effects of osmolytes on rna secondary and tertiary structure stabilities and rna-mg2+ interactions

doi:10.1016/j.jmb.2007.03.080 J. Mol. Biol. (2007) 370, 993–1005

Effects of Osmolytes on RNA Secondary and TertiaryStructure Stabilities and RNA-Mg2+ Interactions

Dominic Lambert and David E. Draper⁎

Department of Chemistry,Johns Hopkins University,Baltimore, MD 21218, USA

Abbreviations used: TMAO, trimeBWYV, Beet Western Yellow Virus; D2,6-diamino-purine; SASA, solvent aE-mail address of the correspondi

[email protected]

0022-2836/$ - see front matter © 2007 E

Osmolytes are small organic molecules accumulated by cells in response toosmotic stress. Although their effects on protein stability have been studied,there has been no systematic documentation of their influence on RNA.Here, the effects of nine osmolytes on the secondary and tertiary structurestabilities of six RNA structures of differing complexity and stability havebeen surveyed. Using thermal melting analysis, m-values (change in ΔG° ofRNA folding per molal concentration of osmolyte) have been measured. Allthe osmolytes destabilize RNA secondary structure, although to differentextents, probably because they favor solubilization of base surfaces.Osmolyte effects on tertiary structure, however, can be either stabilizing ordestabilizing. We hypothesize that the stabilizing osmolytes have unfavor-able interactions with the RNA backbone, which becomes less accessible tosolvent in most tertiary structures. Finally, it was found that as a largerfraction of the negative charge of an RNA tertiary structure is neutralized byhydrated Mg2+, the RNA becomes less responsive to stabilizing osmolytesand may even be destabilized. The natural selection of osmolytes asprotective agents must have been influenced by their effects on the stabilitiesof functional RNA structures, though in general, the effects of osmolytes onRNA and protein stabilities do not parallel each other. Our results alsosuggest that some osmolytes can be useful tools for studying intrinsicallyunstable RNA folds and assessing the mechanisms of Mg2+-induced RNAstabilization.

© 2007 Elsevier Ltd. All rights reserved.

*Corresponding author
Keywords: proline; urea; glycerol; trimethylamine oxide; betaine
Introduction

To respond to changes in osmotic pressure, somecells synthesize or take up small organic solutesknown as osmolytes. These osmoregulatory com-pounds have also been shown to help cells cope withother environmental stresses such as high tempera-ture, pressure or desiccation, and to affect thestabilities of proteins and DNA.1,2 Osmolytes belongto diverse chemical families including methyla-mines, amino acids, sugars and polyols. Most os-molytes, those referred to as compatible or protectiveosmolytes, tend to stabilize protein structure, whileurea, the principal non-compatible osmolyte, is a

thylamine oxide;AP,

ccessible surface area.ng author:

lsevier Ltd. All rights reserve

denaturant. The concentrations of these moleculesare apparently regulated so as to maintain theosmolarity of the cell without perturbing macro-molecular functions, for instance by adjusting theratio of protective and denaturing osmolytes tomaintain protein stability3 or by adjusting theintracellular concentrations of osmolytes and ionsto maintain protein – DNA interactions.4

Studies of osmolytes have primarily focused ontheir effects on protein stability and enzyme func-tion.1,5 Structured RNA molecules are also impor-tant components of the cell, and any changes to thestabilities of tRNAs, hairpins controlling transcrip-tion termination, or riboswitches (to name a fewpossibilities) could alter gene expression in dramaticways. In one study, the ratio of denaturing and sta-bilizing osmolytes (urea and TMAO, respectively)needed to maintain the tertiary structure of a tRNAwas found to be about the same as the ratio neededto maintain protein stability.6 But in general, there isno reason to suppose that RNA and proteins, which

d.

mailto:[email protected]

994 Effects of Osmolyte on RNA Stability

are stabilized by very different balances of forces,should respond to osmolytes in the same ways, andthere are few data that bear on this question. Here,we provide a first systematic look at the degree towhich RNA structures are sensitive to osmolytes bysurveying the effects of nine different osmolytes onfive RNAs with increasing structural complexity(Figure 1). A sixth RNA with the same overallstructure but different stability as the RNA in Figure1(d) has also been included.Protein – osmolyte interactions have been quanti-

tatively described in thermodynamic terms. Thecritical factor is the partitioning between water andosmolyte at solvent-exposed surfaces of a protein.While stabilizing osmolytes tend to be excluded fromthe protein surface, forcing the polypeptide to adopta compactly folded structure with a minimum ofexposed surface area, denaturing osmolytes accu-mulate at the surface and promote unfolding.7–10

Accumulation or exclusion occurs primarily at polar

Figure 1. Schematics of the secondary and tertiarystructures of the RNAs used in this study. Horizontal blackbars and black dots represent Watson–Crick and non-canonical base-pairs, respectively. Gray bars representbase–base tertiary interactions, and thin lines with arrow-heads denote 5′-3′ backbone connectivities. (a) A designedshort hairpin. (b) Tar-tar* kissing-loop complex (1KIS).22

(c) BWYV pseudoknot (1L2X).61 (d) 58mer rRNA fragmentbinding L11 protein. The sequence is the U1061A variantof the E. coli rRNA (1HC8).62 Broken arrows indicatefour point mutations in the GACG RNA construct.20 (e) A-riboswitch adapter region (1Y26).29 The adenine ligand isshown in outline typeface.

surfaces, particularly the backbone amides.9,11,12

Thus, the sensitivity of a protein to osmolytesdepends primarily on the degree to which its polarpeptide backbone becomes buried upon folding intothe native structure and the relative strength ofosmolyte versus water interactions with peptidegroups.12

Structured RNAs differ from proteins in tworespects that are relevant to the way these moleculesinteract with osmolytes. First, functional RNAs invivo may be entirely secondary structure or alsoinclude tertiary interactions; in contrast, extensiveprotein secondary structure is usually not observedin the absence of the complete tertiary fold. Theextents to which base versus backbone surfaces areburied upon folding RNA secondary or tertiarystructures differ considerably. It is thus possible thatan osmolyte could have qualitatively different effectson RNAs that rely to different degrees on sets oftertiary interactions (in addition to secondary struc-ture) for function. Second, all RNAs have a highnegative charge density and consequently stronginteractions with ions,13 a factor that is usuallynot as important to protein folding. Osmolytescould therefore affect RNA stability indirectly, bymodulating the thermodynamic activity of theions, the strength of the Coulombic interactionsbetween ions and RNA, and the energetics of iondehydration.With these considerations in mind, the survey of

osmolyte effects on RNA reported here examinesboth RNA secondary structure stability and theformation of several different RNA tertiary struc-tures that differ in their solvent accessible surfaceareas and in their interactions with ions. We find thatall osmolytes destabilize RNA secondary structureto some degree, but many osmolytes stabilizetertiary structure. The latter stabilizing influence is,in most cases, strongly attenuated or even reversedby high Mg2+ concentrations. Besides the relevanceof these results to the in vivo role of osmolytes, ourfindings suggest that some osmolytes could beuseful tools for RNA folding studies.

Results

Choice of RNAs and osmolytes for study

The five RNA structures chosen for our survey ofosmolyte effects on folding transitions are shown inFigure 1. The simple hairpin (Figure 1(a)) wasdesigned to be a representative secondary structurewith both A-U and G-C base-pairs. The other RNAsare all naturally occurring RNAs with tertiary struc-ture, andwere selected on the basis of several criteria.First, in order to measure the effect of an osmolyte onthe RNA stability, the tertiary folding transition hadto be resolvable in the UV melting profile of eachRNA. An example profile of each RNA is shown inSupplementary Data Figure S1. Second, by analogywith proteins, we expected that accumulation orexclusion of osmolytes at RNA surfaces would lead

Figure 2. Solvent-accessible surface area calculations.Average exposures (Å2) were calculated per nucleotide for(a) bases, (b) sugars and (c) phosphates in different RNAconformations and constructs. From left to right in eachpanel the calculations represent an average of 30 single-stranded residues observed in RNA crystal structures; aninternal residue within an A-form single-strand (ss) RNAor double-strand (ds) RNA; the tar and tar* hairpins; thetar-tar* complex; BWYV RNA; 58mer rRNA fragment(U1061A RNA); and A-riboswitch RNA.

995Effects of Osmolyte on RNA Stability

to stabilization or denaturation of the molecules.Thus, RNAs showingdifferent proportions of solventaccessible surface area (SASA) in the folded statewere chosen (see below). Lastly, RNA tertiarystructures tend to be strongly stabilized by Mg2+,but different mechanisms may dominate betweenRNAs.13 At one extreme, “diffuse” ions, whichremain fully hydrated and do not directly contactthe RNA, contribute to the stability of all RNAstructures.14–16 At the other extreme, “chelated” ionsare substantially dehydrated andbind to a specific setof RNA ligands.13,17 Because osmolytes may affectthe free energy of ions in various RNA environmentsdifferently, RNAs stabilized to different degrees bydiffuse and chelated Mg2+ were included.We calculated the SASA of each RNA in Figure

1(b)–(e) from its atomic coordinates, and divided theexposed surface into contributions from base, sugar,or phosphate. The results (Figure 2) are expressed asSASA per nucleotide; smaller values correspond toan overall more compact structure. For reference,the average exposure of an internal nucleotide in anA-form helix or an A-form single strand are shown.A-form geometry maintains a high degree ofstacking between bases and undoubtedly under-estimates the solvent exposure of single-strandednucleotides in unfolded RNAs. To obtain a morerealistic estimate of nucleotide exposure in a singlestrand, we extracted single strand, non-A-formsegments of RNA from several crystal structuresand averaged the SASA of these nucleotides (seeMaterials and Methods). Though the componentsurface areas obtained in this way are much largerthan those calculated for nucleotides in A-formconformations, they are still 55–70% of the calcu-lated maximal surface areas of a fully extendedpolynucleotide.18 Comparing the SASA of singleand double-stranded RNA nucleotides in Figure 2, itis apparent that the major change in SASA uponRNA secondary structure formation is burial of thebases; changes in backbone exposure are relativelyminor.There are distinct differences in SASA among the

selected RNA tertiary structures. The 58mer rRNAfragment (Figure 1(d)) is the most compact structure(smallest total SASA per nucleotide) and has asignificantly higher degree of phosphate burialthan the other RNAs. Both Mg2+ and K+ are foundchelated to buried backbone oxygen atoms in thisRNA and are energtically important for folding.17,19

Two sequence variants of this RNA were studied,U1061A and GACG; U1061A RNA is more stable.20

The Beet Western Yellow Virus (BWYV) pseudoknot(Figure 1(c)) is the least compact RNA. Mg2+ stabi-lizes the structure, but only as a diffuse ion.16 Thetertiary unfolding transition reported here involvesonly the disruption of loop L2 interactions in theminor groove of helix H1 (Figure 1(c))21; changes inSASA per nucleotide are probably small comparedto the other RNAs described here. Formation of thetar-tar* complex (Figure 1(b)), by the interaction oftwo hairpin loops, is accompanied by large change inthe SASA of bases and the creation of an unusual

tunnel in the major groove.22 Finally, the A-ribo-switch (Figure 1(e)) shows the smallest base SASAamong the RNAs. Formation of its tertiary structurerequires binding of an adenine base derivative.Because the ligand interactions with the RNAdepend on base stacking and hydrogen bonding inthe same way as any intramolecular interactions of abase with the rest of an RNA, osmolytes should

Figure 3. Extraction of transition Tms and m-valuesfrom UV melting profiles. (a) Example melting profiles ofGACG in various molalities of TMAO. The temperaturedependence of the absorbance is plotted as the first deriva-tive (dA260/dT) for the following TMAO concentrations: 0(red), 0.46 (blue), 0.92 (cyan), 1.38 (light green) and 1.84 m(dark green). The least squares best fit profile for dA260/dTdata in the absence of TMAO is shown (red line) along withits deconvoluted transitions 1 (black line), 2 (large dash line),3 (small dash line) and 4 (dotted line). (b) Plots of reciprocalTms of each unfolding transition (line-coded as in (a)) versusTMAO molality; slopes of these lines were used incalculating m-values. Error bars, derived from bootstrapanalysis of the melting profiles, are shown for all points; insome cases, the bars are smaller than the data points.


perturb both ligand binding and overall RNAstability by similar mechanisms.The osmolytes used in this survey were selected

to be representative of different chemical classesof naturally occurring osmolytes: amino acids(proline and betaine), methylamines (betaine andtrimethylamine oxide (TMAO)), and polyols oralcohols (sorbitol, sucrose, glycerol, ethylene gly-col, and methanol). Methanol is not a naturalosmolyte but was included in this survey becauseit has been previously observed to stabilize RNAtertiary structure.23

Approach: UV-thermal melting analysis andm-value determination

Bymonitoring theUVmeltingprofiles of anRNA inthe presence of increasing osmolyte concentration, asexemplified by the GACG RNA melting profiles inFigure 3(a), we obtain the Tm and ΔH° of each RNAunfolding transition as a function of osmolytemolality (see Materials and Methods for data analy-sis). Figure 3(b) shows the behavior of all fourunfolding transitions that were fit to the GACGRNA melting profiles, plotted as 1/Tm versus osmo-lyte molality. In this RNA, the first unfoldingtransition represents disruption of tertiary structureand the last corresponds to the melting of the smallhelix H2b. Only the osmolyte dependence of the firsttransition has been compiled for this RNA, because itis not well understood what secondary structures areunfolding in the second and third transitions,24 andthe last transition is poorlydetermined in the presenceof certain osmolytes. The slopes of these plots aremultiplied by an appropriate factor to obtain the freeenergy change as a function of osmolyte concentra-tion (see Materials and Methods), known as the m-value.25 In the case of proteins, the folding free energytends to vary linearly with osmolyte concentration,26a phenomenon that has been attributed to the smallmagnitude of the equilibrium constant for exchan-ging osmolyte and water at the protein surface.27 Alinear free energy dependence is observed for almostall of the osmolyte-RNA combinations reported here,though in some cases (marked by an asterisk inFigures) the free energy of folding reaches aminimumvalue by 1–2 m osmolyte. Such behavior might beexpected if an osmolyte binds specific site(s) on anRNA surface much more strongly than does water.

Osmolytes destabilize RNA secondarystructures

Besides melting experiments with the designedhairpin, three other hairpin unfolding reactionswere resolved as transitions in the melting profilesof the RNAs with tertiary structure; these are thetar and tar* hairpins and the hairpin containinghelix H1 of BWYV RNA (Figure 1(b) and (c),respectively). With one unusual exception, all ofthe tested osmolytes destabilized all of these hair-pins, though to widely varying degrees (Figure 4(a)).Surprisingly, proline is the most efficient denaturant

of RNA secondary structure, not urea (Figure 4(a)).The BWYV H1 hairpin consists entirely of G-Cbase-pairs, which could be a factor in its moreeffective denaturation by proline and betainerelative to the other hairpins. Intriguingly, glycerolgreatly stabilizes the BWYV H1 hairpin, while itacts as a mild denaturant on the other RNAs. Thestabilizing effect of glycerol is also unusual inreaching a plateau at about 1 m (Figure 4(b)). This“saturation” behavior suggests that glycerol formsunusually strong interactions with the H1 hairpin(see Discussion).

Figure 4. Osmolyte effects on RNA secondary struc-ture stability in the absence of Mg2+ (K+ concentrations aredifferent for each RNA; see Materials and Methods.) (a)m-Values obtained for the indicated RNA hairpins in thepresence of 0–2 m of various osmolytes. The m-valuerelates to the folding reaction; hence a positive valueindicates osmolyte-induced destabilization of the RNA.The effect of glycerol on BWVY RNA (indicated by anasterisk) is the maximum stabilization free energy inducedby the osmolyte (see (b)). (b) Effects of glycerol onsecondary and tertiary structure stabilities of BWYVRNA in the absence of Mg2+ (44 mm K+). Reciprocal Tmsfrom secondary (transition 3, square) and tertiary (transi-tion 1, circle) structure melting transitions at differentglycerol molalities are shown with error bars (sometimessmaller than the points) derived from the bootstrapprocedure. The reported stabilization energy (a) iscalculated from the Tm at the intersection of the twoleast squares lines.

Figure 5. Osmolyte effects on RNA tertiary structurestability in the presence of K+ as the only cation (tar-tar*,BWYV, and A-riboswitch RNAs) or Mg2+ and K+ (U1061Aand GACG RNAs) (see Materials and Methods for all saltconcentrations). m-Values are plotted for each RNA in thepresence of the indicated osmolytes, except for the effect ofglycerol on BWVY RNA (indicated by an asterisk), whichis the maximum stabilization free energy observed at asaturating level of ∼1 m osmolyte and is −2.0 kcal/mol m(Figure 4(b)).


m-Values for urea and duplex RNAs have beenreported.28 The value obtained for a six base-pairduplex is about two-thirds larger than we obtain forthe six base-pair hairpin, after making an estimatedcorrection for the difference between molar andmolal units used in the two studies. The discrepancymay arise from the different salt conditions (100 mm

KCl in our study, versus 0.5 M NaCl) and experi-mental methods, which derive m-values at muchhigher urea concentrations than those used here.

Osmolyte effects on RNA tertiary structure canbe either stabilizing or destabilizing

We next examined the effects of osmolytes on theunfolding of RNA tertiary structure (Figure 5). Thesemeasurements were made in the presence of K+ asthe only stabilizing cation, except for the two 58merrRNA fragments, which require some Mg2+ to fold.(The influence of Mg2+ on osmolytes is addressed inthe following section.) Urea and proline are uni-formly destabilizing. The effects of sorbitol, betaine,and sucrose are RNA-dependent, while the remain-ing alcohols and TMAO tend to be stabilizing, insome cases strongly so. There is considerablevariation from RNA to RNA in the effectiveness ofan osmolyte. For example, the U1061A and GACGvariants of the rRNA fragment, which differ by onlyfour nucleotides in sequence (Figure 1(d)), differ bynearly a factor of two in sensitivity to methanol andethylene glycol. Because the tested RNAs differ insolvent exposure of bases and backbone (Figure 2), itis perhaps not surprising that their responses to anosmolyte are variable (see Discussion).

Osmolyte effects on RNA–ion interactions

As pointed out above, RNA tertiary structures tendto be strongly stabilized byMg2+, even in the presenceof a large excess of monovalent cations. Osmolytes

Figure 6. Osmolyte effects on RNA tertiary structurestability in buffers containing either K+ as the only cationor mixtures of K+ and Mg2+. Details of the salt concentra-tions used with each RNA are given in Materials andMethods. m-Values for the tar-tar* complex, BWYV RNA,and A-riboswitch RNA are shown. Asterisks indicate thatΔG° versus osmolyte molality did not remain linear to 2 mosmolyte; the maximum free energy of stabilization isreported instead of an m-value. The stabilization byglycerol of BWVY RNA in the absence of Mg2+ was –2.0kcal/molm. For ethylene glycol and BWYV RNA in bufferwith K+ and Mg2+, the value is −0.86 kcal/mol m.


could indirectly affect RNA stability by altering theactivity or solvation free energy of ions and thus thefree energy of either diffuse or chelated ion – RNAinteractions. If such indirect effects are negligible,thenMg2+-induced stabilization and osmolyte effectsshould be additive. To see if this is the case, we com-pared RNA-osmolyte m-values measured with K+ asthe only cation to measurements made in K+/Mg2+

mixtures (Figure 6). The K+/Mg2+ ratio was adjustedin each case to ensure that Mg2+ was responsible forsubstantial stabilization of the RNA. These compar-isons could only be donewith the tar-tar*, BWYV, andA-riboswitch RNAs, which have stable structures inthe absence of Mg2+.Urea, sorbitol, and methanol are the least sensitive

to the presence of Mg2+ with all three RNAs. Amongsome of the other osmolytes, instances of large,Mg2+-induced increases in m-values are seen (proline,betaine, TMAO). There are fewer cases of a significantMg2+-induceddecrease inm-value (BWYVRNAwith

Figure 7. Effects of osmolytes on Mg2+-dependence ofRNA tertiary stability. The reciprocal Tms for tertiarystructure unfolding in the absence (open) or presence(closed) of osmolytes are plotted for BWYV RNAwith 2 methylene glycol (a), A-riboswitch RNA with 2 m ethyleneglycol (b) or 1.84mTMAO (c), and a 58mer rRNA fragment(GACG RNA) with 2 m ethylene glycol (d) or 1.84 mTMAO (e) are depicted with corresponding bootstraperrors (some are smaller than data points). K+ concentra-tions were 14 mm for BWYV RNA, and 104 mm for theother two RNAs. Reciprocal Tms at 0 mmMg2+ are boxed.

ethylene glycol, sucrose). Two of the most stabilizingconditions for BWYVRNA, glycerol in the absence ofMg2+ and ethylene glycol in its presence, again showsaturation behavior, suggesting relatively strongbinding of the osmolyte to the folded RNA.


It thus appears that Mg2+ and many osmolytes arecapable of either synergistic or competitive effects onRNA stability, depending on the RNA and specificconditions. To characterize the interplay betweenMg2+ and osmolytes further, we measured thestability of several RNAs over wide ranges of Mg2+

concentrations, with or without an osmolyte present(Figure 7). Ethylene glycol and TMAO were chosenfor study, to see if their opposite effects on BWYVRNA stability extend to other RNAs. The principalfinding is that Mg2+ strongly attenuates the stabiliz-ing effects of the two osmolytes (compare Figure7(a)–(c)). At high enough concentrations,Mg2+ eitherrenders the osmolyte ineffective (Figure 7(a) and (c))or converts it to a somewhat destabilizing osmolyte(Figure 7(b)). Similar experiments with the A-riboswitch and proline, betaine, ormethanol showedsimilar trends as seen in Figure 7(b): the stabilizingeffect of Mg2+ was attenuated by the osmolyte, andthe stabilizing osmolytes (betaine and methanol)became destabilizing above 1 mm Mg2+ (data notshown). Some synergism between Mg2+ andethylene glycol with BWYV RNA is observed atvery low Mg2+ concentrations (Figure 7(a)). Pre-sumably the diverse results of the measurements inFigure 7 reflect the strong Mg2+-dependence ofosmolyte–RNA interactions, which may vary fromsynergistic with Mg2+ to competitive as the Mg2+

concentration increases.Mg2+ ions in the presence of BWYV RNA remain

hydrated,16 and it is likely that the same is true forthe A-riboswitch RNA: Mg2+ observed in theriboswitch crystal structure have at least one shellof hydrating water.29 To ask whether Mg2+ has thesame attenuating effect on osmolytes when the RNAis stabilized by both diffuse and chelated ions, weperformed similar experiments on the GACG rRNAfragment. As Mg2+ concentration is increased, thestabilizing effect of ethylene glycol becomes smallerbut is not eliminated (Figure 7(d)). Of most interestis the fact that the stabilizing effect of TMAO isnearly constant over the entire range of Mg2+

concentrations (Figure 7(e)), the only instance weobserved of an osmolyte and Mg2+ acting in anadditive way to stabilize RNA tertiary structure.Finally, we note that m-values for the secondary

structure transitions observed in the melting of the

tar-tar* complex and BWYV RNA were unaffectedby the exchange of K+ for the K+/Mg2+ mixture inthe experiments reported in Figure 6 (data notshown). This result is consistent with Mg2+ having amuch more dramatic effect on RNA tertiary struc-ture stability than on secondary structure.

Discussion

Osmolyte effects on RNA stability: generalconsiderations

A framework for considering the effects ofosmolytes on the stabilities of RNA structure isproposed in Figure 8. Unfolding of native (N state)RNA structures has been described as occurring in ahierarchical fashion.28 Tertiary interactions are dis-rupted first, leading to the formation of an inter-mediate (I state) characterized by the presence ofonly secondary structure. When further destabi-lized, RNAs in this intermediate state melt into acompletely unfolded, single-stranded RNA (Ustate). This unraveling of a complex RNA tertiarystructure is accompanied by a decrease in thenegative charge density and a concomitant reduc-tion in the number of excess monovalent anddivalent cations needed to neutralize the phosphatenegative charges.13 When RNA unfolding is allowedto proceed in the presence of osmolytes, the smallorganic solutes have the potential to shift theequilibria between the different folding states (Nversus I and I versus U) through favorable orunfavorable interactions with one conformationover another. Although the survey of osmolyteeffects on RNA folding transitions presented herereveals a wide range of effects, we suggest that themajority of these observations can be rationalized interms of a few basic considerations as to (i) thepreferential interactions of nucleic acid bases withan osmolyte compared to water; (ii) the unfavorableinteractions of an osmolyte with the ribose-phos-phate backbone (i.e. a preference of the backbone forsolvation by water), and (iii) the attenuation ofosmolyte discrimination between I and N stateRNAs by high concentrations of Mg2+. In a fewcases, relatively strong interactions of an osmolyte at

Figure 8. Representation of thepotential effects of osmolytes onRNA folding equilibria. Unfolded(U), intermediate (I) and native (N)RNA states are depicted. The pre-dominant solvent-accessible sur-faces of each state are indicated inboxes.


specific RNA sites may also be an important factor.These themes are considered in more detail in thefollowing sections.

Osmolytes destabilize RNA secondary structure

The I→U unfolding RNA unfolding transition isaccompanied by a large increase in the SASA ofbases, and relatively little change in ribose or phos-phate exposure to solvent (Figure 2). All osmolytestested destabilize RNA helices (Figure 4), which isconsistent with preferential accumulation of osmo-lyte at the surfaces of bases. To the extent thatliterature data are available, they tend to confirm thefavorable interaction of the osmolytes studied herewith bases or nucleosides. Urea, methanol, andethylene glycol increase the solubility of bases.30,31

Urea and betaine preferentially accumulate aroundsingle-stranded DNA, and are either excluded from(betaine) or are unaffected by (urea) double strandDNA surfaces.32,33

Favorable and unfavorable effects of osmolyteson RNA tertiary stability

In contrast to the I→U transition, the I→N RNAfolding transition is not dominated by changes in theexposure of one type of surface area (Figure 8): some Istate singled-stranded regions may become morestacked, helix groovesmay be filled by bases, and thedevelopment of a more compact structure tends tobury ribose-phosphate backbone. The I→N transi-tion also tends to be more sensitive to the ionic com-position of the solvent, especially the presence ofMg2+, than secondary structure unfolding. Thus,the balance between an osmolyte's affinity forbases (destabilizing N), exclusion from backbone(stabilizing N), and synergism or competition withMg2+, will dictate how an osmolyte affects thefolding of a certain RNA. RNAs in which thesethree factors are weighted differently may respondto the presence of the same osmolyte in disparateways.Two of the osmolytes that we studied, urea and

proline, consistently denature tertiary structure (Fig-ure 5). These osmolytes must either favor backboneexposure as well as base exposure, or the osmolytepreference for exposed base surfaces overwhelms anyfavorable free energy associated with the burial ofbackbone.With the exception of sorbitol, the remaining

osmolytes that we studied (betaine, polyols, metha-nol, and TMAO) are effective stabilizers of at leastsome RNA tertiary structures (Figure 5). Becausethese same osmolytes tend to destabilize secondarystructure, we argue that these compounds favorburial of RNA backbone, that is, favorable osmolyteinteractions with base surfaces must be outweighedby unfavorable interactions with backbone. Thereare few literature data from which the preferentialinteraction of osmolytes with nucleic acid backbonecan be deduced, but we note that most osmolytes areexcluded from the protein peptide backbone, and it

is this unfavorable interaction that largely accountsfor the stabilization of proteins by osmolytes.12,34

(The denaturants urea and guanidine hydrochlorideare exceptions, favorably interacting with the pep-tide unit.) The hypothesis that RNA-stabilizingosmolytes are excluded from the polar RNA back-bone is consistent with their exclusion from polarprotein backbone.

Osmolyte – ion opposition in RNA stabilization

Monovalent salts strongly stabilize both RNA andDNA secondary structure. Changes in water activityor solvent dielectric constant caused by osmolyteaddition could, in principle, modulate the activitiesof ions and their effects on RNA stability. However,the monovalent salt dependence of DNA helixdenaturation is affected by osmolytes (ethyleneglycol, betaine, and urea) to an extent that is at theedge of statistical significance.33,35 Similarly smalleffects of Na+ have been seen with ethylene glycoland the denaturation of a DNA triplex, a systemwhich has a higher charge density approaching thatof an RNA pseudoknot.35 Mg2+ ions interact muchmore strongly than monovalent ions with RNA,especially with compact tertiary structures, but theirpossible modulation of osmolyte – nucleic acidinteractions have not been explored. We havetherefore focused on the possible interplay betweenMg2+ and osmolytes in this survey of osmolyteeffects.Initial experiments comparing the effects of osmo-

lytes on RNA stability in the presence of either K+ asthe only cation or a Mg2+/K+ mixture showed thatthe ionic composition of the buffer increases ordecreases the m-values of many osmolyte-RNAsystems, suggesting that osmolytes and ions maystrongly influence each others' interaction withRNAs, but no clear pattern of effects was apparent(Figure 6). A consistent theme emerged when RNAstability over a wide range of Mg2+ concentrationswas measured in the presence or absence of anosmolyte: the stabilizing influence of the osmolyte isstrongly attenuated with increasingMg2+ concentra-tion or, viewed in a reciprocal way, the stabilizingeffect of Mg2+ is attenuated in the presence ofosmolyte. In individual cases, low concentrationsof Mg2+ may add to the osmolyte-induced stabiliza-tion (Figure 7(a)), or high Mg2+ concentrations maycause the osmolyte to become destabilizing (Figure7(b)).We conclude that the stabilizing effects ofMg2+

and most of the osmolytes studied here are notadditive. Because Mg2+ ions tend to be held close tothe RNA surface,36 it is perhaps to be expected thatstrong interactions would arise between the ions andthe water/osmolyte layers solvating the RNA.Relatively strong interactions between Mg2+ andosmolytes are also likely; for instance, Mg2+ formsion pairs with the carboxylate anions found inproline and betaine.37 But detailed speculations asto why Mg2+ and osmolyte act in opposition cannotbe made without more information about the ac-cumulation (or exclusion) of osmolyte, water, and


ions near the surfaces of I and N state forms of theRNA.As mentioned in Results, BWYVand A-riboswitch

RNAs are probably stabilized predominantly bydiffuse Mg2+, while with GACG RNA a partiallydehydrated Mg2+ at a buried chelation site con-tributes most of the Mg2+-dependent folding freeenergy at lower monovalent ion concentrations.15 Itis therefore interesting that Mg2+ and TMAO areadditive in their stabilizing effects with this RNA(Figure 7(e)). This result implies that the opposingfree energies of Mg2+ dehydration and burial at theelectronegative binding site are either unaffected byTMAO, or affected in parallel fashion to give thesame net free energy advantage.

Sequence and structure-specific effects

Some of the osmolytes studied here are knownto vary in their preferences for G,C or A,U-rich seq-uences. For example, urea has a stronger affinity forA and T33 and betaine is more effective in de-stabilizing G-C base-pairs.38 Such preferences prob-ably account for some of the variation that we see inosmolyte effects between RNAs. For instance,betaine and proline have larger m-values for dena-turation of the BWYV RNA hairpin H1, which isentirely G-C pairs, than with the other hairpinstested.A potential sequence-specific effect of an osmolyte

is the unusual stabilization of the BWYV helix 1 byglycerol, which shows “saturation” behavior sug-gestive of an unusually strong interaction with thehelix (Figure 4(b)). Glycerol has been observed aspart of an RNA minor groove network of hydrogenbonds involving water molecules, 2′-hydroxyls, andthe 2- and 3-aminos of G.39 An accumulation ofhydrogen-bonded glycerol molecules in the helixH1 minor groove, which is entirely G-C, mightaccount for its unusual stabilizing effect. When the2′OH is missing from a helix (i.e. in DNA), gly-cerol is destabilizing and has no base compositionpreferences.33,40

The only other instance of saturation behavior thatwe observed was with ethylene glycol stabilizingthe tertiary fold of BWYV RNA, though in this caseit is the hydrogen bonding of loop 2 bases in thehelix H1 minor groove that is stabilized. We know ofno precedent for specific binding of ethylene glycolto RNA surfaces. The cases of these two osmolyteshaving unusually strong effects with specific RNAssuggest that RNA control elements (such as ribos-witches) could have been devised by naturalselection to sense the concentrations of specificosmolytes.

Comparison of osmolyte effects on proteins andRNA and in vivo implications

m-Values for the folding of small proteins havebeen measured for most of the osmolytes used here.The values obtained for a carboxyamidated form ofribonuclease lacking disulfide bonds (104 residues,

∼5600 Å2 folded surface area) are typical12: urea isdestabilizing, m≈2 kcal/mol M; proline and betaineare moderately stabilizing, m≈−0.6 kcal/mol M;sucrose and sorbitol are more effective (m≈−1.6kcal/mol M) and TMAO is the strongest stabilizer(m≈−2 kcal/mol M). The range of these protein m-values is comparable to the range of RNA m-values,but the specific effects are much different. Withregard to RNA tertiary structure, both betaine andproline can be destabilizing, the latter strongly so, incontrast to their consistent stabilization of proteinstructure. Sorbitol is weakly destabilizing with RNAtertiary structure, but moderately stabilizing withproteins. Only sucrose and TMAO have roughlysimilar effects on protein and RNA structure,though the magnitude of the effects of theseosmolytes on RNAs in cells will depend on thedegree to which RNA structures are neutralized byMg2+ in vivo.It has been assumed that a selective advantage for

cellular accumulation of protecting osmolytes inresponse to water stress is their compatibility withprotein structure. But some of these protectingosmolytes are clearly not compatible with RNAtertiary structure. The function of numerous crucialcellular components depends on the folding ofRNAs into specific tertiary structures and, in manycases, their ability to switch between alternativeconformations. Translational repression,41,42 ribo-switches,43 and the control of plasmid copy num-ber44 are examples of regulatory systems dependenton the proper folding of RNA tertiary structuressimilar to those used here. The spliceosome, ribo-some and other RNA-containing complexes alsorely on RNA tertiary interactions for their function.Although the effects of osmolytes on such com-plexes has not been systematically explored, it isknown that betaine stimulates translation in vitro,45

and both betaine and TMAO stimulate 50 S subunitreconstitution.46

The selective pressures on cellular accumulation ofosmolytes are probably also acting at the level ofRNA secondary structure. Hairpin formation regu-lates transcription termination47 and modulatestranslational efficiency48; stable base-pairing isrequired for the function of a host of short RNAsequences that regulate transcription, translation,and RNA modification.49 All osmolytes appear todestabilize such structures, at least weakly. Itappears that cells must regulate the concentrationsof various ions and small organic molecules in waysthat integrate their sometimes opposing contribu-tions to the stability of proteins, duplex RNA, andRNA tertiary structures.

Potential uses for osmolytes in RNA foldingstudies

In protein studies, osmolytes have been used tomeasure folding free energies,25 to force marginallystable structures to fold,50 and to probe the area andchemical nature of surfaces exposed in folding orbinding reactions.51 Several similar applications of


osmolytes to RNA folding studies are possible. First,urea has been used to measure the RNA surface areaexposed in folding intermediates28,52; its consistentdestabilization of RNA secondary structure (Figure4) and relative insensitivity to the presence ofdivalent ions (Figure 628) are useful features forthis application. Betaine has been used as a probe ofphosphate burial in protein-DNA complexes,51,53

and could potentially be used in a similar way toquantify phosphate burial in the formation of RNAstructure. These applications of osmolytes to RNAfolding are currently limited by the lack of modelsfor base and backbone exposure in U and I stateRNAs, which are needed for calculations of thechanges in SASA taking place in a folding reaction.Second, the apparent differential effect of TMAO onthe energetics of Mg2+ interactions in different RNAenvironments (Figure 7) may be a useful tool forelucidating the mechanisms by which Mg2+ stabi-lizes RNA interactions in complex RNAs. Finally,the stabilizing effects of TMAO and other osmolytescould also be useful for studying RNAs withinherently unstable structures or for extending therange of solution conditions under which an RNAfolds. Methanol has been used in this way tomaintain folding of GACG RNA with a widerrange of ion types and concentrations than other-wise possible.23

Material and Methods

Chemicals and solutions

All solutions were prepared using distilled deionizedwater at 18.3MΩ resistivity. Betaine ((carboxymethyl)trimethylammonium) monohydrate (N99% pure), D-sorbi-tol (N99.5% pure), L-proline (N99.5% pure), urea (99.5%pure), and potassium chloride (N99.5% pure) werepurchased from Fluka. TMAO (N98% pure) and glycerol(N99% pure) and Mops (99.5% pure) were obtained fromSigma. Methanol (N99.8% pure), ethylene glycol (100%)and sucrose (99.9% pure) were purchased from E.M.Science, J.T. Baker and Roche, respectively. Magnesiumchloride (N99.8% pure) was purchased from Aldrich.Because it is difficult to keep the latter salt dry duringstorage, solution concentrations were determined bytitration with EDTA according to described procedures.54All chemicals were used without further purification.The short hairpin RNA (Figure 1(a)), tar and tar* (Figure

1(b)) were purchased from Dharmacon. BWYV RNA(Figure 1(c)), the two 58mer rRNA fragment variants(Figure 1(d)) and the A-riboswitch (Figure 1(e)) wereprepared by in vitro transcription with T7 RNA polymer-ase from double-stranded synthetic DNA template(BWYV RNA) or linear plasmid DNA (58-mer and A-riboswitch RNAs) as described.16,55 RNAs were purifiedeither by chromatography under denaturing conditionson ion-exchange columns or by denaturing polyacryla-mide gel electrophoresis followed by electroelution, asdescribed.16,54

Before use, RNAs were extensively equilibrated withthe appropriate buffers, using Centricon filter units(Millipore, Billerica, MA). Osmolyte studies have var-iously used molar or molal concentration scales. Molalunits (m) are more convenient for our purposes, in which

we wish to keep both water and ion concentrationsconstant as osmolyte concentration increases, and havebeen used throughout. Mops buffer was adjusted to pH7.0 with KOH (KMops). For each RNA studied, buffers of10 mm KMops (pH 7.0) and 2 μm EDTAwith various KClor KCl/MgCl2 concentrations were used unless otherwiseindicated. Hairpin: 0.1 mm EDTA and 100 mm KCl. Tar-tar*: 400 mm KCl or 100 mm KCl and 250 μm MgCl2.BWYV RNA: 0.1 mm EDTA and 40 mm KCl or 14 mm KCland 10 μm MgCl2, except buffers for use with TMAO,which had higher buffer concentrations but the same K+

concentrations: 36 mm KMops (pH 7.0) and 30 mm KCl or10 μm MgCl2. U1061A RNA: 0.1 mm EDTA, 100 mm KCland 1.2 mm MgCl2. GACG RNA: 0.1 mm EDTA, 100 mmKCl and 3.2 mm MgCl2. A-Riboswitch RNA: 5 μm 2,6-diaminopurine (DAP), 250 mm KCl or 100 mm KCl and1.2 mm MgCl2. Experiments on osmolyte effect on Mg2+-induced stability were done using the same KMops/EDTA buffer with the following KCl concentrations:BWYV RNA: 14 mm KCl; GACG RNA:100 mm KCl; A-Riboswitch RNA: 5 μm 2,6-diaminopurine and 100 mmKCl. MgCl2 was added to these buffers as needed.All osmolyte solutions were prepared gravimetrically.

A common buffer was made to which was added preciseweights of EDTA, KCl and MgCl2 as needed. Each osmo-lyte was then weighed and added to this buffer to make astock solution. Precisely 1 ml of each solution wasweighed to determine its density. Densities were used tocalculate the amount of stock needed for the volumetricpreparation of each thermal melt analysis sample.

UV thermal analysis

Melting experiments were performed in a Cary 400spectrophotometer with 1 cm path length cuvettes. Foreach RNA, absorbance data were collected at both 260 and280 nm from 5 to 95 °C (except for the tar-tar* complex,which was collected from 2 to 95 °C) and plotted as thefirst derivative of absorbance with respect to temperature(a melting profile). To simplify data analysis, sequentialtwo-state transitions, defined by Tm, ΔH° (assumed to beindependent from temperature), and absorbance changes,were used and fit globally to both the 260 and 280 nm dataas described.56 Low temperature baselines for RNAs withtertiary structure are frequently small or zero, and only ina few cases were manually adjusted to optimize the fittedcurve. Three unfolding transitions were fitted for theBWYV RNA melting profiles, as previously reported.16,21

Three and four transitions were fitted to the U1061A andGACG 58mer rRNA fragments, respectively.20,23 Char-acteristics of the A-riboswitch in melting experimentshave not previously been reported. We find that the Tm ofthe first unfolding transition depends on the concen-tration of ligand (in this case, DAP) as expected for un-folding of tertiary structure (D. Leippley and D.E.D.,unpublished observations), and, depending on the saltand osmolyte concentrations, have fit the melting profileswith either two or three transitions to extract the Tm andΔH° of the first unfolding transition. Absorbance data forthe small hairpin, which melts in a single transition, wereanalyzed by standard methods incorporating low andhigh temperature baselines as fitted variables.57 SeeSupplementary Data Figure S1 for examples of deconvo-luted melting profiles for each RNA in the absence ofosmolytes.For each RNA, the averages and standard deviations of

Tm and ΔH° were calculated from multiple meltingexperiments in the absence of osmolyte; errors averagedabout ±0.5 deg C for Tm and ±7% for ΔH° (complete lists


are in Supplementary Data Table S1). To determine thefitted parameter errors for individual melts and to confirmthat a deconvolution of the data into unique values of Tmand ΔH° had been obtained, a bootstrap method wasused.58 In this analysis, parameters are fit to a largenumber of artificial data sets generated by a Monte Carlomethod. Statistics compiled on the fitted parametersprovide confidence intervals for each variable and detectcorrelations between variables (see Draper et al.56 forfurther details).The effect of an osmolyte on the stability of an RNAwas

calculated from ΔΔG°=(ΔH°)(T0)(1/Tm – 1/T0), whereΔH° and T0 were the enthalpy and Tm of an RNAtransition in the absence of osmolytes (SupplementaryData Table S1). Alternatively, the slope of a plot of (1/Tm)versus osmolyte molality is multiplied by (ΔH°)(T0) toobtain the so-called m-value, (∂(ΔG°)/∂mosmolyte). Thisequation assumes two-state behavior of the secondary ortertiary folding transition. There is evidence that ΔH° isaffected by some osmolytes,40 but the effects are small andwe did not see significant trends in ΔH° with osmolytemolality, within the error of the experiments. In the case ofthe hairpin, which was analyzed as a single unfoldingtransition, we also recast the melting curves as the fractionRNA folded as a function of temperature, fromwhichΔG°for folding could be obtained for a temperature rangebracketing the hairpin Tm. Thus it was possible to findΔG° versus mosmolyte at a common temperature. Thisapproach gave results in reasonable agreement with theone described above, as long as the transition regions frommelting curves for each osmolyte concentration overlapsufficiently.

Solvent accessible surface area calculations andmolecular modeling

All SASA calculations were performed using theprogram Surface Racer59 with the Richards parametersand a 1.4 Å probe radius. As a rough estimate of theaverage surface accessibility of residues in single-strandedRNA, we summed and averaged the results from fivesegments of RNA that clearly do not adopt A-form helicalstructure (residues from the following PDB files: 1ATO,9–12; 1CX0, 149–158; 1NBS, 183–190; 1U9S, 219–222 and437D, 18–21). Likewise, to evaluate the SASA of aninternal residue in a single or double-stranded RNA in A-form geometry, we used models generated using MC-Sym (3.3.2) for the helix of hairpin RNA (Figure 1(a)).60

SASA calculated for the other RNAs shown in Figure 1included all residues except for the residue at position 1(GTP) in the BWYV RNA, using the following PDBcoordinate files: 1KIS, 437D, 1HC8 and 1Y26. SASAs ofthe tar and tar* hairpins were calculated for theindividual chains extracted from the coordinate file,without attempting to re-model the hairpin loop regions.The solvent exposure of bases in these loops is thereforeunderestimated.

Acknowledgements

We thank Desirae Leippley for the plasmidencoding the A-riboswitch RNA and for samplesof purified A-riboswitch RNA, Dr Ross Shiman forhelpful advice on experimental protocols, and Dr

François Major for access to version 3.3.2 of MC-Sym. This work was supported by NIH grant 1RO1GM58545.

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2007.03.080

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Edited by J. Doudna

(Received 7 February 2007; accepted 19 March 2007)
Available online 5 May 2007

effects of osmolytes on rna secondary and tertiary structure stabilities and rna-mg2+ interactions

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