucleotide phosphates are screened according to electrostatic interactions with cations. Small metal ions, such as Na+ and Mg2+, associate with single strands and even more with basepaired structures because of their greater electrostatic fields. These associations are capable of facilitating the formation of electrostatically condensed structures and exhibiting the catalytic activity of ribozymes. In contrast, large metal ions cannot approach the RNA surface so closely (21), and the property of cation accumulation in RNA pockets with negative electrostatic potentials varies with the ionic radius or the charge density (22). In particular, the ionic radius of metal ions determines the folding equilibrium of the Tetrahymena ribozyme and the reaction pathway of the hepatitis delta virus ribozyme (23, 24). Alkylammonium ions are also reported to have distinct DNA-binding preferences depending on the ion size (25). Because amphiphilic alkylammonium ions are effective for screening the charge of nucleotide phosphates in apolar media (26, 27, 28, 29), applications of nucleic acids to nonaqueous conditions would be improved by understanding the thermodynamic properties of interactions with bulky cations. However, it remains unclear how the molecular environment influences ion binding to nucleic acids. In this study, we investigated the thermal stability of short and long RNA structures in the presence of bulky cations in aqueous mixtures with organic additives, as shown in Fig. 1. We show that the stability and salt concentration dependence of RNA are dependent on the cation sizes and dielectric constants of mixed solutions. Moreover, bulky cations have the ability to change the effects of metal ion salts on RNA. The results elucidate the ion-binding properties of RNA in the presence of organic additives and will be useful for controlling the stability of nucleic acid structures using bulky cations.

Figure 1.

Figure 1

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(A) Chemical structures and abbreviations of the bulky cations examined in this study. (B) Organic additives used to prepare mixed solutions. The PEGs (PEG200, PEG600, PEG2000, and PEG8000) have average molecular weights of 2 × 102 to 8 × 103. EG is the monomer unit of PEG. Glyc, PDO, MME, and DME are structurally related to EG. MeOH, EtOH, and PrOH are typical primary alcohols. Urea and FA are amide compounds. DMF and AcAm are structurally related to FA. AcCN, DMSO, THF, and DOX are aprotic solvent molecules.

Materials and Methods

Preparations of RNA and buffer solutions

Short RNA oligonucleotides of a high-performance liquid chromatography (HPLC) purification grade were purchased from Hokkaido System Science (Hokkaido, Japan). Poly(A)/poly(U) (homopolymer duplex of adenylic and uridylic acids), poly(I)/poly(C) (homopolymer duplex of inosinic and cytidylic acids), and phenylalanine transfer RNA (tRNA) were purchased from Sigma-Aldrich (St. Louis, MO). To eliminate contaminating salts and small molecules from these RNA samples, RNA solutions were passed through a size-exclusion spin column with a molecular weight cutoff ultrafiltration membrane (Microcon YM-3; Merck Millipore, Darmstadt, Germany). This procedure was repeated several times before the solutions were used. Peptide nucleic acid (PNA) molecules with N-(2-aminoethyl)glycine backbones were synthesized using the Fmoc strategy and were purified using HPLC as described previously (11).

The reagents 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and disodium salt of ethylenediamine-N,N,N′,N′-tetraacetic acid (Na2EDTA) were purchased from Dojindo (Kumamoto, Japan). The chloride salt of the tetrahexylammonium (THA) ion and PEG with an average molecular weight of 8 × 103 (PEG8000) were purchased from Sigma-Aldrich, and 2-methoxyethanol (MME) and 1,2-dimethoxyethane (DME) were purchased from TCI (Tokyo, Japan). All other reagents used to prepare buffer solutions were purchased from Wako (Osaka, Japan). Mixed aqueous solutions of 20 wt % were prepared unless otherwise stated.

Measurements of the thermal stability of RNA and PNA structures

The thermal melting curves of the RNA and PNA structures were measured by monitoring absorption at 260 nm using a spectrophotometer (UV1800; Shimadzu, Kyoto, Japan) equipped with a temperature controller. Typically, melting-curve data were obtained using oligonucleotide duplexes and a PNA duplex at a total strand concentration of 2 μM and a heating rate of 1°C min–1, whereas those for poly(A)/poly(U) and poly(I)/poly(C) were examined at a nucleotide concentration of 40 μM and a heating rate of 0.2°C min–1, and tRNA was examined at a strand concentration of 1 μM and a heating rate of 0.5°C min–1. The measurements were performed in a buffer containing 10 mM Na2HPO4 (pH 7.0) and 0.1 mM Na2EDTA using a cuvette sealed with an adhesive sheet to prevent evaporation. In experiments using MgCl2, a buffer solution comprised of 50 mM HEPES (pH 7.0) was used. The melting temperatures (Tm) of polynucleotides and tRNA were determined from the first derivative of a thermal melting curve. Thermodynamic parameters, such as the enthalpy change (ΔH°), entropy change (ΔS°), and free-energy change (ΔG°) for the formation of oligonucleotide duplexes at 37°C (see Tables 1, 2, and 3) were determined from a nonlinear fit of the melting curve to a theoretical curve, and from Tm–1 versus log (Ct/4) plots using Tm values with various total strand concentrations (Ct) (30, 31). The parameters determined from the plot of Tm–1 versus log (Ct/4) were similar to those determined by fitting of the melting curves, indicating the validity of the assumptions of a two-state transition and a temperature-independent change in heat capacity upon duplex formation.

Table 1.

Thermodynamic Parameters for RNA Duplex Formation in the Presence of 100 mM Salt

Salt Ion –ΔH° (kcal mol–1) –ΔS° (cal mol–1 K–1) –ΔG° (kcal mol–1)
5′-CAACGCAAG-3′/5′-CUUGCGUUG-3′
NH4+ 88.9 ± 4.1 252.4 ± 13.0 10.7 ± 0.3
TEA 85.7 ± 1.0 251.7 ± 3.4 7.60 ± 0.13
TBA 82.5 ± 2.2 245.1 ± 7.0 6.47 ± 0.16
TBA(w/PEG)a 86.4 ± 4.0 257.5 ± 13.0 6.45 ± 0.34
5′-CGGCGCGGG-3′/5′-CCCGCGCCG-3′
NH4+ 96.9 ± 0.6 247.9 ± 1.5 19.4 ± 0.4
TEA 89.2 ± 2.6 242.0 ± 8.3 14.1 ± 0.2
TBA 92.5 ± 1.0 255.8 ± 3.0 13.1 ± 0.1
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a

Data were obtained in the presence of 20 wt % PEG8000.

Table 2.

Values of –ΔΔG° and Linear Slopes of Log K versus Log [MCl] Plots

Salt Ion –ΔΔG° (kcal mol−1)a Slope
5′-CAACGCAAG-3′/5′-CUUGCGUUG-3′
Na+ 2.0 1.66 ± 0.05
NH4+ 2.1 1.46 ± 0.08
Choline 0.3 0.53 ± 0.08
GB –0.4 –0.22 ± 0.04
TMAO –0.1 –0.18 ± 0.03
TMA 0.3 0.56 ± 0.08
TEA –1.0 –0.30 ± 0.03
TBA –2.1 –1.00 ± 0.10
TPeA –2.2 –1.11 ± 0.11
5′-CGGCGCGGG-3′/5′-CCCGCGCCG-3′
Na+ 2.0 1.92 ± 0.12
NH4+ 2.3 2.01 ± 0.08
Choline 0.1 –0.05 ± 0.08
GB –0.7 –0.98 ± 0.06
TMAO –0.1 0.10 ± 0.06
TMA –0.5 –0.07 ± 0.06
TEA –3.0 –1.56 ± 0.07
TBA –4.0 –2.20 ± 0.11
TPeA –4.9 –2.60 ± 0.11
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a

Values were calculated by subtracting the –ΔG° value obtained in the presence of 100 mM salt from that obtained without salt (8.6 kcal mol–1 for the 9-mer duplex and 17.1 kcal mol–1 for the GC duplex).

Table 3.

Thermodynamic Parameters for 9-mer Duplex Formation with 100 mM NaCl in the Absence and Presence of Background TBACl

[TBACl] (mM) –ΔH° (kcal mol–1) –ΔS° (cal mol–1 K–1) –ΔG° (kcal mol–1)
0 89.2 ± 3.1 253.5 ± 9.9 10.6 ± 0.2
100 96.1 ± 3.0 278.6 ± 9.8 9.66 ± 0.21
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Determination of the solution property values

The relative dielectric constants of the solutions were calculated according to the equation reported by Oster (32) or experimentally determined using the fluorescent probe 1-anilino-8-naphthalene sulfonate as described previously (33). The water activities of the solutions were determined using osmotic pressure methods with vapor phase osmometry (5520XR pressure osmometer; Wescor, Logan, UT) or freezing-point depression osmometry (Typ Dig.L osmometer; Knauer, Berlin, Germany). The solution viscosities were measured using a viscometer (SV-10 vibro viscometer; A&D, Tokyo, Japan). These parameters were measured using buffer solutions containing 1 M NaCl.

Circular dichroism spectra of RNA

Circular dichroism (CD) spectra were obtained using a spectropolarimeter (J-820; JASCO, Tokyo, Japan) equipped with a temperature controller. All spectra were measured at 5°C in phosphate buffer containing RNA at 20 μM, with the exception of 5′-CCCGCGCCG-3′, which was used at 40 μM because of its low signal level. All RNA solutions were heated to 80°C and cooled at the rate of 2°C min−1 before use.

Results

Influence of tetraalkylammonium ions on the thermal stability of oligonucleotide duplexes

Because a fully matched duplex minimizes structural ambiguity, we prepared a short, basepaired RNA duplex, 5′-CAACGCAAG-3′/5′-CUUGCGUUG-3′, which is referred to as a 9-mer duplex in this study. Initially, the effect of cation size on the thermal stability of the duplex was studied using chloride salts of NH4+, TEA, and TBA ions. The dependences of the duplex stability on the salt concentrations were analyzed to evaluate RNA-ion interactions. Fig. 2 A shows the melting temperatures (Tm) in the presence of varying concentrations of NH4Cl, TEACl, or TBACl. The duplex stability increased with increasing NH4+ concentrations from 20 mM to 1 M, reflecting effective electrostatic screening of the charge of the duplex backbone. In contrast, Tm was decreased by 4.4°C with an almost linear relationship with the logarithm of TBACl concentrations in the 20–200 mM range, and the stability was more markedly decreased at concentrations higher than 200 mM. The TEA ion, which is smaller than the TBA ion (0.345 and 0.417 nm, respectively, as a hydrated radius (34)) decreased the Tm by at most 3°C at high concentrations. This ion-size-dependent stability is consistent with a previously proposed model in which large tetraalkylammonium ions preferentially bind to single strands rather than to duplexes, resulting in an equilibrium shift toward single-strand conformations (25).

Figure 2.

Figure 2

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(A) Tm values for the 9-mer duplex at 2 μM in solutions containing various concentrations of NH4Cl (circles), TEACl (triangles), or TBACl (squares), represented by [MCl]. (B) Tm values for the PNA duplex at 2 μM in solutions containing NH4Cl (circles), TEACl (triangles), or TBACl (squares). (C) CD spectra of the 9-mer duplex (left) and its single strands 5′-CAACGCAAG-3′ (middle) and 5′-CUUGCGUUG-3′ (right) in the absence (black) and presence of TBACl at 100 mM (red), 200 mM (green), 500 mM (blue), 700 mM (purple), or 1 M (orange). To see this figure in color, go online.

To evaluate the significance of the phosphate charge on the effects of alkylammonium ions on RNA, we prepared a PNA with a noncharged backbone. PNA forms a stable double-helical structure through basepairing (35), and the duplex stability is less affected by the presence of metal ions and organic solvents (11, 36, 37, 38). We measured a PNA duplex with the same sequence as the 9-mer duplex except that U was replaced with T. In the experiments, the Tm was not decreased but was slightly increased at high TBACl concentrations (Fig. 2 B). This result suggests that hydrophobic interactions between TBA ions and nucleotide bases are not attributed to destabilization of the RNA duplex.

We studied the effect of TBA ions on RNA helical structures by using CD measurements. It should be noted that it was not possible to obtain the CD spectra of PNA strands because PNA has an achiral backbone. The CD spectra of the 9-mer RNA duplex did not change upon the addition of TBACl; however, the spectra of its single strands, 5′-CAACGCAAG-3′ and 5′-CUUGCGUUG-3′, changed with increasing TBACl concentrations (Fig. 2 C). These results are consistent with TBA ion binding to single strands rather than to duplexes. The binding preference could emerge as the result of a higher flexibility of RNA single strands that allows a close contact of the large ion with the RNA surface through nonspecific electrostatic interactions. We also investigated the effects on the duplex 5′-CGGCGCGGG-3′/5′-CCCGCGCCG-3′, which is comprised only of G•C basepairs and is referred to as a GC duplex. This duplex also showed ion-size-dependent stabilities and changes in the CD spectra of single strands at high TBACl concentrations (Figs. S1 and S2 in the Supporting Material). Analysis of the thermodynamic parameters for the duplex formations revealed that duplex destabilizations in the presence of the alkylammonium ions resulted from less favorable enthalpy changes (Table 1).

Influence of other bulky ions

Further experiments were performed using various monovalent cations and net neutral zwitterions (choline, GB, TMAO, TMA, TPeA, and THA ions; Fig. 1 A). Choline, GB, and TMAO have methylated amino groups, and TMA, TPeA, and THA ions are tetraalkylammonium ions of different sizes. The free-energy changes (ΔG°) for the duplex formations increased or decreased upon the addition of 100 mM salts (Table 2). For the 9-mer duplex, choline and TMA ions increased the –ΔG° value by 0.3 kcal mol–1, but the increments were much smaller than those observed with Na+ and NH4+. GB and TMAO ions slightly decreased the value by 0.4 and 0.1 kcal mol–1, respectively. TEA ions decreased –ΔG° by 1.0 kcal mol–1, and TBA and TPeA ions decreased it by 2.1–2.2 kcal mol–1. Data were not obtainable for the THA ion because it led to phase separation at high concentrations and temperatures. Similar effects of the bulky ions were also observed for the GC duplex, although the degrees of destabilization by large ions were greater (Table 2). In agreement with a previous study on DNA structures (25), the stabilities of the RNA duplexes were negatively correlated with the hydrated radii of NH4+ and tetraalkylammonium ions (TMA, TEA, TBA, and TPeA), as indicated by the solid symbols in Fig. 3 A.

Figure 3.

Figure 3

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(A) Values of –ΔG° for the 9-mer duplex (circles) and GC duplex (triangles) at a salt concentration of 100 mM plotted against the hydrated radii of NH4+ and tetraalkylammonium ions. Data for the 9-mer duplex in the presence of 20 wt % PEG8000 are indicated by open symbols. (B) Slope values of log K versus log [MCl] plots for the 9-mer duplex (circles) and GC duplex (triangles) plotted against the hydrated radii of NH4+ and tetraalkylammonium ions in the absence (solid symbols) and presence (open symbols) of 20 wt % PEG8000.

The duplex stabilities further decreased with increasing concentrations of TBACl and TPeACl. The dependences of log K (= –ΔG°/2.303RT, where R is the gas constant and T is the absolute temperature (310 K)) on the logarithm of salt concentrations (log [MCl]) were almost linear between 20 mM and 100 or 200 mM. However, the stability at higher salt concentrations decreased more than expected from linear relationships (Fig. S2) where the CD spectra of the single strands changed. Table 2 includes values of the slope of log K versus log [MCl] plots in the linear range, which are often used to analyze RNA-ion interactions, as described in the Discussion. The slope values were negatively correlated with the sizes of ions (solid symbols in Fig. 3 B), indicating that ion size is an important determinant of the salt concentration dependence.

Salt concentration dependence in mixed solutions

The stability of nucleic acid structures changes in aqueous solutions mixed with large quantities of neutral molecules, which act as either solutes or solvents (3) and are referred to as cosolutes in this study. The open symbols in Fig. 3, A and B, show the data obtained in the presence of 20 wt % PEG8000. This mixed solution did not significantly affect the duplex stability (thermodynamic parameters in the presence of 100 mM TBACl are given in Table 1), but it did change the salt concentration dependence. In further investigations of the effects of cosolutes, 20 different kinds of mixed solutions, including different molecular weight PEGs, ethylene glycol, and related compounds, primary alcohols, amide compounds, and aprotic solvent molecules (Fig. 1 B), were tested. Fig. 4 A shows the dependence of log K on log [NaCl] for several typical mixtures containing PEG8000, PEG200, DME, EtOH, FA, DMF, DOX, or DMSO, with different dielectric constant, water activity, and viscosity values. Linear plots with positive slopes were obtained for all mixed solutions, except for the high-salt-concentration data for EtOH (due to deviation from a linear fit) and DMF (due to evaporation at a high temperature). We previously reported that the degrees of NaCl concentration dependence for another RNA sequence were correlated with reciprocals of the relative dielectric constants (εr−1) of mixed solutions (39). In agreement with this, the slopes of Fig. 4 A decreased with increasing εr−1 values of solutions (Fig. 4 B; correlation plots for 20 kinds of mixed solutions are presented in Fig. S3). On the other hand, the degrees of NaCl concentration dependence were not strongly correlated with water activity and viscosity values. In addition, no strong correlation was observed between the –ΔG° and εr−1 values (Fig. S3), possibly due to specific interactions with some cosolutes (e.g., FA and DMF) and the water activity effect, as well as the dielectric constant effect, as discussed in our previous study (39).

Figure 4.

Figure 4

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(A) Dependence of log K of the 9-mer duplex on the NaCl concentration in mixed solutions containing PEG8000 (blue), PEG200 (purple), DME (brown), EtOH (orange), FA (red), DMF (green), DMSO (cyan), or DOX (gray) at 20 wt %. Black symbols indicate the absence of cosolutes. Dotted lines were drawn using linear regression analyses with correlation coefficients (r2) of >0.994. (B) Correlations between slopes of the plots in (A) and εr−1 values. (C) Dependence of log K of the 9-mer duplex on the TBACl concentration in mixed solutions. Symbols are the same as those used in (A). Dotted lines were drawn using linear regression analyses with r2 values of >0.970. (D) Correlations between slopes of the plots in (C) and εr−1 values. To see this figure in color, go online.

The slopes of log K versus log [TBACl] plots in a linear range were negative, and many of the mixed solutions decreased the slope to more negative values. In particular, the presence of PEG8000 or DME decreased the slope from –1.0 to about –1.9, whereas FA did not change the slope (Fig. 4 C). As in the experiments with NaCl, the slope values of log K versus log [TBACl] plots were correlated with the εr−1 values of the solutions (Fig. 4 D), but were not strongly correlated with water activity and viscosity values (Fig. S4).

Investigations using polynucleotides

In investigations of a synthetic poly(A)/poly(U) polynucleotide duplex, thermal denaturation caused A•U basepairs to separate in a highly cooperative manner, leading to the generation of bubble structures. As observed for oligomer duplexes, poly(A)/poly(U) showed ion-size-dependent Tm values, and the duplex had relatively low Tm values at high TBACl concentrations (Fig. 5 A). For example, the Tm values in the presence of 100 mM TBACl were 27.3°C for poly(A)/poly(U) and 26.8°C for the 9-mer duplex, although the Tm of the oligonucleotide duplex changed depending on the RNA concentration.

Figure 5.

Figure 5

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(A) First derivatives of the melting curves of poly(A)/poly(U) with NH4Cl (black) or TBACl (red). Arrows indicate increasing salt concentrations from 20 mM to 1 M. Data obtained in the absence of additional salts are shown in blue. (B) Correlations between the dependence of Tm of poly(A)/poly(U) on NaCl (circles) or TBACl (squares) concentrations and εr−1 values of mixed solutions. Colored symbols are the same as those used in Fig. 4. To see this figure in color, go online.

With the exception of the solution containing PEG8000, which caused precipitation, the mixed solutions increased or decreased the Tm of poly(A)/poly(U) (Table S1). Most mixed solutions decreased the slope of Tm versus log [NaCl] plots, whereas solutions containing FA increased the slope. Experiments with TBACl also showed that the solutions, apart from those containing FA, negatively increased the slope. Strikingly, the slope values for both NaCl and TBACl showed strong correlations with the εr−1 values of the solutions (Fig. 5 B).

Metal ion concentration dependence in the presence of background bulky ions

When RNA-binding preferences differ between small and large ions, competition for ion binding to RNA is expected to be limited. In experiments with a background of TEACl and TBACl at 100 mM, the dependence of Tm of poly(A)/poly(U) on NaCl concentrations was increased (Fig. 6 A). As demonstrated in Fig. 6 B, the presence of TEACl or TBACl also increased the NaCl concentration dependence of the stability of a synthetic poly(I)/poly(C) polynucleotide duplex and the stability of tRNA forming the L-shaped tertiary structure that is converted to a random coil at elevated temperatures (40).

Figure 6.

Figure 6

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(A) Dependence of Tm of poly(A)/poly(U) at 40 μM on the NaCl concentration in the absence (black) and presence of background TEACl (blue) or TBACl (red) at 100 mM. (B) Slope values of Tm versus log [NaCl] plots for poly(A)/poly(U), poly(I)/poly(C), and tRNA in the absence (black) and presence of background TEACl (blue) or TBACl (red) at 100 mM. (C) Dependence of log K of the 9-mer duplex on the NaCl concentration in the absence (black) and presence of NH4Cl (gray) or TBACl (red) at 100 mM. (D) Slope values of log K versus log [NaCl] plots for the 9-mer duplex with a background of 100 mM bulky ions. Data were obtained in the absence and presence of 20 wt % PEG8000, indicated by black and gray bars, respectively. Data for the slope values of log K versus log [MgCl2] plots showing a linear range from 0.2 to 20 mM are also presented. To see this figure in color, go online.

We further investigated the influence of background bulky ions on NaCl concentration dependence using the 9-mer duplex. In the presence of 100 mM NH4Cl, the duplex stability was not changed with increasing NaCl concentrations from 20 to 200 mM. In contrast, in the presence of 100 mM TBACl, a linear relationship was observed between the log K and log [NaCl] values in the range of 20 mM to 1 M (Fig. 6 C). Salts of GB, TMAO, and other tetraalkylammonium ions also yielded linear relationships with NaCl concentrations in the same range. Fig. 6 D shows slope values obtained with different background salts at 100 mM. Compared with the slope in the absence of background bulky ions, GB, TMAO, and TMACl decreased the slope, whereas TBACl and TPeACl increased it. Analysis of the thermodynamic parameters revealed an increased entropic penalty by the presence of background TBACl (Table 3). In further experiments, mixed solutions containing PEG8000 were examined for the 9-mer duplex, but they were not feasible for long RNAs due to the production of precipitants. Although PEG8000 did not alter the effects of TBACl and TPeACl on the NaCl concentration dependence, it did reduce the effects of smaller bulky ions (gray bars in Fig. 6 D). Similar effects of background TBACl were also observed for the dependence on MgCl2 concentrations (Fig. 6 D).

Discussion

RNA-binding preferences of bulky ions

The stability of RNA structures is affected by the binding strength and number of cations that are bound to folded and unfolded conformations. Because a folded conformation has a greater electrostatic field than an unfolded conformation, electrostatically bound cations stabilize more condensed structures. However, this is not the case when steric effects affect ion binding to RNA. In this study, we examined fully matched oligonucleotide duplexes, where specific ion-base interactions are not significant, and atmospherically bound ions play a central role in structural stability through electrostatic interactions. Our experimental results showed that NH4Cl stabilized RNA duplexes as effectively as NaCl (Table 2), indicating efficient binding of NH4+ to the duplex. In contrast, choline, GB, TMAO, and TMA ions only slightly changed the stabilities. These observations indicate that the overall charge of these bulky cations with trimethylammonium groups is unable to participate in the screening of the charge of basepaired nucleotides, as calculated based on the Poisson-Boltzmann electrostatic model (41). Furthermore, GB substantially decreased the stability of the GC duplex, consistent with a previous study in which more interactions were observed with G and C surfaces than with A and T surfaces in the single-stranded form (16). The influence of TMAO was also in accord with the small destabilizing effects on RNA secondary structures reported in earlier studies (19, 20). It was previously proposed that TMA and TEA ions fit into the double-helical groove of DNA polynucleotides and increase the duplex stability at high salt concentrations (42, 43). In contrast, the data for RNA listed in Table 1 demonstrate duplex destabilizations by TEACl at 100 mM. Hence, TEA ion binding to the helical groove could be less pronounced for RNA, which is A-form, under the condition of moderate salt concentrations.

Tetraalkylammonium ions with longer alkyl chains significantly destabilized RNA structures. The TBA ion is too large to be accommodated in double-helical grooves, but has been reported to bind to A and T bases of DNA polynucleotides through hydrophobic interactions, and to have limited binding to G and C bases (44). We observed TBA ion-induced perturbations of single-strand conformations of the 9-mer duplex and the GC duplex with concentrations of >200 mM (Fig. 2 C) and >700 mM (Fig. S1), respectively. It appears that the binding preference of TBA ions to A and U bases emerges at higher concentrations and the structural perturbations are related to nonlinear destabilizations observed at high TBACl concentrations (Fig. S2). It is noted that salt conditions of <200 mM used for the slope analyses did not cause the perturbations. In contrast, the PNA duplex was not destabilized even at high TBACl concentrations, despite the presence of the base moiety. It is likely that nonelectrostatic contributions, such as hydrophobic interactions between alkyl groups and nucleotide bases, do not play a crucial role in destabilizing RNA duplexes, and that electrostatic binding to single strands primarily caused the destabilization. Because of the delocalized electrostatic effects of the ion bindings, one may be able to predict the stability of an RNA duplex in the presence of tetraalkylammonium ions at moderate concentrations by considering the cation size and the concentration dependence; however, further studies using other sequences are needed to resolve this issue.

Analyses of ion binding to RNA

Induction of RNA folding by ion binding can be described using the two-state transition model R1 ⇄ R2, where R1 and R2 are less and more condensed states, respectively. In this model, the equilibrium constant for the forward reaction (K) changes depending on the salt concentration ([MCl]). The salt concentration-dependence data for K allow a numerical analysis of ion binding to RNA. The linear relationships between log K and log [MCl] are used to evaluate the degree of net binding or the release of ions (Δn) during the forward reaction. The analysis is expressed by the equation Δln K/Δln [MCl] = αΔn under the assumption of fixed numbers of bound ions, which is almost valid over a wide range of Na+ concentrations (21). The factor α compensates for the nonideality of salt solutions and is close to 0.9 in experiments using NaCl (45). The log K versus log [NaCl] plots shown in Fig. 4 A have positive linear relationships, indicating net Na+ binding during the duplex formation. The number of Na+ ions bound per phosphate of the 9-mer duplex was 0.11 (assuming α = 0.9) and was within the range of values reported for other DNA and RNA sequences (45, 46, 47). However, the analysis of the slope of log K versus log [NaCl] plots assumes ion binding to defined RNA sites according to the stoichiometric mass action law, which is inaccurate. Indeed, the interaction coefficient, 2ΔГMCl (= ΔГM + ΔГCl = Δln K/Δln aMCl), is often applied to the association and dissociation of ions that may not bind stoichiometrically to defined sites. This analysis uses salt activity (aMCl) and considers the degrees of accumulation of cations (ΔГM) and depletion of anions (ΔГCl) surrounding RNA relative to bulk concentrations (48, 49). The analysis using salt activity showed a linear relationship between log K of the 9-mer duplex and log aNaCl, with the slope of 1.80 ± 0.05 (data not shown) differing by ∼10% from the slope of the log K versus log [NaCl] plot (1.66 ± 0.05). Despite the difference between the slope values, the interaction coefficient for NaCl was calculated to be 0.11 per nucleotide phosphate, and this number was the same as that determined from the slope of the log K versus log [NaCl] plot with the assumption of α = 0.9. Because the bulky ion activities and salt activities in mixed solutions were unknown, we performed the analysis using molar salt concentrations. The slope of the log K versus log [MCl] plots decreased as the size of the bulky ions increased, and negative slopes were obtained with large ions. A correlation was found between the slope values and hydrated ionic radii (Fig. 3 B), indicating the absence of specific binding sites for ions of a particular size. The slope analyses indicated a net release of large ions bound to single strands during the duplex formation, consistent with duplex destabilizations caused by unfavorable enthalpy changes (Table 1).

The results of using mixed solutions showed correlations between the dependence of log K on the concentrations of NaCl and TBACl, and εr–1 values, regardless of interactions between the cosolutes and RNA. Mixed solutions with lower dielectric constants decreased the dependence on NaCl concentrations (to less positive slope values) and also decreased the dependence on TBACl concentrations (to more negative slope values). Changes in the salt-concentration dependence in mixed solutions were also observed for intramolecular melting of poly(A)/poly(U) (Table S1). The slope values of Tm versus log [MCl] plots and Tm–1 versus log [MCl] plots (the plots using Tm–1 values were also linear; data not shown) reflect the degree of ion binding, as represented by Δ Tm/Δln aMCl = –2ΔГMClβ and ΔTm–1/Δln aMCl = 2ΔГMClβ′, where β (= RTm2/ΔH°) and β′ (= R/ΔH°) are assumed constants (50, 51). The strong relationships between the dependence of Tm on NaCl concentrations and εr–1 values, and between the dependence of Tm on TBACl concentrations and εr–1 values (Fig. 5 B) indicate that the dielectric constant has a strong influence on the binding of Na+ and TBA ions to RNA. The predominance of the dielectric constant effect on ion binding to RNA is consistent with an electrostatic interaction model, such as the Poisson-Boltzmann treatment, in which ions that associate with the surface of RNA through Coulomb interactions are important for ion-induced stabilization of RNA structures (41, 46, 52).

The efficiency of cation binding to RNA in both less condensed and more condensed states may be enhanced in media with low dielectric constants. We assessed the number of accumulated ions during duplex formation by using a simplified reaction cycle for structural changes from R1 to R2 in a dilute solution (without cosolutes) and a cosolute-containing solution with a lower dielectric constant, as demonstrated in Fig. 7 A. For Na+ ion accumulation derived from the results in Fig. 4 B, process 2 (duplex formation in a cosolute-containing solution) produced fewer bound ions than process 1 (duplex formation in a dilute solution). Accordingly, process 3 (single-strand transfer from a dilute solution to a cosolute-containing solution) produced a greater number of bound Na+ ions than process 4 (duplex transfer from a dilute solution to a cosolute-containing solution). For TBA ion accumulation derived from the results in Fig. 4 D, process 2 produced a greater number of released ions than process 1, and thus process 3 produced a greater number of bound ions if TBA ions are excluded from the duplex. The analysis indicates that binding of both Na+ and TBA ions to single strands is enhanced, compared with binding to duplexes, in media with low dielectric constants. In agreement with this, the dimensions of unfolded nucleic acids have been reported to be reduced in solutions containing PEG or TMAO (20, 47), and the cosolutes used in this study were found to affect the CD spectra of single strands, but not those of duplexes (Fig. S5). Thus, it is likely that the dimensions of single strands, which have a higher flexibility than a duplex, are changed by an increasing number of bound cations, resulting in changes in cation binding numbers during RNA structure formation. This implies that intrinsically disordered residues play a role in enhancing the efficiency of cation binding to RNA under low dielectric constant conditions.

Figure 7.

Figure 7

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(A) Reaction cycle for RNA structural changes from unfolded (R1) to folded (R2) states by ion binding under dilute (superscript d) and high cosolute (superscript c) conditions. (B) Representations of RNA duplex formation in NaCl solutions without (top) and with (bottom) a background of TBA ions.

Competition for metal ion binding and bulky ion binding

In this study, we also investigated the effects of bulky ions on metal ion binding to RNA. The presence of background NH4Cl at 100 mM did not change the stability of the 9-mer duplex in the NaCl concentration range of 20–200 mM, indicating competitive RNA binding between Na+ and NH4+. In contrast, experiments with bulky ions showed linear dependences over the NaCl concentration range from 20 mM to 1 M, reflecting different RNA binding strengths and preferences between Na+ and bulky ions. The presence of GB, TMAO, and TMA ions slightly decreased the NaCl concentration dependence, possibly due to partial displacement of Na+ ions bound to the duplex. These results are in agreement with the Poisson-Boltzmann theory, which predicts that the binding ratio of a bulky ion over Na+ will decrease with increasing ion sizes (41). On the other hand, the presence of TBA and TPeA ions increased the NaCl concentration dependence. This observation suggests that a greater number of Na+ ions are required to replace the large ions bound to single strands during duplex formation (Fig. 7 B), consistent with entropy-driven destabilization by the presence of background TBACl (Table 3). Further experiments with PEG8000 showed a smaller effect on competitive RNA binding between metal and TBA ions (Fig. 6 D). This result indicates that bulky ions are useful for controlling the stability of RNA structures and their sensitivity to metal ion salts even in the presence of organic additives.

Possible relevance to RNA interactions in cells and nonaqueous conditions

The results presented here can be used to consider RNA interactions with intracellular components. Methylamine osmolytes of GB and TMAO accumulate in osmotically stressed cells, and these compounds would not greatly perturb the stability of RNA basepairing and metal ion binding. In contrast, large tetraalkylammonium ions do not accumulate in cells, but they exhibit binding preferences similar to those of single-stranded, nucleic-acid-binding proteins and chaperone proteins that dissociate basepairs during replication, recombination, and folding of DNA and RNA. It may be speculated that the mechanism employed by these proteins to dissociate basepairs partly reflects steric effects that reduce the accessibility to basepaired structures. In addition, the results obtained using mixed solutions imply that RNA structures and their interactions are strongly affected by the media inside cells, where relatively low dielectric constant values of ∼50–60 or lower have been reported (53, 54, 55, 56, 57, 58). The significant effects of dielectric constants on cation binding will further inform the development of nucleic-acid-based technologies for medical and technological applications. In particular, amphiphilic alkylammonium ions are effective for screening the charge of nucleotide phosphates in apolar media, allowing the use of nucleic acids under nonaqueous conditions (26, 27, 28, 29). It is remarkable that some alkylammonium ions are components of ionic liquids; thus, our results may be applicable to investigations of the behavior of nucleic acids in ionic liquids.

Conclusion

In this study, we investigated cation-size-dependent effects on the thermal stability of RNA structures. Our results provide a basis for developing methods to predict RNA structures in aqueous mixtures with organic additives, which should aid in the design of RNA molecules with certain desired properties in nonaqueous media. The analysis of the salt-concentration dependence of RNA stability revealed that mixed solutions with low dielectric constants decreased the binding of Na+ ions and increased the release of TBA ions during the formation of RNA structures, which was attributed to the enhancement of electrostatic interactions between RNA and the cations. Furthermore, background large ions increased the dependence of RNA stability on the concentration of metal ion salts. These unique properties of bulky cations could help improve the characteristics of many RNA structures, including catalytic RNAs that function in the presence of metal ions. The results presented here warrant the use of bulky ions and mixed solutions to control the stability of nucleic acid structures, and should facilitate the investigation of interactions between nucleic acids and alkylammonium ions in biotechnology and nanotechnology applications.

Author Contributions

S.N. designed and performed research, analyzed data, and wrote the manuscript. Y.T. and H.H. performed research. N.S. analyzed data.

Acknowledgments

We thank Junpei Ueno for technical assistance.

This work was supported in part by Grants-in-Aid for Scientific Research, Japan Society for the Promotion of Science (JSPS KAKENHI grant number 15K05575), and the Ministry of Education, Culture, Sports, Science and Technology-Supported Program for the Strategic Research Foundation at Private Universities, 2009–2014.

Editor: Tamar Schlick.

Footnotes

Five figures and one table are available at http://www.biophysj.org/biophysj/supplemental/S0006-3495(16)30753-6.

Supporting Material

Document S1. Figs. S1–S5 and Table S1
mmc1.pdf (574.5KB, pdf)
Document S2. Article plus Supporting Material
mmc2.pdf (2.3MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figs. S1–S5 and Table S1
mmc1.pdf (574.5KB, pdf)
Document S2. Article plus Supporting Material
mmc2.pdf (2.3MB, pdf)

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