Divalent Cation-Aided Identification of Physico-Chemical Properties of Metal Ions that Stabilize RNA G-Quadruplexes Sumirtha Balaratnam and Soumitra Basu Department of Chemistry and Biochemistry, Kent State University, Kent, OH 44242 Received 26 September 2014; revised 16 February 2015; accepted 17 February 2015 Published online 23 March 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22628

ABSTRACT: DNA and RNA sequences rich in guanosines (G) can form a four-stranded secondary structure known as a

Keywords: RNA G-quadruplex; divalent metal cations; stabilization of G-quadruplex; properties of metal ions; monovalent cations

G-quadruplex (GQ), which plays a role in regulation of gene expression at the transcription and translation level. Both DNA and RNA GQs typically use the monovalent K1 ion for stabilization of the structures. However, the fundamental reasons for K1 acting as the most stabilizing metal ion for RNA GQs are not known. To identify the properties of a metal ion that stabilizes an RNA GQ we investigated the effect of alkaline earth metal cations and a set of divalent transition metal ions on two previously identified highly stable RNA GQs. Our results based upon circular dichroism and RNase T1 structure mapping data reveal that the RNA GQs are destabilized in the presence of the tested divalent metal cations. The destabilizing effect of a divalent metal cation is reversible upon increasing K1 concentration. Results show that ionic radius, hydration energy, and binding strength towards the hard ligand (guanine O6) are important factors that determine a metal ion’s ability to stabilize an RNA GQ. Additionally, the tested set of divalent metal cations incongruously affects RNA and DNA GQs. C 2015 Wiley Periodicals, Inc. Biopolymers 103: 376– V

386, 2015.

Additional Supporting Information may be found in the online version of this article. Corespondence to: Soumitra Basu; e-mail: [email protected] Contract grant sponsor: KSU

This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of any preprints from the past two calendar years by emailing the Biopolymers editorial office at [email protected].

INTRODUCTION

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NA and RNA sequences adopt various noncanonical secondary structures with regulatory roles. Gquadruplex (GQ) is part of a set of such secondary structures that is formed by G-rich sequences found in DNA and RNA molecules.125 They are known to play a role in replication, epigenetic regulation, recombination, meiosis, and regulation of gene expression at the transcriptional and the translational level.628 The basic subunit of GQ structure is the G-quartet, comprised of a planar cyclic array of four Hoogsteen base paired guanine residues.4,9 The repulsion between the guanine bases is stabilized by coordination of metal cations with the guanine O6 atoms. The formation of GQ structures depends on various factors such as enthalpy of dehydration of metal cations, loop length and composition, sequence length, and conformational polymorphism.10212 The stabilization of GQ structures mostly depend on stacking interaction of G-quartet, hydrogen bonding, cation coordination, and solvent interaction.3 Unlike most other DNA/RNA secondary structures, metal cations localize within the GQ structures by direct cationoxygen lone pair coordination3,13 and thus the formation and the stability of GQ structures are directly linked to the cation species and its concentration.14,15 Besides K1 and Na1, NH41 and Tl1 ions have also been investigated as monovalent cations

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that can stabilize GQ structures.16,17 The Crystallography studies have shown the directed coordination of dehydrated monovalent cations, primarily K1 and Na1 in the central quartet cavity.18 Cation coordination within the GQ structures varies depending on the ionic radius and cation-cation repulsion.3 Based upon a wide range of cations that have been studied for their role in DNA GQ formation and stability, the following general pattern emerged, K1oNa1Rb1>Cs1Li1 and Sr21Ba21>Ca21>Mg21>Mn21>Co21>Zn21 respectively.19 Also, the DNA GQs exhibit change in conformation in presence of some divalent metal cations and given that RNA GQs only adopt one conformation, it would be valuable to study the effect of such ions on RNA GQs. To the best of our knowledge GQs are the most well known noncanonical structure adopted by DNAs that harbor a specific monovalent cation binding site, while such binding sites in complex non-GQ RNA molecules have been reported before, understanding the role of such ions in RNA GQs will be important given the prevalence of such structures. RNA GQs are more stable than their DNA counterparts and RNA G-rich sequences may be more prone to formation of GQ structures in vivo than DNA due to the lack of a complementary strand.20 RNA GQs are involved in the telomere maintenance, pre-mRNA processing, RNA turnover, mRNA targeting and translation.7,8,21 The stability of the RNA GQs also depend on the presence of monovalent cations, mainly K1 ions, because the melting temperature of RNA GQs increased by 10230  C in the presence of K1 compared with Na1 under similar conditions.22 In addition, the stability of RNA GQs increases with increasing K1 ion concentration and at close to the physiological K1 concentration some RNA GQs are resistant to denaturation even at 90  C.23 Most of the studies on RNA GQs based on metal ion dependency had involved monovalent cations and predominantly K1 ions, although the fundamental characteristic that make it the most stabilizing ion are not clearly defined. To address this issue we have systematically investigated the role of divalent metal cations on the stability of RNA GQs. We rationalized that testing a set of divalent cations with diverse characteristics would lead to determination of the key properties of a metal ion needed for stabilization of an RNA GQ. It has been previously shown that GQs located in the 50 -UTR of M3Q mRNA and NRAS mRNA forms an extremely stable parallel GQ structure and inhibits the translation of a reporter gene.7,23 For our studies we chose to use the M3Q and NRAS RNA sequences primarily because of their high stability. Our data revealed that RNA GQs are differentially destabilized by various divalent metal cations and identified a set of key parameters required for metal ion-dependent stability of RNA GQs. Biopolymers

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MATERIALS AND METHODS Preparation of Oligonucleotide Sequences The RNA sequence 50 -GAGGGAGGGAGGGAGAGGGA-30 (M3Q) was purchased from Dharmacon, Inc. The NRAS RNA sequence (50 - GUGGGAGGGGCGGGUCUGGG-30 ) was transcribed in vitro by using synthetic DNA templates.24 The RNA products were purified, harvested and quantitated using previously published methods.23

50 -End Radiolabeling of RNA Oligonucleotides The RNAs were 50 -end radiolabelled by methods published previously.23

Circular Dichroism (CD) Studies All circular dichroism (CD) measurements were recorded at room temperature. RNA was folded by heating the samples at various concentrations of KCl in 10 mM Tris-HCl and 0.1 mM EDTA (pH 7.4) at 95  C for 5 min, followed by slow cooling to room temperature over a 90-min period. The CD spectra were recorded using a Jasco J-810 spectropolarimeter with a 0.1 cm cell at a scan speed of 50 nm/min with a response time of 1 s. The spectra were averaged over three scans and the spectrum from a blank sample containing only the buffer was subtracted from the average of the sample scans. After the collection of the initial spectrum, the samples were titrated with increasing concentrations of various divalent metal cations (alkaline earth metal ions and a set of transition metal ions). For each addition, the divalent metal cation was added to the sample in the cuvette and was mixed several times and incubated for 15 min at room temperature before obtaining a new spectrum.

Thermal Melting Experiments CD melt spectra were recorded using a 0.1 cm path-length cell. Samples were prepared by heating oligonucleotides (5 mM RNA) in 10 mM KCl, 10 mM Tris-HCl, and 0.1 mM EDTA (pH 7.4) at 95  C for 5 min and then slowly cooled to room temperature. Then various concentration of MgCl2 were added into the samples and incubated for 15 min at room temperature. For other divalent metal cations, 2 mM of the metal ions were added into the prefolded RNA GQ and incubated for 15 min at room temperature. Mineral oil was placed on top of the sample to prevent evaporation. The melting curves were obtained by monitoring a 263 nm CD peak. Thermodynamic parameters and Tm values were calculated using the van’t Hoff method.25,26

Structure Mapping by RNase T1 The 50 end-radiolabeled RNA was folded by heating the samples in the presence of 10 mM of KCl, 10 mM Tris-HCl and 0.1 mM EDTA (pH 7.4) at 95  C for 5 min and then slowly cooled to room temperature, following which 20 mM of various divalent metal cations were added into the samples and incubated for 15 min at room temperature. After the incubation, the RNA was digested with 0.25 units of RNase T1 (Ambion) for 5 min at room temperature. The reaction was terminated by using an equal volume of stop buffer as described previously.23

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Statistical Analysis Data are presented as mean 6 sem calculated from three replicates.

following order: 0 mM Mg21 o 5 mM Mg21  8 mM Mg21 > 20 mM Mg21  50 mM Mg21 (Table I). These results confirmed the destabilizing effect of Mg21 ions on the ultrastable M3Q RNA GQ structure.

RESULTS Magnesium (II) Ions Unfold RNA G-Quadruplex Structures and Inhibit Their Formation To determine the effect of divalent metal cations on the RNA GQ we first studied Mg21 ions, since it is the most prevalent intracellular divalent metal cation. We used circular dichrosim (CD) spectroscopy to measure the effect of Mg21 on the RNA GQ stability. Initially Mg21 was titrated in presence of 10 mM of the monovalent metal ion K1. The M3Q RNA generates a CD spectrum with a peak at 263 nm and a trough at 240 nm which can be ascribed to a parallel GQ structure.23 However, when the GQ samples were titrated with increasing amount of Mg21 ions, a progressive decrease in the CD signal at 263 nm was observed (Figure 1a), reflecting a disappearance of the GQ structure presumably due to its unfolding. A similar progressive decrease in CD signal intensity was observed during unfolding of M3Q in presence of the cationic porphyrin TmPyP4.27 Different concentrations of Mg21 were plotted against change in the CD signal intensity, and we assumed that the signal at 263 nm for M3Q in the absence of Mg21 corresponds to the fully folded structure (presumed that intensity of CD signal at 263 nm is equivalent to the fully formed and stabilized GQ structure). As shown in Figure 1b, the data fit well with an exponential function, which allowed us to calculate the Mg21 concentration that causes 50% unfolding of the fully folded M3Q RNA (D50 5 7.98 6 0.04 mM). We tested another stable RNA GQ, a G-rich sequence from the 50 UTR of NRAS mRNA7 to determine the generality of the Mg21 mediated destabilization. The NRAS RNA was folded in the presence of 10 mM KCl and a CD spectrum was obtained. The spectrum showed a positive peak at 263 nm and a negative peak at 241 nm, which are characteristics CD signal for a parallel GQ structure.7 However, when the NRAS GQ samples were titrated with increasing amount of Mg21 ions, decrease in CD signal at 263 nm was observed as was observed previously with the M3Q RNA GQ. As shown in Figure 1c, the data fit with an exponential function and the D50 value was calculated to be 12.71 6 0.52 mM for the NRAS RNA. To further determine the stability of the GQ structure at different Mg21 ion concentrations, the changes in CD spectra at 263 nm were monitored in the presence of 10 mM K1. In the presence of different Mg21 ion concentrations, M3Q showed the expected trend for GQ destabilization with Tm values in the

FIGURE 1 Destabilization of the RNA G-quadruplex in the presence of Mg21 ions. a: CD spectra of 5 mM prefolded (10 mM KCl) M3Q in the presence of increasing concentration of Mg21 ions. Finally all the values were corrected for dilution effect. The arrow indicates the decrease in CD signal as a function of increasing Mg21 concentration. b: Plot of calculated percentage of stabilization of M3Q RNA GQ structure versus Mg21 concentration. c: Plot of calculated percentage of stabilization of NRAS RNA GQ structure versus Mg21 concentration. The 5 mM NRAS RNA was prefolded in the10 mM KCl and was titrated with increasing Mg21.

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Table I Tm Values for M3Q RNA in the Presence of Various Mg21 Ion Concentrations MgCl2 (mM) 0 5 8 20 50

Tm ( C) 86.66 6 0.51 56.00 6 0.69 51.73 6 0.52 45.06 6 0.62 40.56 6 0.30

Magnesium (II) Perturbs K1 Induced Stabilization of the M3Q RNA G-Quadruplex To investigate the ability of Mg21 ions to overcome the stabilizing effect of K1 ions, the M3Q was folded in the presence of various K1 ion concentrations and titrated with increasing Mg21 ions. The D50 values were calculated as described previously. The D50 values for Mg21 changed with K1 concentrations that were used to form GQ and expectedly showed larger values with increasing K1 concentrations (Table II). The stability of RNA GQ increases with increase in K1 concentration, which means a proportionally higher amount of Mg21, would be required to unfold the GQ. As shown in Figure 2a, there is in fact a very strong correlation (R2 5 0.98) between the change in K1 concentration versus the corresponding Mg21 concentration needed to unfold the RNA GQ. Possibly the divalent ions bind to the phosphate backbone peripheral to the quartet and negatively affect the stability of RNA GQ. Intracellular magnesium concentrations range from 5 to 20 mM of which 1–5% is ionized, the remainder is bound to proteins, negatively charged molecules and ATP.28 However the localized concentration of magnesium in the cell can vary. We found that 8 mM magnesium causes 50% destabilization of GQ structure that was formed in 10 mM KCl. This suggests that not all of the GQ structures are unfolded, more so because the physiological intracellular concentration of K1 is 120 mM. However, based on the localized concentration of

Table II The D50 (50% Destabilization) Values of M3Q RNA GQ Structures Which Were Formed at Different K1 Ion Concentrations and Destabilized by Mg21 Ions KCI (mM) 1 5 10 25 50 100

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D50 Value (mM) 4.92 6 0.05 6.65 6 0.09 7.98 6 0.04 10.09 6 0.17 15.83 6 1.10 22.54 6 0.37

FIGURE 2 a: The Correlation between concentrations of K1 ions used to form the GQs and the amount of Mg21 required for obtaining 50% destabilization of GQs (D50 values of Mg21). b: Rescue of Mg21 induced destabilization of the GQ structure by EDTA.

K1 and Mg21 ions, one may speculate that GQs can undergo unwinding by Mg21 ions.

A Common Chelator Rescues the Destabilizing Effect of Mg21 To establish the destabilizing effect of Mg21 a rescue experiment in the presence of a chelator was performed. We reasoned that if sufficient amount of Mg21 gets sequestered by chelation its destabilizing effect will diminish and the already present K1 ions will begin to exert their effect, which should result in reformation of the GQ structure. The M3Q was folded in the presence of 10 mM KCl and to achieve  50% unfolding, 8 mM of Mg21 ions (D50 5 7.98 6 0.04 mM at 10 mM K1) were added into the sample and about 50% unfolding was observed as monitored by decrease in CD signal intensity (Figure 2b). To reverse the destabilizing effect, EDTA (ethylenediaminetetraacetic acid) a well-known divalent metal ion chelator was added. We rationalized that EDTA would chelate and engage the Mg21 ions which then would not be available and the GQ structure should be restored. Indeed with the increase in amount of EDTA it starts sequestering the Mg21 ions from the solution via chelation, the stabilizing effect of K1 ions begins to dominate with a concomitant increase in CD signal at 263 nm, reflecting a reversion of unfolded form of the GQ

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NRAS RNA was folded in the presence of 10 mM KCl followed by titration with increasing amount of Zn21 ions. The D50 value was calculated to be 1.99 6 0.15 (data shown in SupportAlkaline Earth Metal Ions D50 Value (mM) ing Information Figure 2S). The low destabilization (D50) values of Zn21 for both M3Q and NRAS RNA GQs show that 21 7.98 6 0.04 Mg transition metal cations especially Zn21 is a more potent destaCa21 11.14 6 0.05 bilizer compared to alkaline earth metal cations. 12.96 6 0.03 Sr21 In order to understand the difference in effect of the divaBa21 13.59 6 0.09 lents on RNA and DNA GQs, we tested the deoxyribo version of M3Q sequence (50 -dGAGGGAGGGAGGGAGAGGGA-30 ), structure. We performed a similar study with KCl instead of which folded into a parallel GQ structure in the presence of EDTA. The GQ formed in 1 mM KCl was destabilized by add- divalent metal cations (Supporting Information Figure 3S), ing 5 mM Mg21 (D50 = 4.92 6 0.05 at 1 mM KCl) and was fol- although the M3Q ribo sequence fails to fold into a GQ struclowed by addition of increasing amount of K1. The intensity ture in the presence of only divalent metal cations. This clearly at 263 nm kept rising with the increase in K1, which suggested indicates that the effects of divalent cations on RNA and DNA that the destabilizing effect of Mg21 can be overcome by GQs are not the same. increasing the K1 probably by eliminating Mg21 ions’ ability To further investigate the stability of RNA GQ in the presto stabilize the unfolded GQ (data shown in Supporting Infor- ence of divalent metal cations, CD melting was performed in mation Figure 1S). These data also suggest that RNA GQ struc- the presence of different divalent metal cations. The M3Q tures are reversibly destabilized by Mg21. RNA GQ was folded in 10 mM KCl, 2 mM divalent cations were added in the sample and the changes in CD spectra at Alkaline Earth and a Set of Divalent Transition Metal 263 nm were monitored. Thermodynamic parameters from the melting curves (Supporting Information Figure 4S) were Cations Are RNA GQ Destabilizers calculated (Table V) by considering the two state model.25,26 To investigate the effect of divalent metal cations besides Mg21 The melting temperatures (Tm) of divalent metal cations on RNA GQ stability we chose to study the other alkaline earth strongly correlated with their calculated D50 values (Support21 21 21 metal cations, such as, Ca , Sr , and Ba and some of the ing Information Figure 5S) and confirmed the unfolding of transition metal cations Mn21, Co21, Ni21, Zn21, and Cd21. GQ structures with respect to the amount and the identity of Circular dichroism spectra were monitored as a function of divalent cations. The Tm values of divalent cations obtained divalent ion concentration for any change in the 263 nm peak. from the melting curves follow the same trend (except Sr21) The M3Q RNA was folded in the presence of 10 mM KCl to with respect to their propensity to unfold the GQ structure ensure GQ formation followed by titration of each of the metals value): Sr21> Ba21> Ca21> Mg21> (D50 into the solution. The D50 values were calculated as described 21 21 21 21 21 Mn >Co >Ni >Cd >Zn . In addition, there was a previously (Tables III and IV). All of the divalent metal cations correlation between the Gibbs free energy (DG) of unfolding of that were studied caused destabilization of the RNA GQ strucM3Q RNA GQ structure and melting temperature (Tm) in the ture. Based upon the D50 values the following order emerged, presence of divalent metal cations (Supporting Information beginning with the strongest destabilizer (smallest D50 value) of Figure 6S). An interesting observation was that DG value 21 21 21 21 the GQ structures: Zn > Cd > Ni > Co  decreased by 50% in the presence of 2 mM Sr21 compared 21 21 21 21 21 Mn > Mg > Ca > Sr > Ba . To further confirm the to only K1 was, although the Tm values differed by only 5  C. effect of Zn21 ions, we tested it on the NRAS RNA GQ. The The DG at 37  C decreased in the presence of all of the divaTable IV The D50 (50% Destabilization) Values of a Set lent cations tested compared with the DG of only in presence of Transition Divalent Metal Cations of K1, suggesting less thermal stability of RNA GQ structure Transition Metal Ions D50 Value (mM)a due to unfolding of the GQ. Furthermore the DG values also correlate well with the D50 values of divalent metal cations, the Mn21 6.55 6 0.07 weaker destabilizers have more favorable DG compared with 2.97 6 0.02 Co21 the more potent ones. Ni21 2.49 6 0.04 On the basis of the enthalpy (DH) and entropy changes Zn21 2.03 6 0.09 (DS) of the GQ in the presence of each of the divalent metal Cd21 2.24 6 0.02 cations (in presence of 10 mM K1) compared with the values a 1 of such parameters only in presence of K1 (10 mM) indicated The M3Q RNA GQ was formed in 10 mM K . Table III The D50 Values (50% Destabilization) of M3Q RNA GQ Structure by Different Alkaline Earth Metal Cations in Presence of 10 mM K1 Ion

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Table V Tm Values and Thermodynamic Parameter [Gibbs Free Energy (DG), Enthalpy Change (DH) and Entropy Change (DS)] for Unfolding of RNA GQ in the Presence of Divalent Metal Cations Calculated from CD Melting Experiments by Van’t Hoff Plot and First Derivative Analysis Metal Cations K1 Ba21 Sr21 Ca21 Mg21 Mn21 Co21 Ni21 Cd21 Zn21

Tm ( C)

DG37  C (kJ/mol)

DH (kJ/mol)

DS (kJ/mol.K)

85.66 6 0.51 72.44 6 1.16 80.05 6 0.04 64.81 6 0.73 61.18 6 0.81 56.83 6 0.61 51.28 6 0.43 48.07 6 0.42 46.65 6 0.20 38.86 6 0.86

240.05 6 0.72 213.62 6 0.81 227.72 6 0.27 210.67 6 0.62 27.77 6 0.15 26.90 6 0.50 25.51 6 0.42 25.53 6 0.85 24.53 6 0.58 20.20 6 0.38

2295.18 6 1.57 2132.94 6 7.69 2227.37 6 2.04 2128.71 6 4.65 2107.58 6 1.37 2115.96 6 6.40 2126.01 6 12.72 2177.72 6 15.89 2130.02 6 19.56 2132.88 6 11.42

20.823 6 0.004 20.385 6 0.023 20.644 6 0.006 20.381 6 0.013 20.322 6 0.005 20.352 6 0.020 20.389 6 0.040 20.556 6 0.050 20.405 6 0.061 20.426 6 0.037

more favorable enthalpy. In the presence of K1 alone the overall enthalpy is highly favorable. However, when different metal cations are added there was a loss in enthalpy which may contribute to the unfolding. Then we sought to investigate the characteristics that may cause the observed differences in the D50 values. We correlated stabilization of GQ (for a metal ion, higher the D50 value lower its ability to destabilize) to different characteristic features of metal ions, such as ionic radius, hydration enthalpy, and binding strength of coordination ligand to cation. Plot of the ionic radii of the alkaline earth metal ions with the D50 values (Figure 3a) exhibited a strong correlation (R2 5 0.96). Thus the larger the ionic radius the greater its ability to stabilize, which is not unexpected since the ionic radius of the most stabilizing ion K1 is 1.39 A˚ which is close to the ionic radius of Ba21 (1.35 A˚) the least destabilizing divalent cation. However, in case of the tested transition metal divalent cations the ionic radii did not show any correlation with the D50 values (data shown in Supporting Information Figure 7S). Next we determined the correlation between the hydration enthalpy and the D50 values of the divalent cations tested. When not engaged in coordination with other ligands metal ions obviously remain hydrated in aqueous solution and when they coordinate, the new interactions must be energetically more favorable than the hydration energy penalty it has to expend to dehydrate. As shown in Figure 3b, there is a good correlation between the hydration enthalpy and the D50 values. Thus hydration enthalpy does play a role in the stabilizing effect of metal ions. The third parameter we investigated is the ability of the metal ions to coordinate to hard-soft atoms29 and how that correlates to the calculated D50 values. As shown in Figure 3c, Ba21 is the poorest destabilizer of the RNA GQ among the divalents, which were tested, but its value in the HL scale is still higher (more positive) than K1 and thus still acts as a destabilizer relative to K1. Thus optimum ionic radius, hydration Biopolymers

enthalpy, and ability to coordinate to hard-soft ligand are the key set of required characteristics for an ion to be able to stabilize the RNA GQ.

RNase T1 Structure Mapping in Presence of Divalent Cations Shows Destabilization of M3Q and NRAS G-Quadruplexes To biochemically probe the destabilization of GQ structures by divalent cations, the M3Q molecule was subjected to ribonuclease T1 (RNase T1) mediated cleavage. RNase T1 cleaves at guanosine residues in single stranded RNA or unpaired guanosines, but not when they are present in the context of secondary or tertiary structure.30 In GQ structures, guanosines that directly participate in quartet formation are protected from RNase T1 mediated cleavage compared with guanosines that are in the loop or in the unstructured regions.31 In the M3Q molecule G1 and G15 are within the loop regions while the other Gs are engaged in the quartet formation.23 As shown in Figure 4a, in the absence of divalent metal cations (Lane 2), guanosines that are involved in the quartet formation were protected, which matches with the earlier report from our laboratory.23 However, in the presence of divalent metal cations (Lanes 3–5) all of the guanosines in the M3Q sequence were unprotected. It should be noted that in presence of Ba21 M3Q is slightly better protected from RNase T1 mediated cleavage compared to, for example, the level observed in presence of Mg21. This observation can be rationalized, given that in all of the correlation studies Ba21 emerged as the poorest destabilizer of RNA GQ among the tested divalent cations. In the NRAS RNA G1 is within the loop region while the other Gs are engaged in the quartet formation. As shown in Figure 4b, in the absence of divalent metal cations (Lane 2) guanosines that are involved in the quartet formations were protected. However, in the presence of 20 mM Mg21 ions (Lane 3) all of the

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DISCUSSION RNA and DNA G-Quadruplexes Are Differentially Affected by Divalent Metal Cations DNA GQs have been shown to fold in presence of some divalent cations; however, the RNA GQs cannot fold solely in the presence of divalent cations. Crystallographic studies on DNA and RNA GQ structures have shown that the metal cations coordinate with the similar set of functional groups in DNA and RNA GQs.13,18,33,34 However, the recent crystallographic structural studies on both RNA and DNA quadruplexes formed by the human telomeric repeats of r[UAGGGUUAGGGU]35 and d[TAGGGTTAGGGT]14,33,36 have shown that even though overall structure of RNA and DNA Gquadruplex appears to be similar, there are differences in Hbond interaction and bondings with water molecules. In the RNA G-quadruplex C20 -hydroxyl group makes more intramolecular interactions with several different hydrogen bond acceptors, such as O40 atoms of the ribose, the phosphate backbone oxygens, and the N2 groups of guanines involved in G-quartet formation. This preferred interaction of hydroxyl groups results in a reduction in ordered water molecules within RNA quadruplex grooves, compared to the equivalent DNA structure. In addition, the C20 -hydroxyl group in RNA

FIGURE 3 Plots representing the correlation between the D50 values of divalent metal cations and their various physico-chemical properties. a: The correlation between the D50 values of alkaline earth metal cations and their ionic radii.57 b: The correlation between D50 value of divalent metal cations and hydration enthalpy.57 c: The correlation between D50 values of the divalent metal cations and their binding strength towards the coordinating hard atom O6 of Guanine according to the HL Scale.47

guanosines in the NRAS sequence were unprotected. The overall lack of protection from RNase T1 cleavage confirmed that in the presence of divalent cations both M3Q and NRAS GQs were unfolded. Some of the divalent metal cations used in this study are known potent inhibitors of RNase T1.32 Therefore, we were unable to perform RNase T1 structure mapping in the presence of those metal cations.

FIGURE 4 Images of RNase T1 structure mapping showing the unfolding of RNA GQ structure in presence of divalent cations. a: RNase T1 structure mapping of M3Q in presence of divalent cations. Lanes: M3Q without RNaseT1 (Lane 1), M3Q with 10 mM KCl (Lane 2), M3Q with 10 mM KCl and 20 mM of MgCl2, CaCl2, and BaCl2 (Lanes 3, 4, and 5, respectively). b: RNase T1 structure mapping of NRAS in the presence of Mg21 ions. Lanes: NRAS without RNase T1 (Lane 1), NRAS with 10 mM KCl (Lane 2) and NRAS with 10 mM KCl and 20 mM MgCl2 (Lane 3).

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makes a steric hindrance on glycosidic torsion angle favoring the anti-glycosidic conformation, which favors an all-parallel folded topology. However, DNA has equal access to both anti and syn glycosidic torsion angles that support parallel as well as anti-parallel folded topologies. Also, RNA preferentially adopts the C30 -endo sugar pucker which results in alteration in the phosphate backbone thus accommodating more Hbinding network. This difference can lead to preferential binding of certain small molecule ligands.37 The facts stated above have significant implication on metal binding to RNA and DNA GQs because in presence of divalent cations DNA GQs can undergo conformational change while RNA GQs are unable to undergo such switch because they are restricted to adopting parallel conformation only. For example, in an earlier study the authors observed the formation of intramolecular DNA GQ structure in the presence of divalent metal cations and they concluded that compared to K1 ions, Sr21 ions formed more stable DNA GQ structure.38 However, we observed opposite effect of Sr21 as well as other divalent metal cations on the RNA GQ structures. The divalent metal cations not only didn’t support the formation of GQ structure but also destabilized the RNA GQ structure formed in presence of K1. This again strongly indicates that divalent metal cations affect DNA and RNA GQs differentially including GQs formed by identical nucleotide sequences.

Ionic Radius, Hydration Enthalpy, and Binding Strength to Hard Ligands Are Key Determinants for RNA G-Quadruplex Stabilization Monovalent cations play a dual role by imparting stability and helping with the formation of the GQ structure. They induce the formation of GQ structure by direct coordination with guanine O6 atom within the G-quartet and stabilize the GQ by screening the electrostatic repulsion within the quartet and they act as “diffuse ions” to neutralize the negative charge on the surface of the GQ.3,39 There are a number of factors that determine the cation coordination within the quartet cavity. For a stable GQ formation it is important that the size of the quartet cavity is compatible with the ionic radius of the cation in such a way that the cation can participate in stable coordination. Previous studies have shown that Na1 ion (ionic radius 0.95 A˚) is suitable to fit within the plane of the cavity, whereas K1 (ionic radius 1.39 A˚) is too large to coordinate within the plane of a quartet and instead resides between two quartet planes. Monovalent cations adopt multiple coordination geometries in DNA GQ.3 Factors that are relevant in selecting the appropriate DNA GQ stabilizing ions may include the ionic radius, hydration energy and the nature of the coordinating ligand or atom. We analyzed our data to define the key characBiopolymers

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teristics of a cation that contribute to the stability of RNA GQs. Initially we checked the ionic radius of the divalent metal ions since in majority of the studies it correlates with GQ stability. We discovered that the D50 values of the alkaline earth metal cations correlate very well with the ionic radius. As shown in the Figure 3A, when the ionic radius increases it results in higher D50 values indicating least destabilization of the RNA GQ structures by Ba21 and Sr21 in comparison to Mg21. The ionic radius of a Ba21 ion is 1.35 A˚, which is very similar to the radius of a K1 (1.39 A˚) ion. This suggests that the ionic radii of Ba21 and K1 ions can both contribute to the stabilization of RNA GQ, but Ba21 ions also caused the destabilization of RNA GQ which indicates that ionic radii is not the sole determining factor. The high resolution crystal structure of d(TG4T) in the presence of Na1 and Ca21 have shown that only Na1 ions are coordinated and reside in the G-quartet planes even though radius of both metals (0.95 A˚ and 0.99 A˚, respectively) are approximately the same.40 There was no evidence to indicate that the Ca21 substituted for Na1 ions at any coordination site. The crystal structure of RNA GQ formed by the sequence r(UGGGGU) in the presence of Sr21 ions described the tetramolecular GQ structure formation with Gquartets stacked on one another and Sr21 ions were associated with eight carbonyl oxygen atoms of a set of guanines.34 The M3Q used in this study is known to form intramolecular GQ structure and our data show that Sr21 destabilizes the GQ structure. Thus intramolecular GQs may have less “flexibility” in terms of accommodating divalent metal ions than intermolecular GQs. Additionally, the destabilization effect of transition metal cations did not correlate well with their ionic radii. This can be due to the presence of d-orbitals which makes their ionic radii smaller compared to the alkaline and alkaline earth metal ions. In general, ionic radii of the transition metals show progressive decrease with increasing atomic number across a row in the transition series. This is because as the new electron enters a d-orbital, the effective nuclear charge increases by unity and since the shielding effect of a d-electron is relatively less effective compared to the s and p orbitals, the net electrostatic attraction between the nuclear charge and the outermost electron increases causing a contraction in size. Thus, from the above discussion it is clear that ionic radius cannot be the sole determinant and other factors, such as, the dehydration enthalpy, coordination number and coordination ligands involved in the GQ formation must also be considered. Energetically divalent metal cations are about four times more costly to dehydrate compared to monovalent ions of the same radius.39,41 In the GQ structure dehydrated cations are coordinated within the G-quartet. Therefore, low energy cost may favor the monovalent cations over the divalent cations. It has

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by strongly binding to guanine O6 atoms in the quartet and induce formation of RNA GQ structure. As shown in the Figure 3C, our data fit nicely with hard ligand binding strength Alkaline Hydration Enthalpy 47 Metal Ions Radius (A) (kJ/mol) HLScale (HLScale). Transition metal cations have more positive values (lower negative values) in the HLScale compared with alkaline K1 1.39 2519 21.75 earth metal cations. Therefore, it can be argued that the transiNa1 0.95 2406 21.71 tion metal cations’ weak coordination to the guanine O6 atoms 0.68 2322 21.57 Li1 in the quartet caused more destabilization effect on the GQ structure than alkaline earth metal cations. been previously reported that K1 and not Mg21 stabilizes a tertiary RNA structure found within an independently folding Coordination Environment of the Cavity Within an domain of the Tetrahymena group I intron ribozyme where RNA G-Quadruplex Dictates the Choice of the Metal Ion size and hydration energy were suggested to play key roles.42 In While DNA GQs have specific metal binding sites the canonithis work we found out that hydration enthalpy of divalent cal B-form DNA does not harbor any such sites. However, 21 metal cations fitted with our data (D50 value) well. The Ba RNA secondary and tertiary structure formation and function and Sr21 ions have lower energy cost for dehydration than the are very much dependent upon metal ions, specifically on transition metal cations tested, thus they are weaker destabildivalent metal cations. Thus among the RNA secondary strucizers (higher D50 value) of the RNA GQ structure than the tures GQ provides a unique class of structural context where other divalent metal ions. Moreover compared to all divalent monovalent metal cations, particularly the K1 acts as the stabimetal cations, K1 ion has lower hydration energy that aids in lizer of such structure. However, It has been known for more RNA GQ stabilization. Even though Li1 ion has lower cost for than several decades that activities of both ribosomal subunits energy of hydration compared to K1 ions, the significantly depend upon K1 or NH41 and reversibly loses activity in pres1 smaller ionic radius of Li (Table VI) precludes it from being a ence of Na1 or other alkali metal ions49,50 suggesting a specific good stabilizer. Our results match well with a previous report requirement of size and hydration enthalpy. A 58 nucleotide on the effect of divalent metal cations on the stability of DNA ribosomal RNA fragment has also been shown to be stabilized GQ structures19,38,43,44. DNA GQs stabilized by alkaline earth most strongly by K1.51 The most functionally and structurally 21 21 21 21 metal cations including Sr and Ba while Ca and Mg well-defined monovalent metal ion-binding site is perhaps the ions either caused conformational transitions or destabilized one found in the AA-platform of the Tetrahymena group I the GQ, however, transition metal divalent cations including intron RNA.42 Then the question that needs to be answered is: Zn21, Co21 and Mn21 destabilized the DNA GQ.19,45,46 what is unique about RNA GQ structure that distinguishes Because RNAs can only form parallel GQ structure8,35, they are itself from most other types of RNA structures in terms of their unable to undergo conformational transition like the DNA metal binding environment? To address this question we need GQs in the presence of divalent metal cations. Also, the M3Q to take into account chemical characteristics of the metal bindand NRAS sequences form intramolecular GQ structures and ing sites. It is clear that when the metal ion-binding site has a the sequences could not adopt any other secondary structure, number of charged ligands, for example, charged phosphates, such as the hairpin. Therefore, we can conclude that the divadivalent metal ion Mg21 is the choice because of its high lent metal cations indeed destabilized the folded RNA GQ. charge density and high hydration energy (more than five In GQs metal ions coordinate to the guanine O6 atoms, times higher than that of K1). The GQs have multiple polar which is a hard ligand. The polarizability of an acid or base carbonyl oxygen atoms coordinated to the monovalent cation plays a role in its reactivity. Hard acids and bases are nonpolarcreating a chemical environment very similar to the AAizable, while soft acid and bases have more diffused distribution platform with its carbonyl oxygens.42 Thus a chemical environof electrons. Hard acids strongly bind with hard bases compared ment within an RNA that is polar but poor or low in formal with soft bases. Alkaline metal ions (Group I) and alkaline earth charge and that has relatively large pocket will dictate selection metal cations (Group II) are hard acids, but most of the transiof K1 as the metal ion as is observed in RNA GQs. tion divalent metal cations are either soft acids or are on the border line.29 In addition, compared to the alkaline earth metal cations (data shown in Supporting Information Table 2S) alka- Mechanism of Divalent Metal Cation Mediated line metal cations such as K1, Na1 have higher binding Unfolding of GQ Structure strength (K1 5 21.79 and Na1 5 21.71) towards the hard It has been shown that divalent metal cations such as Mg21, ligands.47,48 Therefore, K1 and Na1 ions can act as stabilizers Ca21, and Zn21 have high propensity to bind with N7 position Table VI The Required Ionic Properties of Monovalent Alkaline Metal Cations to Stabilize RNA GQs57

Biopolymers

Metal Ions that Stabilize RNA G-Quadruplexes

FIGURE 5 Schematic depicting a few putative mechanisms of divalent metal cation mediated unfolding of RNA G-quadruplex (GQ) structure. ssRNA (single stranded RNA).

of the Guanine and stabilize the Watson-Crick base pairing.52 Moreover X-ray structure studies indicated that in GG sites, Mn21 ions specifically interact with N7 and O6 position of the guanine directly or via the water molecules in their first coordination sites.53 Besides NMR studies on oligodeoxyribonucleoties have shown that divalent metal cations, specifically Mn21, Zn21, Co21, and Ni21 have preference to bind with N7 position of 50 end guanine.54,55 However, in the GQ structure guanine N7 is involved in the Hoogsteen hydrogen bonding. Therefore, in the presence of divalent metal cations N7 position of guanines coordinate with divalent metal cations and thus prevent the formation of Hoogsteen hydrogen bonding that leads the unfolding of GQ. In addition, it has been shown that hydrated (five water molecules) divalent metal cations such as Mg21, Ca21, Sr21, Ba21, Zn21, Cd21, and Hg21 specifically coordinate with N7 position of adenine in rAA and TA basepairs.56 Furthermore in the purine-purine-pyrimidine triple helix, hydrated Zn21 and Mg21 ions are coordinated to N7 position of purine bases involved in Watson-Crick base pairing.55 Thus, the divalent metal cations have the ability to coordinate to N7 position of adenine and guanine nucleotides when they are in hydrated state. However, in the GQ structure, metal ion should be in the dehydrated form to coordinate in the G-quartet cavity. Because of the low cost of dehydration, monovalent metal cations specifically K1 stabilize the GQ structure. However divalent metal cations in spite of the higher energy cost of dehydration can bind with N7 position of purine bases in the hydrated state and potentially cause the unfolding of GQ structure. Because they can remain partially hydrated the cost of dehydration energy is significantly lower in comparison to if they had to be fully dehydrated. Moreover based on Biopolymers

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the HSAB (Hard Soft Acid Base) theory, N7 is a soft base and have high affinity for binding to soft acid. Therefore, partially hydrated transition metal divalent metal cations can bind to N7 position of purine bases and disrupt the GQ formation. Thus, a possible mechanism of divalent cation aided unfolding of GQ is by their coordination to guanine N7 of the GQ structure causing disruption of the Hoogsteen hydrogen bonding and unfolding of the GQ structure (Figure 5). However, this argument gets weakened by the fact that the DNA can still form GQs in presence of divalent cations. Thus, the metal-N7 interaction can be a minor contributing factor and not a major cause in case of RNA GQ unfolding. Additionally, divalent cations because of their (i) ability to coordinate with N7 and O6 and (ii) higher charge density, and (iii) ability to form metal clusters can adopt local structure resulting in some form of RNA secondary structure, which can be a potential mechanism of unfolding of the GQ structure (Figure 5). Based on the overall findings it can be concluded that the set of divalent metal ions tested destabilize two highly stable RNA GQ structures. The destabilization can be reversed by addition of the monovalent K1 ions and a chelating agent, suggesting a competition of divalent metal cations with the monovalent cation K1. Aptness of a metal cation to stabilize an RNA GQ is dictated by its ionic radius to allow a snug fit into the quartet cavity, higher hard ligand (guanine O6 atoms) binding strength and favorable enthalpy of dehydration. For optimum stabilization favorable values of one or two of these parameters is insufficient and all three of the characteristics must be favorable. Additionally, this work shows how the structural and chemical environment of a folded RNA dictates the choice of the metal ion for its stabilization by discriminating between the highly prevalent usage of the divalent Mg21 and in relatively rare instances where the clear choice is the monovalent cation K1; the later being exemplified overwhelmingly in the G-quadruplexes.

ACKNOWLEDGEMENTS The authors thank Dr. Nicola Brasch for her comments on the manuscript and thank the Basu Lab for many helpful discussions.

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Reviewing Editor: Sarah Woodson

Biopolymers

Divalent cation-aided identification of physico-chemical properties of metal ions that stabilize RNA G-quadruplexes.

DNA and RNA sequences rich in guanosines (G) can form a four-stranded secondary structure known as a G-quadruplex (GQ), which plays a role in regulati...
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