Article

Thermal Stability of RNA Structures with Bulky Cations in Mixed Aqueous Solutions Shu-ichi Nakano,1,* Yuichi Tanino,1 Hidenobu Hirayama,1 and Naoki Sugimoto1,2 1 Department of Nanobiochemistry, Faculty of Frontiers of Innovative Research in Science and Technology and 2Frontier Institute for Biomolecular Engineering Research, Konan University, Kobe, Japan

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 (4–14). 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 (15–18). 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

Submitted July 12, 2016, and accepted for publication August 29, 2016. *Correspondence: [email protected] Editor: Tamar Schlick. http://dx.doi.org/10.1016/j.bpj.2016.08.031 Ó 2016 Biophysical Society.

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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 Naþ and Mg2þ, 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 (26–29), 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

Thermal Stability of RNA Structures

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. 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,N0 ,N0 -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.

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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 mM 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 mM and a heating rate of 0.2 C min–1, and tRNA was examined at a strand concentration of 1 mM 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 (DH ), entropy change (DS ), and free-energy change (DG ) 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.

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.

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FIGURE 1 (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.

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Nakano et al. TABLE 1 Thermodynamic Parameters for RNA Duplex Formation in the Presence of 100 mM Salt –DH (kcal mol–1)

–DS (cal mol–1 K–1)

–DG (kcal mol–1)

TABLE 3 Thermodynamic Parameters for 9-mer Duplex Formation with 100 mM NaCl in the Absence and Presence of Background TBACl

50 -CAACGCAAG-30 /50 -CUUGCGUUG-30

[TBACl] (mM)

–DH (kcal mol–1)

NH4þ TEA TBA TBA(w/PEG)a

0 100

89.2 5 3.1 96.1 5 3.0

Salt Ion

88.9 5 85.7 5 82.5 5 86.4 5

252.4 5 13.0 251.7 5 3.4 245.1 5 7.0 257.5 5 13.0

4.1 1.0 2.2 4.0

10.7 5 0.3 7.60 5 0.13 6.47 5 0.16 6.45 5 0.34

50 -CGGCGCGGG-30 /50 -CCCGCGCCG-30 NH4þ TEA TBA a

96.9 5 0.6 89.2 5 2.6 92.5 5 1.0

247.9 5 1.5 242.0 5 8.3 255.8 5 3.0

19.4 5 0.4 14.1 5 0.2 13.1 5 0.1

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

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 mM, with the exception of 50 -CCCGCGCCG-30 , which was used at 40 mM because of its low signal level. All RNA solutions were heated to 80 C and cooled at the rate of 2 C min1 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, 50 -CAA TABLE 2 Values of –DDG and Linear Slopes of Log K versus Log [MCl] Plots Salt Ion

–DDG (kcal mol1)a

Slope

50 -CAACGCAAG-30 /50 -CUUGCGUUG-30 Naþ NH4þ Choline GB TMAO TMA TEA TBA TPeA

2.0 2.1 0.3 –0.4 –0.1 0.3 –1.0 –2.1 –2.2

1.66 5 0.05 1.46 5 0.08 0.53 5 0.08 –0.22 5 0.04 –0.18 5 0.03 0.56 5 0.08 –0.30 5 0.03 –1.00 5 0.10 –1.11 5 0.11

50 -CGGCGCGGG-30 /50 -CCCGCGCCG-30 Naþ NH4þ Choline GB TMAO TMA TEA TBA TPeA

2.0 2.3 0.1 –0.7 –0.1 –0.5 –3.0 –4.0 –4.9

1.92 5 0.12 2.01 5 0.08 –0.05 5 0.08 –0.98 5 0.06 0.10 5 0.06 –0.07 5 0.06 –1.56 5 0.07 –2.20 5 0.11 –2.60 5 0.11

Values were calculated by subtracting the –DG 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). a

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–DS (cal mol–1 K–1)

–DG (kcal mol–1)

253.5 5 9.9 278.6 5 9.8

10.6 5 0.2 9.66 5 0.21

CGCAAG-30 /50 -CUUGCGUUG-30 , 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 NH4þ, 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 NH4þ 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). 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 (11,36–38). 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, 50 -CAACGCAAG-30 and 50 -CUUGCGUUG-30 , 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

Thermal Stability of RNA Structures

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B

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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 50 -CGGCGCGGG-30 / 50 -CCCGCGCCG-30 , which is comprised only of GC 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 (DG ) 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 –DG value by 0.3 kcal mol–1, but the increments were much smaller than those observed with Naþ and NH4þ. GB and TMAO ions slightly decreased the value by 0.4 and 0.1 kcal mol–1, respectively. TEA ions decreased –DG 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,

FIGURE 2 (A) Tm values for the 9-mer duplex at 2 mM 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 mM in solutions containing NH4Cl (circles), TEACl (triangles), or TBACl (squares). (C) CD spectra of the 9-mer duplex (left) and its single strands 50 -CAACGCAAG-30 (middle) and 50 -CUUGCGUUG-30 (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.

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 NH4þ and tetraalkylammonium ions (TMA, TEA, TBA, and TPeA), as indicated by the solid symbols in Fig. 3 A. The duplex stabilities further decreased with increasing concentrations of TBACl and TPeACl. The dependences of log K (¼ –DG /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

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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 highsalt-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 (εr1) of mixed solutions (39). In agreement with A

C

FIGURE 3 (A) Values of –DG for the 9-mer duplex (circles) and GC duplex (triangles) at a salt concentration of 100 mM plotted against the hydrated radii of NH4þ 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 NH4þ and tetraalkylammonium ions in the absence (solid symbols) and presence (open symbols) of 20 wt % PEG8000.

this, the slopes of Fig. 4 A decreased with increasing εr1 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 –DG and εr1 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). 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

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FIGURE 4 (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 εr1 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 εr1 values. To see this figure in color, go online.

Thermal Stability of RNA Structures

with the εr1 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 AU 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. 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 εr1 values of the solutions (Fig. 5 B).

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 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). A

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

B FIGURE 5 (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 εr1 values of mixed solutions. Colored symbols are the same as those used in Fig. 4. To see this figure in color, go online.

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effectively as NaCl (Table 2), indicating efficient binding of NH4þ 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), respec-

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FIGURE 6 (A) Dependence of Tm of poly(A)/ poly(U) at 40 mM 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.

tively. 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

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

Bulky cations are used to develop nucleic-acid-based technologies for medical and technological applications in which nucleic acids function under non...
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