the osmolyte tmao stabilizes native rna tertiary structures in the absence of mg2+: evidence for a...

doi:10.1016/j.jmb.2010.09.043 J. Mol. Biol. (2010) 404, 138–157

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Journal of Molecular Biologyj ourna l homepage: ht tp : / /ees .e lsev ie r.com. jmb

The Osmolyte TMAO Stabilizes Native RNA TertiaryStructures in the Absence of Mg2+: Evidence for aLarge Barrier to Folding from Phosphate Dehydration

Dominic Lambert1, Desirae Leipply2 and David E. Draper1,2⁎1Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA2Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA

Received 10 July 2010;received in revised form13 September 2010;accepted 17 September 2010Available online25 September 2010

Edited by A. Pyle

Keywords:hydration;dimethylphosphate;RNA folding;linkage relations

*Corresponding author. DepartmenHopkins University, Baltimore, MDAbbreviations used: TMAO, trim

SAXS, small-angle X-ray scattering;osmometry; TLR, tetraloop receptorpotassium dimethylphosphate; SASsurface area; EDTA, ethylenediamin

0022-2836/$ - see front matter © 2010 E

The stabilization of RNA tertiary structures by ions is well known, but theneutral osmolyte trimethylamine oxide (TMAO) can also effectively stabilizeRNA tertiary structure. To begin to understand the physical basis for theeffects of TMAO on RNA, we have quantitated the TMAO-inducedstabilization of five RNAs with known structures. So-called m values, theincrement in unfolding free energy per molal of osmolyte at constant KClactivity, are ∼0 for a hairpin secondary structure and between 0.70 and1.85 kcal mol−1 m−1 for four RNA tertiary structures (30–86 nt). Furtheranalysis of two RNAs by small-angle X-ray scattering and hydroxyl radicalprobing shows that TMAO reduces the radius of gyration of the unfoldedensemble to the same endpoint as seen in titration with Mg2+ and that thestructures stabilized by TMAO and Mg2+ are indistinguishable. Remark-ably, TMAO induces the native conformation of a Mg2+ ion chelation siteformed in part by a buried phosphate, even though Mg2+ is absent. TMAOinteracts weakly, if at all, with KCl, ruling out the possibility that TMAOstabilizes RNA indirectly by increasing salt activity. TMAO is, however,strongly excluded from the vicinity of dimethylphosphate (unfavorableinteraction free energy, +211 cal mol−1 m−1 for the potassium salt), an ionthat mimics the RNA backbone phosphate. We suggest that formation ofRNA tertiary structure is accompanied by substantial phosphate dehydra-tion (loss of 66–173 water molecules in the RNA structures studied) and thatTMAO works principally by reducing the energetic penalty associated withthis dehydration. The strong parallels we find between the effects of TMAOand Mg2+ suggest that RNA sequence is more important than specific ioninteractions in specifying the native structure.

© 2010 Elsevier Ltd. All rights reserved.

t of Chemistry, Johns21218, USA.ethylamine oxide;VPO, vapor pressureRNA; K-DMP,A, solvent–accessibleetetraacetic acid.

lsevier Ltd. All rights reserve

Introduction

Osmolytes are small organic molecules that,together with ions, are accumulated or synthesizedby cells to maintain an appropriate turgorpressure.1,2 The propensity of many osmolytes toaffect protein stability has been extensivelystudied.3,4,5 So-called protecting or compatibleosmolytes tend to be excluded from solvation layersaround the protein backbone, which creates anenergetic penalty for exposing peptides to solvent

d.

http://dx.doi.org/10.1016/j.jmb.2010.09.043

139RNA Stability in TMAO-KCI Solutions

when high concentrations of osmolyte are present.These osmolytes, therefore, favor protein foldinginto more compact structures with buried (solvent-inaccessible) backbone.6–9 The protecting osmolytetrimethylamine oxide (TMAO) is particularly effec-tive in forcing thermodynamically unstable pro-teins to fold into native, functional structures.10,11In a survey of osmolyte effects on RNA stability,we found that TMAO effectively stabilized all theRNA tertiary structures examined, but had a smalldestabilizing effect on secondary structure.12 Wehypothesized that TMAO stabilizes RNA by ananalogous mechanism as deduced for proteins, viz.,unfavorable interactions between TMAO and thesugar–phosphate backbone stabilize more compactconformations of an RNA.Here, we quantitate the stabilization of four

different RNA tertiary structures by TMAO in KClsolutions and, in closer examination of two of theseRNAs, find strong parallels between the effects ofMg2+ and TMAO: both promote more compactconformations of partially unfolded RNAs andultimately stabilize the identical tertiary structure.While Mg2+ favors compact RNA conformations byreducing electrostatic repulsion between phos-phates, we suggest that TMAO reduces the energeticpenalty associated with phosphate dehydration.This suggestion is supported by our measurementof a strong exclusion of TMAO from the vicinity ofan RNA backbone mimic, dimethylphosphate, andestimates of the number of hydrating water mole-cules that are released upon folding of RNA tertiarystructures. Our conclusions emphasize the role ofphosphate desolvation in the formation of RNAtertiary structure and the dominant role of sequence(rather than specific ion interactions) in specifyingthe native structure.

Background

Linkage relations for salt and osmolyte effectson RNA stability

RNA stability is very sensitive to salt concentra-tion. Osmolytes therefore have the potential to shiftRNA folding equilibria in two ways: directly, byfavorable or unfavorable interactions with the RNAitself, and indirectly, via osmolyte–ion interactionsthat change the effective concentration of the ions. Inthis section, we summarize the thermodynamicframework we use to distinguish the effects of saltand osmolyte on RNA folding. Note that we usemolal concentrations throughout this work, ratherthan the more familiar molar units. One reason fordoing so is that water concentration is a constant inmolal units. Titration of osmolyte into a salt solutionof constant molality therefore does not change themole ratio of salt to water.

We define an observed equilibrium constant forRNA unfolding as Kobs=mU/mF, where mF and mUare molal concentrations of folded and “unfolded”RNA conformations, respectively. The equilibriummay be between a duplex and a single-strandedRNA or between the native tertiary structure and apartially unfolded RNA with substantial secondarystructure. (Partially unfolded states of an RNA havesometimes been called intermediate or I states todistinguish them from a single stranded, fullyunfolded conformation.13 For convenience, we use Uto refer to either partially or fully unfolded RNAs,depending on the folding reaction under consider-ation.) The solutes of interest here, salt and osmolyte,affect Kobs because of their differential interactionswith folded and unfolded states of the RNA. All ionsinteract with RNA by long-range electrostatic forces,attractive for cations and repulsive for anions,although other factors (such as hydration changes)may also come into play for cations. Osmolytescompete with water for solvation of the RNA; theymay be either accumulated or excluded from theRNAsurface depending on whether the affinity of differentRNA groups (bases, sugars, phosphates) is greater forosmolyte or water, respectively. For either ions orosmolytes, it is convenient to characterize the RNA–solute interaction in terms of an interaction coefficient,

Gs =Ams

AmRNA

� �As

ð1Þ

wherems is the molality of a solute,mRNA is the RNAmolality, and μs is the solute chemical potential. [InEq. (1) and all following partial derivatives, temper-ature and pressure are also held constant.] The solute smay be the cation, anion, or osmolyte (here abbrevi-ated +, −, or TMAO). Each Γs corresponds to thechange in molal concentration of solute necessary tomaintain its chemical potential (μs) at a constant valuewhen the concentration of the RNA changes.Linkage relations14,15 equate changes in the

interaction coefficient with the sensitivity of thefolding reaction to solute activity (as). For solutionscontaining TMAO and KCl, the relations are

aTMAOð Þ Aln Kobsð ÞAaTMAO

� �aKCl

= GUTMAO − GF

TMAO =DGTMAO

ð2aÞ

Aln Kobsð ÞAlnaF

� �aTMAO

= GUþ + GU

−� �

− GFþ+ GF

−� �

= 2DGF

ð2bÞwhere a± is the mean ionic activity of KCl and thesuperscripts F andUsignify foldedandunfolded formsof an RNA, as defined above. [Kobs for a nucleic acidconformational change tends to follow a power lawwith respect to its dependence on salt activity, which is

140 RNA Stability in TMAO-KCI Solutions

conveniently expressed as the log–log relation in Eq.(2b); osmolyte effects on ln(Kobs) tend to be approxi-mately linear in osmolyte activity.] The ΔΓ terms maybe thought of as the uptake or release of TMAO or KClthat accompanies the RNA folding reaction. Note thatboth cations and anions participate in the foldingreaction; the term 2ΔΓ± is used to emphasize that theremust be identical uptake or release of cations andanions to maintain charge neutrality.16

RNA stability may be measured as a function ofTMAO concentration at a constant KCl concentration,or vice versa, but Eqs. (2a) and (2b) are in terms ofTMAOandKCl activities. To use the linkage relations,we have to first relate changes in concentration [e.g.,∂ln(mKCl)] to changes in activity [∂ln(aKCl)] and thentake into account any mutual interaction betweenTMAO and KCl that would cause the activity of KClto change when its concentration is held constantand TMAO is titrated [Eq. (2a); there is a reciprocalproblem in maintaining constant aTMAO in Eq. (2b)].There are limiting cases in which we are able to applythe linkage relations to experimental data. One is thesalt dependence in the absence of TMAO:

2DGF=Aln Kobsð ÞAln mKClð Þ

� �mTMAO =0

Aln mKClð ÞAlnðaFÞ

� �=

SKobs

1+eF

� �

ð3ÞSKobs is the experimentally determined dependence

of ln(Kobs) on ln(mKCl); it is constant within experi-mental error with the RNAs and salt concentrationranges used here (0.025–0.8 m). ɛ± is related to thedependence of the KCl activity coefficient on KClconcentration; from literature data, a value of −0.106 isappropriate for KCl concentrations up to ∼0.5 m,and −0.107 for concentrations between 0.1 and 0.8m.16

Two useful quantities are derived from a limitingvalue of ΔΓTMAO. We first assume (following thework of Hong et al.17,18) that the activity coefficientof TMAO is constant with respect to TMAOmolalityin the limit of low TMAO concentration (but stilllarge excess over RNA). The activity coefficient thenfactors out of Eq. (2a) when mTMAO→0,

DGTMAO

mTMAO=

Aln Kobsð ÞAmTMAO

� �aKCl

ð4Þ

It is still necessary to relate the derivative at constantsalt activity to one at constant concentration. Anadditive correction factor (derived in Refs. 17 and 18)accounts for any effect of TMAO on the KCl activity:

DGTMAO

mTMAO=

Aln Kobsð ÞAmTMAO

� �mKCl

+SKobsμKCl�TMAO

2RT 1 + e Fð Þ� �

ð5aÞADG0

obs

AmTMAO

� �aKCl

=ADG0

obs

AmTMAO

� �mKCl

−SKobsAKCl�TMAO

2 1 + e Fð Þ� �

ð5bÞ

μKCl–TMAO is the chemical potential derivative thatquantifies interactions between KCl and TMAO;its measurement by osmometry is described below.(The limit mTMAO→0 applies to the value used forμKCl–TMAO.) Equation (5a)wasmultiplied by−RT (R isthe gas constant, T the temperature) and combinedwith Eq. (4) to put the expression in terms of theobserved folding free energy ΔG°obs [Eq. (5b)]. Thisform of the equation is the same as the “m value”frequently used to report the effects of osmolytes onprotein stability, with the exception that molalconcentrations are used here instead of the morecommon molarities.5,8,19

Protecting osmolytes such as TMAO tend to beexcluded from the surface of a protein or RNA (Γs isnegative). If an osmolyte is so strongly excluded thatthe concentration of osmolyte within a solvationlayer around the macromolecule is negligiblecompared to osmolyte concentration in bulk solvent,then it is simple to relate the number of excludedosmolyte molecules Γs to the number of watermolecules from which the osmolyte is excluded, B1:

Gsc − B1 ms=mwaterð Þ ð6ÞThis formula has been derived in several different

ways (see Ref. 15 for references and comments).Combining Eqs. (5a) and (6), the release or uptake ofwater of solvation associatedwith a folding reaction is

DB1c − 55:5ð Þ DGTMAO

mTMAO

� �ð7Þ

where 55.5 is the molality of water. As with Eq. (4),Eq. (7) applies in the limit mTMAO→0. ΔB1 will belarger than calculated by Eq. (7) if TMAO is notcompletely excluded from solvation layers aroundthe RNA.

Measurement of TMAO–solute interactions byvapor pressure osmometry

Vapor pressure osmometry (VPO) measures thepartial pressure of water in equilibrium with asolution. The result of the measurement is reportedas the solution osmolality,

Osmu − m•waterlnðawaterÞ ð8Þ

where m·water≡55.5 mol/kg, the molality of water,and awater is the thermodynamic activity of water inthe solution being measured, as derived from theratio of the partial pressure of the solution to that ofpure water. Osmolality is identical to molality if thesolutes present in solution behave ideally; that is, theactivity coefficient of each solute γi =1. According tothe Gibbs–Duhem equation for a two-componentsolution, any deviation from ideal behavior of onecomponent is accompanied by a reciprocal deviationby the other component. Thus, the dependence of

Fig. 1. Schematics of the secondary and tertiary struc-tures of the RNAs used in this study. Horizontal black barsand bullets representWatson–Crick and noncanonical basepairs, respectively. Gray lines symbolize base–base tertiaryinteractions and black lines with arrowheads represent 5′–3′ backbone connectivity. (a) A designed hairpin. (b) The


the solution osmolality on solute concentration canbe used to calculate the solute activity coefficient.20

In solutions with several solutes, the deviation ofthe measured osmolality from ideality containsinformation about interactions between all thedifferent solutes. In principle, the strengths of all theinteractions can be quantified from water vaporpressure measurements alone, as long as a largeenough matrix of solute concentrations is measuredto high enough accuracy.21 As a practicalmatter, onlypairwise interactions in a three-component system(water plus two solutes) can reliably be extracted. Thedata analysis needed to obtain the relevant para-meters has been discussed extensively.22–24 Briefly,the osmolality (Osm) of solutions with varyingconcentrations of two solutes is compared with theosmolalities of solutions containing only one solute toobtain the difference ΔOsm,

DOsm = Osm m2;m3ð Þ − Osm m2ð Þ − Osm m3ð Þ ð9Þwhere the subscripts 2 and 3 refer to the two solutes(water is component 1). The chemical potentialderivative μ23 is defined and related to ΔOsmmeasurements by24,25

l23uAl2Am3

� �m2

cRTDOsmm2m3

ð10Þ

μ23 can be decomposed into two terms,

lex23 = l23 − lmix23 ð11Þ

lmix23 is derived from the ideal mixing entropy and is

independent of the chemical nature of the solutes; theexcess potential lex23 originates in the interactionsbetween solutes that cause the solution to deviatefrom thermodynamic ideality.23,25 For the concentra-tion ranges of solutes used in the VPO experimentsreported here, the contribution of the mixing entropyis small, lmix

23 /RT=-0.0174 for neutral solutesand −0.0342 for the potassium salt of dimethylphos-phate (K-DMP) [see Eq. (5) of Ref. 25].

tar–tar⁎ kissing loops complex. (c) Nucleotides 1051–1108of the Escherichia coli 16S rRNA, with a mutation (U1061A)that stabilizes tertiary structure37 (58mer RNA). (d) Theaptamer domain of the adenine-binding riboswitch withadenine ligand depicted in outline typeface (A-riboswitch).(e) The tetraloop–receptor RNA (TLR).

Results

RNAs selected for this study

Five RNAs (Fig. 1) were chosen to investigate theeffects of TMAO on structure stability. A shorthairpin was designed to be a representative secon-dary structure with both A:U and G:C base pairs(Fig. 1a). The other four RNAs all contain naturallyoccurring tertiary-structure elements and wereselected on the basis of two criteria. First, in orderto measure the effect of various concentrations ofTMAO and KCl on the RNA stability, the tertiaryfolding transition had to be resolvable in the UVmelting profile of each RNA. Second, the RNAs

were chosen to demonstrate a range of foldingstrategies as suggested by differences in theirstructural complexity, their ionic requirements forfolding, and the degree to which the phosphatebackbone is solvent-inaccessible in the native struc-ture. Three of the RNAs adopt native structuresunder moderate monovalent salt conditions in theabsence of Mg2+: the tar–tar* kissing loop is a smallcomplex of two RNA hairpins (Fig. 1b);26 a homo-dimeric tetraloop–receptor complex (TLR RNA)

Fig. 2. Salt and TMAO dependences of tertiary struc-ture stability of the A-riboswitch aptamer domain, plottedas (1/Tm) obtained from melting experiments. The twopanels show the same set of 30 different conditions. Thedrawn curves are least-squares best fits all derived fromthe same three-dimensional surface specified by six fittingparameters (see Materials and Methods). (a) KCl depen-dence at fixed TMAO concentrations: blue, no TMAO;green, 0.4 m; orange, 0.8 m; brown, 1.284 m; dark brown,1.6 m; red, 2.0 m. (b) Data from (a) replotted to showTMAO dependence at fixed K+ concentrations: blue,54 mm; green, 104 mm; orange, 204 mm; brown, 304 mm;red, 404 mm. Each melt sample contained 50 mm K-Mops(pH 7.0), 5 μm 2,6-diaminopurine, and 2 μm EDTA alongwith additional KCl and TMAO to give the indicatedconcentrations. Error bars are smaller than the size ofdata points.


reproduces a common tertiary structural motif (Fig.1e)27,28 and is selectively stabilized by K+ binding ata chelation site;29 and the aptamer domain of theA-riboswitch contains two sets of tertiary inter-actions (Fig. 1d),30,31 one ofwhich is formed around apurine ligand. The fourth RNA, a 58-nt fragment ofribosomal RNA (58mer RNA, Fig. 1c) is stabilized bychelation of both K+ and Mg2+ and stronglydependent on Mg2+ for stability.32,33 This rRNAfragment is also themost compact RNA in this study,in the sense that it exposes less surface area to solvent(per nucleotide) than any of the others.12

TMAO and salt dependences of foldingfour RNAs

We used UV melting profiles to follow the effectsof TMAO on RNA stability12 (see Materials andMethods for details). To allow for the possibility ofsynergistic or antagonistic effects between salt andTMAO, we performed melting experiments in thepresence of increasing KCl molality at various fixedTMAO concentrations (Fig. 2, and see Supplemen-tary Information). To keep the analysis confined to atwo-dimensional matrix of salt versus TMAO con-centrations, we did not include Mg2+ in any of theseexperiments. Four of the RNAs in Fig. 1 wereinvestigated this way. More extreme TMAO andsalt concentrations are needed to fold the 58merrRNA in the absence of Mg2+; it is consideredseparately below.After the melting temperature (Tm) of the first

(tertiary) melting transition from the melting pro-files was extracted, data for the A-riboswitch show atypical semilogarithmic dependence of (1/Tm) onthe KCl molality (Fig. 2a). The dependence of (1/Tm)on TMAO molality is best described by a second-order polynomial (Fig. 2b), with higher TMAOconcentrations being proportionately less stabiliz-ing, per molal of osmolyte. The entire TMAO–saltdata set was globally fit to one set of six parameters(see Materials and Methods). In the analysis thatfollows, (1/Tm) was transformed into ln(Kobs) usingan average ΔH° of folding obtained by analysis ofthe melting curves (Table 1; see Materials andMethods). Kobs is the observed equilibrium constantfor an unfolding transition; therefore ln(Kobs)decreases as an RNA becomes more stable. In thelimit of low TMAO concentrations, three empiricalquantities derived from the parameter set are ofinterest (Table 1): the dependence of ln(Kobs) on saltconcentration in the absence of TMAO, called SKobs[Eq. (3)]; the dependence of ln(Kobs) on TMAOmolality at a fixed 200 mm KCl concentration, [∂ln(Kobs)/∂mTMAO]mKCl

; and an interaction term thatgives the effect of TMAO (in the limit of lowconcentrations) on SKobs. Results for the tar–tar* andTLR RNAs (Supplementary Fig. S1) were qualita-tively similar to those shown for the A-riboswitch;

all three of these RNAs are stabilized by TMAO(Table 1). TMAO had only a slight destabilizingeffect on the hairpin, and the salt dependence of thehairpin stability was virtually unaffected by TMAO(Table 1).Two thermodynamic quantities listed in Table 1

are derived from the empirical parameters anddescribe the sensitivity of the RNA folding reactionto either salt or TMAO. One is 2ΔΓ±, the number

image of Fig. 2

Table 1. Parameters for KCl and TMAO effects on RNA unfolding reactions

RNA

UnitsHairpin A-riboswitch Tar–tar⁎ TLR

SKobsa −0.59±0.08 −4.03±0.49 −2.13±0.19 −3.99±0.40 —

[∂ln(Kobs)/∂mTMAO]mKCl

b 0.09±0.06 −3.12±0.41 −1.19±0.14 −2.47±0.27 m−1

Salt–osmolyte interactionc — 0.90±0.13 0.38±0.06 1.00±0.11 m−1

ΔH°d 54.7±3.9 63.2±7.7 29.2±2.6 30.0±3.0 kcal/mol2ΔΓ±

e −0.66±0.08 −4.50±0.55 −2.38±0.21 −4.47±0.45 ions per RNA(∂ΔG°/∂mTMAO)aKCl (m value)f −0.05±0.04 1.85±0.25 0.70±0.08 1.46±0.16 kcal mol−1 m−1

ΔB1g −5±3 173±23 66±8 137±15 H2O per RNA

The first three rows of tabulated parameters were derived from sets of Tm values derived frommelting curves done in amatrix of KCl andTMAO concentrations. The hairpin RNA parameters were obtained by linear least-squares fitting at various fixed TMAO concentrationsand the assumption of a zero salt–osmolyte interaction term; data sets for the other RNAs were globally fit to a single equation. SeeMaterials and Methods for further details. Errors include the error associated with the global fit analysis and the uncertainty in ΔH°.

a ∂ln(Kobs)/∂ln(mKCl) evaluated in the absence of TMAO (coefficient a0 multiplied by (ΔH°/R); see Eq. (12) of Materials and Methods).b Derivative evaluated at 200 mm KCl in the limit of zero TMAO [Eq. (14)].c Coefficient a1 multiplied by (ΔH°/R); see Eq. (12) and Materials and Methods.d Unfolding transition ΔH° and the associated error were obtained by averaging values returned in fitting two-state transitions to

melting profiles (see Materials and Methods); the exception is the TLR RNA, for which ΔH° from scanning calorimetry experiments waspreviously reported.12

e Derived from SKobs by Eq. (3).f Dependence of the folding free energy on TMAOmolality at constant KCl activity in the limit of low TMAO concentration, called the

m value in the text [see Eqs. (4) and (5)].g ΔB1 is the minimum number of water molecules that become inaccessible to TMAO upon unfolding of the RNA [Eq. (7)].


of ions released upon unfolding [Eq. (3)] in theabsence of TMAO. The other quantity, (∂ΔG°obs/∂mTMAO)aKCl, is similar to the m value standardlyused to characterize the effects of osmolytes onprotein folding [see Background, Eq. (5b)], butdiffers in being expressed in molal instead ofmolar concentration units. In calculating the mvalue, an additive correction term is needed totransform the TMAO dependence of the RNAstability measured at constant KCl concentration tothe same dependence taken at constant KCl activity[Eqs. (5a) and (5b)]. Measurement of the TMAO–KClinteraction energy necessary to calculate this correc-tion is considered below; the term makes anegligibly small contribution to the reported mvalues, on the order of 0.001 kcal mol−1 m−1.(About 10% of the reported m value errors reportedin Table 1 comes from estimated errors in themeasurement of the TMAO–KCl interaction energy.)The m values we measure are qualitatively differentbetween the hairpin secondary structure (∼0 kcalmol−1 m−1) and the RNAs with tertiary structure(0.70 to 1.85 kcal mol−1 m−1; Table 1). For compar-ison, somemolarm values for TMAO stabilization ofproteins are 1.64 kcal mol−1 m−1 (reduced ribonu-clease A, 124 residues) and 2.37 kcal mol−1 m−1

(staphylococcal nuclease, 149 residues).10 On a perresidue basis, TMAO is a more effective stabilizer ofRNAs than proteins.In all three RNAs with tertiary structure, the

positive salt–TMAO interaction factor (Table 1)means that TMAO concentration reduces the mag-nitude of ion release (2ΔΓ±). We attribute thisreduction to an effect of TMAO on the extension ofthe partially unfolded RNA in solution, bringing it

closer to the native-state charge density (see Discus-sion). Lastly, the estimated number of solvatingwater molecules that are taken up when the RNAunfolds is derived from the limiting value of ∂ln(Kobs)/∂mTMAO by Eq. (7) (Table 1). The calculationassumes that TMAO is strongly excluded from thesewater molecules, a point that is considered in theDiscussion.

Isothermal determination of the A-riboswitchm value

The derivation of the thermodynamic parametersin Table 1 from Tm data assumes that the parametersare temperature independent. The ΔH° values forRNA unfolding derived from the melting profiles donot show any trend with Tm or TMAO concentra-tion, within error, but the possibility remains thatTMAO could become a more (or less) effectivestabilizer at higher temperatures. For the A-riboswitch, we found salt (50 mm K+) and ligand(10 μm adenine) concentrations at which the RNAis predominantly in the unfolded form, but folds tothe native structure upon titration with TMAO atconstant temperature (22.5 °C) (Materials andMethods and Supplementary Fig. S2). The m value(0.69±0.04 kcal mol−1 m−1) at the midpoint of thetitration (1.47±0.31 m TMAO) was determined bystandardmethods.19 (The large error in themidpointcomes from uncertainties in baselines overthe accessible range of TMAO concentrations.)From the global fit analysis of Tm data (Fig. 2), them value at the same salt and TMAO concentrations is0.92±0.36 kcal mol−1 m−1, which is higher than theisothermally determined value but within

Fig. 3. Melt profiles of 58mer RNA in various TMAOand salt concentrations. (a) Data sets were collected at 260(blue) and 280 (red) nm in buffer containing 400 mm K+, 2μm EDTA, and either 100 mm K-Mops (pH 6.8) with 4 mTMAO (light shades), or 160 mm K-Mops (pH 6.8) and 6mTMAO (dark shades). (b) Data were collected at 260 (blue),280 (red), and 295 (purple) nm in the presence of 52.4 mmK+, 2 μm EDTA, 182 mm K-Mops, and 7.7 m TMAO.Arrows indicate the change in ratio of absorbance at 260/280-nm (A) or 295-nm signal (b) corresponding to meltingof tertiary structure.


experimental error. The overall range of Tm dataused in the global fit analysis is 17–54 °C, althoughthe interpolated m value for 50 mm K+ and 1.47 mTMAO is most strongly affected by Tms near 30 °C.We conclude that the m values compiled in Table 1are not very strongly biased by temperature effects,although it is still possible that m values varysignificantly over the full temperature range coveredby the data in Fig. 2.

TMAO dependence of 58mer RNA stability

Among the RNAs in Fig. 1, the 58mer rRNAfragment (Fig. 1c) is unique in having a chelatedMg2+ buried within its structure.34 In the absence ofMg2+, the RNA folds only under extreme conditions,for example, 1.6 M NH4Cl.

35,36 We find that highconcentrations of TMAO can substitute for Mg2+ inpromoting the native 58mer RNA structure (Fig. 3).The tertiary unfolding transition in this RNA ischaracterized by an increase in absorbance at260 nm, a minimal change at 280 nm, and a decreaseat 295 nm.33,37 In 400 mm K+ and 4 m TMAO, thetertiary-structure unfolding is apparent as a re-solved transition at ∼30 °C (Fig. 3a). Raising theTMAO concentration to 6 m simultaneously raisesthe Tm of the tertiary transition and decreases thestability of the higher-temperature secondary-struc-ture transitions. The tertiary structure no longerunfolds as a distinct transition, although the smalldisplacement of the peaks at 260 and 280 nmindicates that tertiary unfolding precedes subse-quent unfolding steps. At higher concentrations ofTMAO, most of the RNA unfolds in a singletransition (Fig. 3b); the presence of a hypochromicchange at 295 nm confirms the existence of thetertiary structure under these conditions (7.7 mTMAO, 52.4 mm K+). As the K+ concentration israised, less TMAO is needed to induce formation ofthe tertiary structure; for example, at 1 m K+, only∼3 m TMAO is needed to see the tertiary unfoldingtransition (Tm ∼35 °C, data not shown).By assuming that the enthalpies of the unfolding

transitions do not change with salt or osmolyteconcentration, we extracted ΔG° for tertiary-structure unfolding from melting data obtainedbetween 4 and 6 m TMAO (see Materials andMethods). At a constant 400 mmK+, ∂ΔG°/∂mTMAOfor RNA unfolding is approximately 1.1 kcal mol−1

m−1; at 600 mm K+, it is 1.4 kcal mol−1 m−1. Thesem values are not exactly the same as the free-energy derivative reported for the other RNAs inTable 1, as they have not been extrapolated to thelimit of low TMAO concentrations. Because wehave seen with other RNAs that higher concentra-tions of TMAO tend to be less effective on a permolal basis, these numbers may underestimate themagnitude of the osmolyte stabilizing effect withthe 58mer RNA.

TMAO–solute interactions

To ask if the observed stabilizing effect of TMAOon RNA tertiary structures can be interpreted interms of TMAO interactions with specific groups,we used VPO to measure the interactions betweenTMAO and several small molecules mimickingRNA bases, ribose sugar, or phosphate backbone.The methodology is described in the Backgroundsection and developed in detail elsewhere (see Ref.24 and references therein). Figure 4a shows typicalVPO data obtained with K-DMP, which wassynthesized to reproduce an isolated phosphodie-ster of the RNA backbone (see Materials andMethods). The VPO instrument reports the

image of Fig. 3

Fig. 4. VPO measurements and analysis. (a) Osmolalityof K-DMP with (from bottom to top) 0, 0.5, or 1.0 mTMAO. Fitted curves are second-order polynomials. Errorbars are shown, but are generally smaller than the datapoints. (b) The nonadditivity of TMAO and soluteosmolalities (ΔOsm) plotted according to Eqs. (7) and(8). Lines are linear least-squares fits; the slopes arereported in Table 3 as μ23/RT. Blue, TMAO and K-DMAP;green, TMAO and KCl; red, TMAO and glycerol.


osmolality of a solution, which is the concentrationof an ideal solute that would be required to producethe observed water vapor pressure. For K-DMP,there is a nearly linear dependence of the osmolalityon the actual solution molality (Fig. 4a). WhenTMAO is included, the slope of the plot becomesslightly steeper, which indicates a repulsive (unfa-vorable) interaction between TMAO and the K-DMPions; that is, TMAO and K-DMP interact morestrongly with water than with each other. Aconvenient way to show these small differencesand quantify the strength of the interaction is theplot in Fig. 4b; the slope of the plot is the quantityμ23/RT as defined in Eq. (10) (Table 2). Theinteraction free energy between TMAO andK-DMP is given by the related quantity lex23, theexcess chemical potential derivative [Eq. (11)].

TMAO is found to have a strongly unfavorableinteractionwith K-DMP, 211 calmol−1m−1 (Table 2).This positive free energy is especially notable incomparison to the weak overall interactions ofTMAO with KCl (Table 2 and see below). TMAOmust have a much more unfavorable interactionwith the DMP− anion than with Cl−.Additional VPO measurements were made with

compounds representative of the base and sugarcomponents of an RNA (Table 2). A base solubleenough for VPO measurements is purine,38 whichhas a significant unfavorable interaction withTMAO. The nucleosides cytidine and uridine alsoshow positive interaction free energies (Supplemen-tary Fig. S3). Lastly, glycerol was examined as amodel for TMAO interactions with the ribose sugar;it has small but favorable interactions with TMAO(Table 2 and Fig. 4b).For comparison, free energies for the transfer of

amino acid side chains from water to 1 m TMAO,based on solubility measurements reported byAuton and Bolen,8 are included in Table 2. Thesefree energies are similar to the chemical potentialderivatives obtained from VPO experiments. Inter-actions with aliphatic and aromatic carbon atomsare weak, but there are strongly favorable interac-tions with tryptophan and tyrosine side chains. Allthe data in Table 2 can be qualitatively rationalizedby the assumptions that net favorable interactionstake place between TMAO and hydrogen bonddonors, while TMAO is strongly excluded (unfa-vorable, positive free energies) from the vicinity ofhydrogen bond acceptors. For instance, tryptophanand tyrosine side chains and purine all have onehydrogen bond donor, but purine also has threehydrogen bond acceptors and, thus, a net unfavor-able interaction with TMAO. The relation betweenthis pattern of TMAO interactions and RNA helixstability is elaborated in the Discussion.

TMAO–KCl interactions are weak

RNA stability is strongly dependent on thethermodynamic activity of salt present in solution;therefore, any interaction of TMAO with salt willshift the RNA folding equilibrium simply because ofthe change in salt activity. Based on VPO measure-ments (Fig. 4a), μ23 for TMAO–KCl interactions isnot significantly different from zero (Table 2), andno correction need be made to (∂ΔGobs/∂mTMAO)mKCl

to obtain the m value [Eq. (5b)]. This result isconsistent with the weak interactions measuredbetween glycine betaine, an osmolyte that ischemically similar to TMAO, and KCl (28.4 calmol−1 m−1) or NaCl (−43.8 cal mol−1 m−1).24 Weconclude that essentially all the effects of TMAO onRNA stability that we measure (Table 1) originatefrom favorable and unfavorable interactions of theosmolyte with the RNA.

image of Fig. 4

Table 2. TMAO–solute interaction free energies

Solute μ23/RT (m−1)lex23 (25 °C)

(cal mol−1 m−1)

Purine 0.1425±0.018 94.7±10.6Cytidine 0.161±0.024 106±14Uridine 0.145±0.017 96±10Glycerol −0.0503±0.0072 −17.3±4.2K-DMP 0.322±0.027 211±16KCl −0.0018±0.015 —

Amino acid side chain ΔGtransfer(cal mol−1 m−1)a

Ala −13.6Leu 10.8Phe −8.65Trp −141.8Tyr −106.0His 39.0Arg −101.4

μ23/RT is the slope of ΔOsm [Eq. (7)] plotted as in Fig. 4b. lex23 iscalculated from μ23/RT at 25 °C and includes a correction for theideal entropy of mixing. See Background [Eqs. (7)–(9)] andMaterials and Methods for details.

a Transfer free energies based on relative solubilities of aminoacids in water and 1 M TMAO, taken from Ref. 8. The transfer freeenergy of glycine has been subtracted, so ΔGtransfer applies to theamino acid side chain. The partial molar volume of TMAO68 wasused to transform ΔGtransfer from molar to molal units.


TMAO reduces the overall dimensions ofpartially unfolded RNAs

Protecting osmolytes such as TMAO reduce theradius of gyration (Rg) of denatured proteins.39 TheRg of the ensemble of RNA structures from whichfolding takes place is also sensitive to solventconditions, notably to the concentration of Mg2+,but also to monovalent ions.40–44 To determinewhether TMAO also affects the overall dimensionsof partially folded RNAs, we made small-angleX-ray scattering (SAXS) measurements on two of theRNAs in Fig. 1, the A-riboswitch and the 58mer

Fig. 5. Dimensions of A-riboswitch and 58mer RNAs atvarious TMAO andMg2+ concentrations, as determined bySAXS. (a) Rg of the A-riboswitch wild-type sequence (red)and two folding-impaired variants, C60G (orange) andG28C (green), in 182 mm K-Mops (pH 6.8), 2 μm EDTA,50 mm K+, and various molalities of TMAO. For compar-ison purposes,Rg in the same buffer containing 1mMMg2+

(black point) is also indicated. (b) Distance distributionfunctions of theA-riboswitchwith 8mTMAO (red) or 1mmMg2+ (black) and of the G38C (green) and C60G variants(orange) with 8m TMAO. Other buffer components are thesame as in (a). (c) Rg of the 58mer RNA (blue) in 182 mmMops (pH 6.8), 2 μm EDTA, 50 mm K+, and variousmolalities of TMAO. For comparison purposes, Rg in thesame buffer containing 0.1 mm Mg2+ (black) is alsoindicated. In both (a) and (c), error bars are smaller thanthe size of the data points. (d)Distancedistribution functionof the 58mer rRNA in 2 m TMAO (light blue), 4 m TMAO(medium blue), 8 m (dark blue), and 0.1 mm Mg2+ (black).Other buffer components are the same as in (c).

rRNA fragment. In the absence of TMAO, the Rg ofthe A-riboswitch is nearly 25 Å. Increases in TMAOconcentrations reduce Rg to 20.6 Å, nearly the sameradius as observed when 1 mM MgCl2 is addedunder the same buffer conditions (50mMK+; Fig. 5a)and close to the Rg calculated from the A-riboswitch

image of


crystal structure (20.3 Å). (Because scattering mea-surements were made in the absence of ligand, thecompact A-riboswitch structures we observe cannothave the native conformation of the purine-bindingpocket. The tertiary contacts between the twohairpin loops nevertheless form when millimolarconcentrations of Mg2+ are present.31) Two A-riboswitch variants were also assayed, G38C andC60G (see sequence in Fig. 1d). These mutationsdisrupt the formation of a base pair between thehairpin loops and strongly destabilize the tertiarystructure (Ref. 45 and unpublished data). In theabsence of the osmolyte, these two variants displayradii of gyration identical to that of the wild-typeRNA (25 Å), but even at 8 m TMAO their radii areunchanged (G38C) or only slightly reduced (C60G).Thus, it appears that TMAO only reduces thedimensions of the A-riboswitch RNA if the correcttertiary structure can form. TMAO also reduces thedimensions of the 58mer rRNA fragment fromaround 25 Å to 18.2 Å, a radius similar to the oneobserved in the presence of 100 μmMg2+ in the samebuffer (60 mm K+) (Fig. 5b).The distance distribution profile derived from the

scattering profile contains more information aboutthe shape of the RNAs than Rg, which is derivedfrom only the smallest scattering angles. The wild-type A-riboswitch profiles are very similar whethera compact form was induced by the presence ofTMAO or Mg2+; the variant sequences show abroader distribution of distances (Fig. 5c andSupplementary Fig. S4). Similarly, the 58mer rRNAstructures stabilized by 8 m TMAO or 100 μm Mg2+

are nearly indistinguishable (Fig. 5d).

Hydroxyl radical probing of TMAO-stabilizedtertiary structures

To further compare the Mg2+- and TMAO-stabilized structures of the A-riboswitch and 58merRNAs, we probed the accessibility of the backbone tohydroxyl radical. This free radical reacts with DNAand RNA sugar moieties and serves as a probe ofsolvent accessibility to the backbone at nucleotideresolution; tertiary-structure formation is usuallyassociated with reduced ribose reactivity.46,47 In thecase of the A-riboswitch, sugar moieties at positionsassociated with the interacting hairpin loops (33, 37,38) and the adenine-binding pocket (46–49, 52 and53) all become similarly protected from reactionupon addition of either 5 mm Mg2+ or 8 m TMAO;5m TMAO is almost identically effective (Fig. 6a andb). (Because ligand was not included in theseexperiments, the organization of the binding pocketregionmay not be the same as observed in the crystalstructure.) In a similar way, the 58mer rRNAfragment shows protection of a number of ribosesassociated with tertiary structure (18–21, 35–37, 45–47) (Fig. 6c and d). There is a clear trend of increasing

protection as a function of TMAO concentration(most easily observable for residues 18–21); anessentially identical reactivity pattern is seen witheither 8 m TMAO or 5 mm Mg2+ (60 mm K+).

Monovalent ion specificity of 58mer RNA folding

The 58mer RNA is selectively stabilized by K+ inpreference to larger or smaller group I ions,36 mostlikely reflecting the specificity of the K+ chelation siteseen in the crystal structure (Fig. 7a).32 The bindingpocket for this ion is part of an unusual tertiarystructure in which four phosphates are turnedtoward the RNA interior and line cavities containingchelated Mg2+ and K+ ions; A1073 contributes oneanionic oxygen to each chelation site. Thus, it was ofinterest to know whether the monovalent ionspecificity would persist when the tertiary structureis stabilized by TMAO, or whether the lack of achelated Mg2+ would cause a rearrangement of theburied phosphates. We observe a similar preferencefor K+ over most other group I ions, whether thestructure is stabilized by TMAO or byMg2+ (Fig. 7b).(Na+ is significantly more effective with the TMAO-stabilized structure; perhaps the ion is able to bindspecifically in or near the Mg2+ chelation site.) Thisresult is consistent with the same protection of A1073from hydroxyl radical reaction in either TMAO- orMg2+-stabilized RNAs.

Discussion

Effects of TMAO on RNA structure and stability

TMAO is an effective stabilizer of protein nativestructure;6,9 it also favors more compact (smaller Rg)conformations of unfolded proteins.39 In this work,we find that TMAO has similar effects on RNAswith tertiary structure. Before discussing theseeffects, we note an important control observation:the activity of KCl is negligibly perturbed by TMAO(Table 2). Therefore, the effects of TMAO on RNAstability are not an indirect effect of changes in the“effective concentration” (thermodynamic activity)of the salt, and we do not expect TMAO to alter thedistribution of monovalent ions around an RNA.With this convenient simplification in mind, weconsider three aspects of the effects of TMAO onRNA structure and stability.First, we confirmed our previous observation12

that TMAO perturbs the stability of RNA secondarystructure to only a small degree. The Tm of asynthetic hairpin was nearly unaffected by TMAO(Table 1), and slight destabilization of secondary-structure transitions was seen in all of the RNAstested (e.g., Fig. 3).We attribute this lack of effect to afortuitous cancellation of favorable and unfavorableinteractions, primarily with base hydrogen bond

Fig. 6. Normalized data for the reactivity of the A-riboswitch (a and b) and 58mer rRNA fragment (c and d) towardhydroxyl radical. Gel band intensities have been normalized as described (Materials and Methods). (a) Reactivity of theA-riboswitch was assayed in K-Mops buffer (pH 6.8), 50 mm K+ plus no TMAO (purple); 5 m TMAO (yellow); 8 m TMAO(green); or 5mmMgCl2 (blue).Mops anion concentrationswere 20mm (noTMAOorMgCl2), 125mm (5mTMAO), or 182mm(8 m TMAO). Regions of increased protection compared to the unfolded conformation are indicated by colored barscorresponding to residues depicted in (b). (b) Residues protected fromhydroxyl radical displayed on theA-riboswitch crystalstructure (1Y26) where residues 33, 37, and 38 are shown in red, 46 to 49 are displayed in blue, and 52 and 53 are shown inyellow. The adenine ligand is depicted in green. (c) Reactivity of the 58mer RNA obtained in K-Mops buffer (pH 6.8), 60mMK+ plus no TMAO (purple); 4 m TMAO (red); 5 m TMAO (yellow); 8 m TMAO (green); or 5 mm MgCl2 (blue). Mops anionconcentrations were 20 mm (no TMAO or MgCl2), 100 mm (4 m TMAO), 125 mm (5 m TMAO), or 182 mm (8 m TMAO).Regions of increased protection compared to the unfolded conformation are indicated by colored bars corresponding toresidues depicted in (d). (d) Representation of residues protected from hydroxyl radical on the 58mer rRNA crystal structure(1HC8)where residues 18 to 21 are shown inblue, 35 to 37 are depicted in yellowand 45 to 47 are indicated in red.Residues 14in the 58mer and 66 in the A-riboswitchwere impossible to quantify accurately andwere not plotted. Protection assessmentsfor residues at the 3′ end, which correspond to nucleotides at the bottom of the gel, were also unreliable.


acceptors and donors. TMAO is a very polarmolecule and should be a good hydrogen bondacceptor;48 it is therefore unsurprising that hydrogenbond donors (e.g., glycerol; Table 2) have somepreference for interacting with TMAO over water.Conversely, TMAO is incapable of donating a

hydrogen bond and should not compete well withwater for hydrogen bond acceptors such as uridinecarbonyls. Although other factors undoubtedlycontribute, we suppose that a major reason for thenet exclusion of TMAO from purine and the nucleo-sides we tested is the presence of more hydrogen

image of Fig. 6

Fig. 7. The 58mer RNA ion-binding pocket confers aspecific monovalent ion requirement for optimal stability.(a) View of the crystal structure of the 58mer RNA (1HC8)regions surrounding chelated K+ (violet sphere) and Mg2+

(green sphere) ions. Anionic oxygen atoms of A1073contact either the K+ or Mg2+ ion. (b) Relative free-energychanges for folding the 58mer rRNA fragment with either0.5 mM MgCl2 (blue) or 6 m TMAO (red) added to bufferwith different group I ions [1 m M+, 150 mm M-Mops(pH 6.8), 2 μm EDTA]. The ionic radii are taken fromPauling.69


bond acceptors than donors on the bases. Detailedstudies of glycine betaine, another trimethylamineprotecting osmolyte, have similarly found a favor-able interaction of the compound with hydrogenbond donors (e.g., amide nitrogen) and unfavorableinteraction with hydrogen bond acceptors (e.g.,amide oxygen).24 (The balance of favorable andunfavorable interactions is such that glycine betainedestabilizes RNA secondary structure.12,49) Dena-turation of an RNA duplex to single strands isaccompanied by the exposure of equal numbers ofbase hydrogen bond acceptors and donors tosolvent. We hypothesize that favorable TMAO

interactions with base hydrogen bond acceptors areapproximately canceled by unfavorable interactionswith hydrogen bond donors, leaving RNA duplexstability nearly unaffected.Hydrogen bond surfaces exposed upon unfolding

of tertiary structures may include ribose 2′ OH aswell as base acceptors and donors. The rather weak,favorable interaction of TMAO with hydroxyl(glycerol; Table 2) may not cancel the stronglyunfavorable interactions that probably take placewith base hydrogen bond acceptors. However,exposure of 2′ OH to solvent is unlikely to accountfor the effects of TMAO on the tertiary structuresexamined here. The TLR motif, for example, has asingle 2′ OH hydrogen-bonded to a donor N1 andacceptor N6 of two adenine bases. Exposure of thesethree hydrogen-bonding groups to TMAO shouldhave little net effect on the stability of the RNA andcertainly cannot account for the m value we observe.Second, TMAO reduces the dimensions of par-

tially unfolded RNAs. X-ray scattering experimentsshow that the average Rg of the unfolded form ofeither the A-riboswitch or 58mer RNAs is graduallyreduced as TMAO is added, until the RNA hasnearly the same dimensions as seen with Mg2+

present. This observation is consistent with thefinding that the unfolding reaction becomes less saltdependent (fewer ions are released) as TMAO isadded (Table 1; the salt–TMAO interaction factor ispositive). The less extended the unfolded state, thecloser it is in charge density to the native conforma-tion and the smaller the overall uptake of ions uponfolding. The lack of any effect of TMAO on theaverage extension of mutant RNAs unable to formthe native tertiary structure shows that TMAO doesnot indiscriminately restrict the RNA to morecompact conformations. Instead, the data suggestthat the RNA transiently samples conformationswith native tertiary contacts and that TMAO shiftsthe distribution of conformations toward ones withnative-like structures. In a similar way, Mg2+

promotes more compact conformations of theA-riboswitch RNA, but has little effect on thedimensions of the C60G or G38C mutants (D.Leipply and D.E.D., unpublished data).Third, TMAO stabilizes the correct RNA tertiary

structure. By two criteria, the native structuresstabilized by TMAO are the same as those observedin the presence of Mg2+: the distance distributionprofiles from SAXS experiments are essentially iden-tical whether the structure has been stabilized byTMAO orMg2+ (Fig. 5), and hydroxyl radical probinggives the same pattern of nucleotide reactivity (Fig. 6).

TMAO exclusion from RNA phosphates andchanges in RNA hydration

The propensity of TMAO to stabilize nativeprotein structures has been attributed to the strong

image of Fig. 7


exclusion of TMAO from the peptide backbone(83.5 cal mol−1 m−1), which favors the burial ofpeptides in the protein interior over their exposureto solvent.8 The even stronger exclusion of TMAOfrom backbone phosphate (211 cal mol−1 m−1; Table2) suggests that a similar mechanism could be atwork in RNA: the energetic cost of phosphatedesolvation is reduced when TMAO is part of thesolvent. We argue that this mechanism is the onlyplausible way TMAO could be stabilizing RNAtertiary structures: as pointed out in the abovesection, the net interactions of TMAO with basesurfaces must change very little when RNA struc-tures form, and we find no suggestion of strongTMAO interactions with backbone sugars (Table 2).If TMAO–phosphate interactions drive the RNAstabilizations we measure, to what extent mustphosphate hydration be changing?The unfolding of an RNA tertiary structure

increases the volume of water from which TMAOis excluded [both ∂ln(Kobs)/∂mTMAO, Table 1, andΔΓTMAO, Eq. (5a), are negative]. The value ofΔΓTMAO sets a lower limit on the uptake of waterthat accompanies RNA unfolding, which is listed inTable 1 as ΔB1 [see Eq. (7)]; it ranges from 66 to 173water molecules per RNA. Based on the aboveargument about the stabilization of RNA tertiarystructure by TMAO exclusion from phosphate, wesuggest that these ΔB1 values primarily report theincrease in phosphate hydration that accompaniesdisruption of RNA tertiary structure. Although ΔB1is nominally a lower limit on water uptake, it will beclose to the actual value if TMAO is completelyexcluded from the water of hydration. It is reason-able to assume that this is the case. TMAO exclusionfrom phosphate anionic oxygen is comparable tothat of the related osmolyte glycine betaine, forwhich measurements with double-stranded DNA50

and phosphate24 are consistent with completeexclusion. The same measurements imply that ahydration layer two to three water molecules thickaround anionic oxygen atoms, or about 17 watermolecules per nucleotide of duplex DNA, is inac-

Table 3. Changes in phosphate SASA accompanying RNA un

RNAPO2 SASA(native) (Å2)

PO2 SASA(unfolded) (Å

58mer RNA 3136 3534–4007Tar–tar⁎ 1968 1860–2108TLR 722 (receptor) 758 (recepto

391 (GAAA) 388 (GAAAA-riboswitch 4768 4401–4991

SASAs of backbone phosphate (phosphorus and the two nonbridging ofiles specified in the legend to Fig. 8. For TLRRNA, surface areas correspA; U48–G52 and C77–G82 for chain B) and the tetraloop hairpin (nucseparately. The unfolded TLR RNA surface areas are derived from mosubunit, whereas the total change in SASA is for the complex. The valuethe per-nucleotide SASA previously calculated for canonical A-form RNof phosphate exposure to the m value for RNA unfolding in TMAO w

cessible to the osmolyte. With this number as aguide to what might be expected for phosphatehydration at the surfaces of partially unfoldedRNAs, the three RNAs with tertiary structure inTable 1 have totals of 510 to 1462 hydrating watermolecules, which are reduced by 10–15% bytertiary-structure formation. Because some fractionof the nucleotides in these RNAs are in helicalsegments that are unperturbed by tertiary-structureformation (e.g., the stems of the tar and tar*hairpins), some phosphates must be much moreextensively dehydrated than this average value. Ourresults therefore imply that significant changes inphosphate hydration accompany RNA tertiary-structure formation.

Solvent accessibility of phosphates in RNAtertiary structures

As a another way to evaluate phosphate hydra-tion in RNA tertiary structures, we have calculatedthe solvent-accessible surface area (SASA) of back-bone phosphates (the sum of anionic oxygens andphosphorus, excluding ester oxygen) for each RNAby standard methods (see Materials and Methods;total surface areas are listed in Table 3 and plots ofindividual residues are shown in Fig. 8). Followingstudies of the effects of TMAO and other osmolyteson protein folding and protein–DNA bindingequilibria,8,24 we ask whether the free energy ofstabilization contributed by TMAO is proportionalto the change in the phosphate SASA that accom-panies folding. Based on our measurement ofTMAO–K-DMP interactions and a value of 85 Å2

for the SASA of the DMP phosphorus and anionicoxygen atoms, we estimate a proportionality factorof 2.48 cal Å−2 m−1 to use in calculating an m valuefrom changes in phosphate SASA (Table 3). (Thiscalculation assumes that TMAO interactions withboth K+ and methyl groups are negligible.*)Of the four RNA tertiary structures examined,

only the 58mer shows dramatic (and nearly com-plete) burial of several phosphates, most of which

folding

2) Δ(SASA) (Å2)Estimated m value(kcal mol−1 m−1)

398 or 871 0.99 or 2.16−92 or 141 −0.23 or 0.35

r) −67 −0.17)

−367 or 223 −0.91 or 0.55

xygen atoms) for native RNA structures were calculated fromPDBonding to the receptor (nucleotides U5–G9 andC34–G39 for chainleotides G19–C24 for chain A; G62–C67 for chain B) are reporteddels as described in the text. Note that these numbers are for eachs given for unfolded tar–tar⁎, A-riboswitch, and 58mer RNA usedA (the smaller value) or single-stranded RNA.12 The contributionas calculated using the proportionality 2.48 cal Å−2 m−1.

Fig. 8. SASAs of phosphate groups for four RNAs withtertiary structure (Fig. 1). (a) 58mer rRNA (1HC8), (b) thetwo monomers from the tetraloop–receptor RNA complex(2JYF), (c) A-riboswitch (1Y26), and (d) Tar–tar⁎ (1KIS).Blue data points indicate SASA obtained with a proberadius of 1.4 Å, whereas red points correspond to a proberadius of 2.8 Å.


contact chelated ions (Fig. 8a). To estimate thechange in solvent-accessible phosphate surface area,we use average values for either single-strandedor double-helical RNA (previously computed inRef. 12) as surface areas that should bracket theextent of phosphate exposure in an unfolded RNA.The range of calculated m values (0.99–2.16 kcalmol−1 m−1, Table 3) overlaps the range of theestimated m values (1.1 to 1.4 kcal mol−1 m−1,corresponding to 102–130 water molecules takenup). As previously mentioned, these estimates mayunderestimate the limiting sensitivity of the RNA toTMAO. Nevertheless, the extent of phosphate burialadequately rationalizes the m value.None of the other three RNAs shows enough

burial of phosphate surface area to account formeasuredm values when a proportionality based onmodel compound (K-DMP) interaction energies isused. The most egregious case is the TLR RNA,which docks a very stable hairpin structure, theGAAA tetraloop, with a receptor. Contacts betweenthe two RNA segments consist entirely of hydrogenbonding and stacking between bases with noinvolvement of the backbone.51–53 As the structureof the tetraloop probably does not change upondocking27 and the structure of the receptor in theabsence of tetraloop is known from NMR studies,52

we have a good model for the undocked structure.There is actually a small decrease in phosphateexposure of the separate components compared tothe complex, which predicts a negative m value(Table 3). The calculatedm values for tar–tar* and A-riboswitch RNAs, based on single-strand andduplex models for the unfolded RNAs, also under-estimate the measured m values significantly.We suspect that, in these three RNAs, the

accessibility of phosphate to water is not a goodmeasure of the extent of phosphate dehydration.Dimerization of the TLR RNA places two helices inclose proximity (Fig. 1e), where several phosphatescome fairly close together (2.6 to 4.5 Å betweenanionic oxygen atoms). If the water excluded toTMAO extends over a layer two to three moleculesthick around phosphate anionic oxygen atoms,24,50

then bringing two phosphates into a position inwhich they share hydrating water molecules mustsubstantially decrease the volume of water that isexcluded to TMAO. When a sphere twice thediameter of water is used as a more realisticassessment of solvent changes around phosphates,six phosphates now appear “inaccessible” (Fig. 8b).With the A-riboswitch RNA, tertiary structureformation brings the backbones of two helicalsegments close together (nucleotides 26 with 68–69); a turn of the backbone in the adenine-bindingpocket brings two phosphate oxygen atoms within3.6 Å (nucleotides 21–22). These two regions areidentified by their lack of accessibility to a 2.8 Åsphere (Fig. 8c). The two backbones of the tar–tar*

image of Fig. 8

Fig. 9. Contrasting mechanisms by which Mg2+ andTMAOmay stabilize the same RNA tertiary structure. Therelative free energy (chemical potential) of folded (F) andunfolded (U) forms of a hypothetical RNA tertiarystructure are diagrammed. In buffer, the U form of theRNA is represented as more stable than the F form (blackboxes; the observed folding free energyΔG°obs is positive).Mg2+ interacts strongly (red arrows) with both U and Fforms of the RNA, but preferentially stabilizes the nativestructure (red boxes; ΔG°obs is now negative). TMAOinteractions with both U and F forms are stronglyunfavorable (blue arrows), but the U form is more stronglyaffected because of its more extensive exposure ofphosphates to solvent. Therefore, ΔG°obs becomes nega-tive (blue boxes).


RNA also bring several phosphate oxygen atoms lessthan a water molecule diameter apart (Fig. 8d).26,54

From these examinations of RNA structures, itseems that tertiary-structure formation is frequentlyaccompanied by the formation of phosphate–waternetworks, in which adjacent phosphates might sharean inner shell of water but lose outer shells. Theisolated phosphate of K-DMP may not be anappropriate compound for modeling the energeticsof TMAO exclusion from these kinds of networkedstructures. The one case in which we calculated areasonable m value on the basis of SASA calcula-tions, the 58mer RNA is also an RNA in whichseveral phosphates become completely buried anddesolvated. In this situation, the exclusion of TMAOfrom K-DMP might be expected to reproduce theenergetics of phosphate burial.Lastly, we note a parallel between protein and

RNA folding studies: both are limited by theavailability of good models for the ensemble ofunfolded or partially unfolded conformations fromwhich folding takes place.55,56 Among the RNAsdiscussed here, only for the TLR RNA do we have agood model based on experimental data.

Parallels between TMAO- and Mg2+-inducedRNA folding

It is interesting to note that Mg2+ stronglyinfluences RNA folding, with exactly the sameoutcomes as seen with TMAO: it favors compact or“collapsed” forms of partially unfolded RNAs41 andstabilizes RNA tertiary structure much more effec-tively than secondary structure.57 However, themechanisms bywhich a divalent cation and a neutralosmolyte affect RNA must be completely different,as diagrammed in Fig. 9. Mg2+ interacts favorablywith both folded and unfolded forms of an RNA,primarily by strong attraction to the negativeelectrostatic field created by phosphates. However,interactions with the more compact native structureare stronger and lower its free energy relative to thatof an unfolded state.33 In contrast, TMAO interactsunfavorably with both unfolded and folded RNAstructures, but unfolded conformations expose morephosphate to solvent and have a more positiveinteraction free energy with TMAO. In a sense, Mg2+

“pulls” the folding equilibrium toward the nativestate by virtue of favorable interactions with thenative RNA, while TMAO “pushes” the sameequilibrium because of repulsive (unfavorable)interactions with partially unfolded RNA.The case of the buried Mg2+ in the 58mer RNA is

an example of how Mg2+ and TMAO can promotethe same RNA structure by entirely differentmechanisms. Four phosphates create two ionchelation sites in this RNA, one for K+ and one forMg2+ (Fig. 7a). The same selectivity for K+ and thesame hydroxyl radical sensitivity is seen whether

the Mg2+ site is occupied by ion or simply stabilizedby TMAO (Figs. 6c and 7b). In other work, we findthat high concentrations of L11 protein (normallyassociated with this RNA domain in ribosomes) trapthe RNA in its native conformation in the absence ofMg2+, with the sole exception of nucleotide A1073,which retains the reactivity of unfolded RNA tohydroxyl radical. K+ selectivity has been lost in thiscomplex, presumably because of the nonnativestructure at A1073 (D. Leipply and D.E.D., manu-script submitted).We infer that there is a strong driving force for the

A1073 phosphate to stay exposed to solvent, bothbecause of electrostatic repulsion from nearbyphosphates and from the energetic cost of phosphatedehydration, and suggest that the substantial free-energy cost of burying this phosphate can be paid intwo ways. One way is for Mg2+ to occupy thechelation site and reduce electrostatic repulsionamong the phosphates; the favorable electrostaticenergy of ion binding must be enough to overcomethe dehydration penalty. The other way is forTMAO to reduce the dehydration penalty; theelectrostatic repulsion between phosphates in the

image of Fig. 9


unoccupied site still remains, but is now insufficientto disrupt the native conformation. The surprisingimplication is that TMAO exclusion from this sitegenerates stabilizing free energies as large as Mg2+

binding.In summary, the parallel effects of Mg2+ and

TMAO suggest that RNA phosphates present twodifferent kinds of barriers to formation of tertiarystructures: strong electrostatic repulsion betweenphosphates and strong interactions with water(hydration) both favor nonnative, extended confor-mations of an RNA. Those barriers may be reducedbecause of net favorable interactions of ions with thenative structure or because of unfavorable interac-tions with protecting osmolyte by the partiallyunfolded RNA (Fig. 9). In either case, the samenative structure is adopted, implying that the nativetertiary structure is primarily specified by the RNAsequence. Ions and osmolyte facilitate RNA foldingbut do not organize the native structure in the casesexamined here.

Materials and Methods

Chemicals and solutions

All solutions were prepared using distilled deionizedwater at 18.3 MΩ resistivity. Mops, potassium chloride,sodium chloride, and rubidium chloride (all N99.5% pure)were purchased from Fluka. Lithium chloride, cesiumchloride, and ethylenediaminetetraacetic acid (EDTA)(also N99.5% pure) were obtained from Sigma-Aldrich.Lithium hydroxide, sodium hydroxide, potassiumhydroxide, rubidium hydroxide, cesium hydroxide (all99.99% pure), and trimethyl phosphate (N99% pure) werepurchased from Aldrich. TMAO dihydrate (≥99% pure)was purchased from Fluka; contaminating amines wereremoved by treating ∼10 m TMAO (∼175 g total) forseveral hours at room temperature with approximately5 g of granular activated carbon (Darco, 20-40 mesh,Aldrich), followed by vacuum filtration using 0.22-μmDurapore membrane filters (Millipore). This procedurewas repeated until no amine smell was detectable. Thesolution was then deionized with 2 g of mixed bed resin(AG 501-X8, 20-50 mesh, Bio-Rad), divided into aliquotsand stored at −80 °C until used. By atomic absorption,the stock solution was less than 0.16 μM in Mg2+, whichafter dilution contributes less than 0.06 μm Mg2+ to a 2 mTMAO solution. This amount of Mg2+ is far less than theRNA concentrations typically used in melting experi-ments (∼1 μm). The final concentrations of TMAOsolutions were determined by stoichiometric titrationwith standardized HCl. We also confirmed that thevapor pressure of our potassium chloride solutionscorresponded to literature values.25,58

K-DMP was synthesized by mixing equimolar amountsof trimethyl phosphate (Sigma-Aldrich) and KOH (Fluka)in 80% ethanol/water solution as described,59 with theexception that NaOH was substituted by KOH. Hydroly-sis was allowed to proceed for at least 4 h. The solutionwas then concentrated under vacuum, redissolved in

water, and subsequently passed through an ion-exchangecolumn (DOWEX 50WX8, Dow) to ensure that all thedimethylphosphate was in the K+ salt form. The resultingsolution was concentrated under vacuum until solid. Noimpurities were detected by phosphorus or proton NMRspectroscopy. Due to the hygroscopic nature of this salt, itwas first stored in preweighed microtubes in a desiccatorcontaining phosphorus pentoxide powder (98+%, ACSReagent, Sigma-Aldrich). Tubes were weighed under thedry atmosphere of a glove box every few days over thecourse of 3 weeks until the weight stabilized. A 10 m stockwas obtained by adding an appropriate weight of water toeach tube, and the samples were stored at −80 °C.The short hairpin oligonucleotides tar and tar* (Fig. 1a

and b) were purchased from Dharmacon and deprotectedaccording to the manufacturer's directions. Oligonucleo-tide length homogeneity was confirmed by denaturingpolyacrylamide gel electrophoresis. The 58mer rRNAfragment (Fig. 1c), the tetraloop–receptor complex (Fig.1e), and the A-riboswitch aptamer domain (Fig. 1d) wereprepared by in vitro transcription with T7 RNA polymer-ase from linearized plasmid DNA and purified bydenaturing polyacrylamide gel electrophoresis followedby electroelution, as described.12,33 Before use, RNAs wereextensively equilibrated with the appropriate buffers,using Amicon Ultra centrifugal filter devices (Millipore,Billerica, MA). Buffers for UV melting, VPO, and SAXSmeasurements are specified in the figure legends. Mopsbuffer was adjusted to pH 7.0 or 6.8 with KOH (K-Mops)or, for experiments done with other group I ions, theappropriate alkali metal hydroxide. The total K+ ionconcentration was noted; it is the sum of K+ added fromthe K-Mops buffer plus KCl.All solutions with osmolyte were prepared in molal

concentration units (moles solute per kilogram solvent) sothat osmolyte and salt concentrations could be variedindependently. Stock solution densities were used tocalculate the volume of each needed to prepare samplesfor experiments. To determine densities, we first calibra-ted pipettors using the density of water at 20 °C(0.997 g/mL). Then, precisely 1 mL of each solution wasweighed on an analytical balance (Mettler Toledo).

UV thermal analysis

Melting experiments were performed in a Cary 400spectrophotometer with 1-cm path length cuvettes. Absor-bance data were collected at 260 and 280 nm (as well as295 nm in the case of the 58mer rRNA fragment) from 5° to95 °C for the hairpin and the A-riboswitch, and from 2° to95 °C in the case of the tar–tar* and the tetraloop–receptorcomplexes. Absorbance data for the small hairpin, whichmelts in a single transition, were analyzed by standardmethods incorporating low and high temperature baselinesas fitted variables.60 Data for the other RNAs studied wereplotted as the first derivative of absorbance with respect totemperature (a melting profile). For simplification of dataanalysis, sequential two-state transitions, defined by Tm,ΔH° (assumed to be independent from temperature), andamplitude of absorbance changes, were fit globally to boththe 260- and 280-nmor 260- and 295-nmdata as described.61

Low temperature baselines for RNAswith tertiary structureare frequently small or zero, and only in a few cases weremanually adjusted to optimize the fitted curve. Three


transitions were fit to the tar–tar* and tetraloop–receptordata. Four transitions were used to fit the profile of the58mer rRNA as reported.29,36 For this RNA, the enthalpiesof the unfolding transitions obtained from experiments at4 m TMAO were used to fit the data at 6 m TMAO, inaccord with the observation that ΔH° does not changewith salt or osmolyte concentration.12 Characteristics ofthe A-riboswitch in melting experiments have recently beenreported.31 TheTmof the first unfolding transitiondependsonthe concentration of ligand (in this case, 2,6-diaminopurine)as expected for unfolding of tertiary structure; depending onthe salt and osmolyte concentrations, we fit the meltingprofiles with either two or three transitions.The averages and standard deviations of Tm and ΔH°

for each RNA in the absence of osmolytes were previouslydetermined;12,29 ΔH° values agree within error with thevalues found in the presence of TMAO. Errors in Tm forindividual melting curves were determined by a bootstrapmethod,61,62 and were generally smaller than the plottedpoints (Figs. 2 and S1). Global fitting of (1/Tm) as afunction of both mTMAO and ln(mKCl) was performed withPro Fit 6.1 using the following polynomial equation withsix independent variables:

1=Tm¼ a0ln mKClð Þþb0ð Þþ a1ln mKClð Þþb1ð Þ mTMAOð Þþ a2ln mKClð Þþb2ð Þ mTMAOð Þ2 ð12Þ

The six fitted coefficients were then multiplied by ΔH°/Rto obtain derived parameters in terms of ln(Kobs). Errorsreported in Table 2 were propagated using both theuncertainties in the fitted coefficients and in ΔH°; thelatter were larger. The salt dependence at a specifiedTMAO concentration [cf. Eq. (3)] is given by:

Aln Kobsð Þ=AmKCl¼a0þa1 mTMAOð Þþa2 mTMAOð Þ2 ð13Þand in the absence of TMAO is just a0 (SKobs). Thedependence of ln(Kobs) on osmolyte concentration at aspecified KCl [cf. Eq. (5a)] concentration is

Aln Kobsð Þ=AmTMAO¼ a1ln mKClð Þþb1ð Þþ2 a2ln mKClð Þþb2ð Þ mTMAOð Þ ð14Þ

and the salt–TMAO interaction term in the limit of lowTMAO concentration is a1.A global fit of hairpin RNA 1/Tm values using Eq. (10)

was unreliable because the salt–osmolyte interaction term(a1) was poorly determined. We therefore report para-meters derived from the average slope of (1/Tm) versusmTMAO or (1/Tm) versus ln(mKCl) for this RNA in Table 2.

Isothermal titrations

Titrations of the A-riboswitch with TMAO were moni-tored by circular dichroism. The titrated solution had aninitial concentration of 130mmK-Mops (pH 7.0), 50mmK+,1 μm A-riboswitch RNA, and 10 μm adenine ligand. Thetitrant was the same, but with TMAO at a final concentra-tion of 8.5 m. The RNA sample was first denatured for 10min at 65 °C, and brought to room temperature forapproximately 10 min before titration was initiated.Titrations were performed at precisely 22.5 °C with anAviv Model 400 CD Spectrometer. A Hamilton Microlab500 automated titrator was used to perform 37 injections.

Data were collected at 268 nm with a bandwidth of 2 nmand averaging time of 30 sec. Titration data were fit to anequation with six variables specifying the slopes andintercepts of baselines for the unfolded and folded RNAsand a TMAO-dependent equilibrium constant expressed interms of the midpoint of the titration and the m value.

Vapor pressure osmometry

A Wescor VAPRO 5520 (Logan, UT) at ambienttemperature (22.5 °C) was used for all measurements.Three identical sample solutions in microtubes wereassembled from water and stock solutions of knownmolality and density. Triplicate measurements of osmolal-ity were made on each sample for a total of nine readings.To minimize the concentration of protonated TMAO, weincluded 50 mm K-Mops (pH 7.0) in all samples. Controlsdone in the presence andabsence of buffer showeda strictlyadditive effect of K-Mops on the measured osmolalities.Data obtained in the presence and absence of TMAO wereanalyzed as described in Background [Eqs. (7)–(9)].

Hydroxyl radical footprinting

RNA for footprinting experiments was dephosphory-lated for 5′ labeling by reaction with calf intestinalphosphatase at 37 °C for 1 h, followed by phenol–chloroform extraction and ethanol precipitation to removethe enzyme and recover the RNA. RNA (80 pmol) was thenend-labeled in 20-μL reactions with 10 μL of [γ-32P]ATP(6000 Ci/mmol; Perkin Elmer) and 10 U of polynucleotidekinase in the appropriate kinase buffer (NEB). Samplesweregel-purified after labeling on 12% or 16% denaturingacrylamide gels, and RNA was recovered by freeze–thawand ethanol precipitation. Each footprinting reactioncontained 106 cpm of RNA in K-Mops buffer and thedesired amount of TMAOorMg2+. On ice,H2O2was addedto 0.03%, and the Fe–EDTA complex to 1 mM ferrousammonium sulfate and 2 mM EDTA, after degassing thesolution. Reactions were quenched after 1 min withthiourea. RNA was recovered by ethanol precipitationand resuspended in 10μLof formamide loading buffer. Fivemicroliters of each sample, along with unreacted, alkalinehydrolysis, and T1 digest controls, were loaded on adenaturing sequencing gel (12% for A-riboswitch RNA,16% for 58mer RNA) and run for 3 h at 70 W. The gel wasthen transferred to Whatman filter paper, dried, andexposed to a phosphorimager screen for 18 h before beingimaged. The cleavage products in the sequencing gels werequantified using SAFA.63 Five invariant residues (C1064,A1067, C1075, U1083, G1091, and G1099) were chosen tonormalize the band intensities in the 58mer. Four invariantresidues (U36, G43, U62, and G78) were chosen tonormalize the band intensities in the adenine riboswitch.

Small-angle X-ray scattering

SAXS experiments were performed at beamlines 12-IDand 18-ID of the Advanced Photon Source at ArgonneNational Laboratory. The wavelength of incident X-rayradiation was set to 1.033 Å (a beam energy of 12 keV), theexposure timewas 0.15 s, and sampleswere flowed throughan X-ray flow cell while in the path of the beam to avoid


radiation damage. RNA samples for the measurementswere extensively exchanged into the appropriate K-Mopsbuffer supplemented with the salt and osmolyte concentra-tions of interest. The ambient temperature of the experimentwas ∼29 °C; samples were allowed to temperatureequilibrate for an hour prior to beam exposure.Twenty images were collected for each sample or buffer

solution in order to obtain good statistics. Aberrant datasets were removed, and the remaining two-dimensionalimages from the charge-coupled device were reduced toone-dimensional scattering profiles using the programMarDetector (from Dr. David Tiede, unpublished). Theresulting data sets were averaged prior to backgroundsubtraction of the buffer. The scattering profile of the RNAwas then calculated using the following equation:

IRNA qð Þ = Isample qð Þ − a4Ibuffer qð Þ ð15Þwhere I(q) is the appropriate scattering intensity atmomentum transfer vector q=4π sin(θ/λ) (where 2θ isthe scattering angle) and α is the scaling factor that takesinto account the partial molar volume of RNA. The radiusof gyration (Rg) was obtained from linear fits of the datawith q*Rgb1.2), using the Guinier approximation:

lnI qð Þ = ln I 0ð Þð Þ − R2gq

2 = 3 ð16ÞThe program GNOM was used to compute distancedistribution functions, P(r), from the scattering profiles.64

The Dmax parameter was systematically varied untilconsistent solutions were attained. The Rg values calculat-ed from integration of P(r) agreed well (within 10%) withthose obtained fromGuinier analysis, validating the choiceof Dmax. CRYSOL65 was used to calculate the expected Rgof native A-riboswitch (1Y26) and 58mer RNA (1HC8)from the indicated Protein Data Bank (PDB) files.

SASA calculations

Surface area calculations were performed using theprogram Surface Racer66 with the Richards parameters67

and probe radii of 1.4 or 2.8 Å.Supplementary data can be found online at

doi:10.1016/j.jmb.2010.09.043. The file includes plots ofthe dependences of ln(Kobs) on salt or TMAO concentra-tions for the hairpin, tar-tar† complex, and TLR RNA

† It is not possible to measure l23 for individual ions,only the sum of the anion and cation contributions. Incalculating the proportionality factor for backbone phos-phate, we are assuming that the measured l23/RTc0 forKCl results from a 0 value for both K+ and Cl-. In anextensive study of glycine betaine with various com-pounds and salts, Capp et al.24 derived a self-consistentset of l23 values in which l23/RT was assigned to 0 forNa+; l23/RT then took on the values –0.03 for Cl- and +0.09 for K+. As TMAO is chemically similar to glycinebetaine and has a net weaker interaction with KCl, itseems reasonable to assume that interactions with eitherK+ or Cl- will be small compared to the magnitude of theinteraction with DMP-. We have also neglected theexclusion of TMAO by the two methyl groups of DMP-;aliphatic carbons interact with glycine betaine an order ofmagnitude more weakly than anionic oxygen.24

(Figure S1), isothermal titrations monitored by CD for theA-riboswitch (Figure S2), osmolality versus m2m3 plots foruridine, cytidine, and purine with TMAO (Figure S3); andthe distance distribution function of the A-riboswitch atvarious TMAO concentrations (Figure S3).

Acknowledgements

This work was supported by NIH grants RO1GM58545 and T32 GM008403 (Program in Molecu-lar Biophysics). We thank Sean Campbell andKenny Xu for assistance with VPO measurementsand Drs. Xiaobing Zuo and Sonke Seifert for helpwith collection and analysis of the scattering data.

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the osmolyte tmao stabilizes native rna tertiary structures in the absence of mg2+: evidence for a...

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