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Effect of Urea on G-Quadruplex Stability Lusine Aslanyan, Jordan Ko, Byul G. Kim, Ishkhan Vardanyan, Yeva B. Dalyan, and Tigran V. Chalikian J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 15 Jun 2017 Downloaded from http://pubs.acs.org on June 16, 2017

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The Journal of Physical Chemistry

Effect of Urea on G-quadruplex Stability

Lusine Aslanyan,1 Jordan Ko,2 Byul G. Kim,2 Ishkhan Vardanyan,1 Yeva B. Dalyan,1 and Tigran V. Chalikian2*

1

Department of Molecular Physics, Faculty of Physics, Yerevan State University, 1 Alex Manoogian Street, Yerevan 375025, Armenia

2

Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario M5S 3M2, Canada

RUNNING TITLE: Effect of Urea on G-quadruplex Stability

Tel: (416) 946-3715 Fax: (416) 978-8511 E-mail: [email protected] *To whom correspondence should be addressed

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ABSTRACT: G-quadruplexes represent a class of noncanonical nucleic acid structures implicated in transcriptional regulation, cellular function, and disease. An understanding of the forces involved in stabilization/destabilization of the G-quadruplex conformation relative to the duplex or single-stranded conformation is a key to elucidating the biological role of G-quadruplex-based genomic switches and the quest for therapeutic means for controlled induction or suppression of a G-quadruplex at selected genomic loci. Solute-solvent interactions provide a ubiquitous and, in many cases, the determining thermodynamic force in maintaining and modulating the stability of nucleic acids. These interactions involve water as well as water-soluble cosolvents that may be present in the solution or in the crowded environment in the cell. We present here the first quantitative investigation of the effect of urea, a destabilizing cosolvent, on the conformational preferences of a G-quadruplex formed by the telomeric d[A(G3T2A)3G3] sequence (Tel22). At 20 mM NaCl and room temperature, Tel22 undergoes a two-state urea-induced unfolding transition. An increase in salt mitigates the deleterious effect of urea on Tel22. The urea m-value of Tel22 normalized per change in solvent-accessible surface area, ∆SA, is similar to those for other DNA and RNA structures while being several-fold larger than that of proteins. Our results suggest that urea can be employed as an analytical tool in thermodynamic characterizations of G-quadruplexes in a manner similar to the use of urea in protein studies. We emphasize the need for further studies involving a larger selection of G-quadruplexes varying in sequence, topology (parallel, antiparallel, hybrid), and molecularity (monomolecular, bimolecular, tetramolecular) to outline the advantages and the limits of the use of urea in G-quadruplex studies. A deeper understanding of the effect of


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solvent and cosolvents on the differential stability of the G-quadruplex and duplex conformations is a step towards elucidation of the modulating influence of different types of cosolvents on duplex-G-quadruplex molecular switches triggering genomic events.



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INTRODUCTION In a suitable environment, guanine-rich DNA and RNA sequences fold into one of the various G-quadruplex conformations in which guanines are collected into stacks of Gquartets 1-7. A G-quartet is a planar structure formed by guanine bases pairwiseinterlinked by Hoogsteen hydrogen bonds and additionally stabilized by a mono- or divalent cation of a proper size coordinated to the O6 carbonyls of the guanines within the central cavity of the core of a G-quadruplex 1, 3-4, 8. Guanine-rich sequences potentially capable of folding into G-quadruplex have been identified in hundreds of thousand loci in the human genome including telomeres, centromeres, and promoter regions of oncogenes with clear connections between G-quadruplexes and cellular function and disease 2, 8-14. Human telomeric DNA consists of tandem repeats of the 5'TTAGGG-3' sequence with a 3'-end single-stranded overhang of 100-200 nucleotides 7, 11, 15

. The structural, kinetic, and thermodynamic properties of monomolecular G-

quadruplexes containing four repeats of 5'-TTAGGG-3' have been extensively studied 5, 16-27

. In the atmosphere of Na+ ions, the 22-meric telomeric sequence d[A(G3T2A)3G3]

(Tel22) forms a basket structure with antiparallel strands and a diagonal and two lateral loops 16. In an aqueous solution, the Tel22 G-quadruplex is fully formed at ~20 mM NaCl and becomes further stabilized with an increase in the concentration of Na+ ions 25

. Among all interactions governing the stability and conformational preferences of G-

quadruplex-forming DNA sequences, solute-solvent interactions (solvation) stand out as a ubiquitous thermodynamic force prominently present in all major structural transitions involving nucleic acid structures (e. g., duplex-to-single strand) while also participating 4 ACS Paragon Plus Environment

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in fine tuning of more subtle transitions between conformational substates (e. g., A-to-B duplex transition or transitions between G-quadruplex topoisomers) 28-32. Solvation should be viewed to include not only solute-water interactions but also interactions between DNA and water-miscible cosolvents that may be present in solution. The conformational preferences of guanine-rich DNA sequences have been shown to be strongly modulated by water-soluble organic compounds 30-31, 33-35. Urea is a quintessential, widely used cosolvent with a strongly denaturing action on both proteins and nucleic acids 36-37. It has been employed for over a century in protein studies as a means to disturb and direct folded/unfolded equilibria 38-39. Remarkable progress has been made in developing an understanding of the action of urea on protein stability at the molecular level 36, 40-41. Studies of the effect of urea on the stability of nucleic acid structures have been relatively scarce compared to proteins. While the influence of urea on the stability of DNA and RNA structures have been studied to some extent, quantitative investigations of the effect of urea on conformational equilibria involving G-quadruplexes are lacking 37, 42-51. The deficiency is unfortunate for a number of reasons. Firstly, G-quadruplexes are unusual nucleic acid structures sharing a number of common features with globular proteins; they are globular in shape, less charged compared to other nucleic acids, and compressible owing to the presence of the central cavity. Thus, urea may interact with Gquadruplexes in a manner that is distinct compared to canonical DNA and RNA structures. Secondly, given the polymorphism of G-quadruplexes, urea may prove to be an effective and subtle tool in modulating the equilibria between various G-quadruplex conformations. Thirdly, an understanding of urea-G-quadruplex interactions may form



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the basis for subsequent studies of molecular forces underlying the duplex-to-Gquadruplex and single strand-to-G-quadruplex conformational switches within the genome in the crowded environment of the cell. Finally, urea is a natural cosolvent present in large concentrations in various living organisms which makes its effect on Gquadruplexes of biological significance. In this work, we explore the conformational states of Tel22 as a function of temperature and the concentrations of urea and sodium ions. Our results represent the first systematic investigation of the stability of a G-quadruplex within the temperatureurea-salt conformational space, while also producing the necessary experimental basis for further investigations of the preferential interactions of this important cosolvent with G-quadruplex-forming DNA sequences in their folded and unfolded states.

MATERIALS AND METHODS Materials. The 22-mer, d[A(G3T2A)3G3] (Tel22), containing four repeats of the human telomeric DNA sequence was synthesized and purified by Integrated DNA Technologies (Coralville, IA). Sodium chloride, phosphoric acid, and urea were purchased from Sigma-Aldrich Canada (Oakville, ON, Canada). EDTA (free acid) was purchased from Fisher Biotech (Fair Lawn, NJ, USA). These reagents were used without further purification. All solutions were prepared using doubly distilled water. All measurements were performed in a pH 7.0 buffer containing 10 mM monosodium phosphate/disodium phosphate, 0.1 mM EDTA, NaCl between 20 and 1000 mM, and urea at concentrations between 0 and 8 M. Solutions of urea with concentrations of 1, 2, 4, 6, and 8 M were prepared by weighing urea and adding pre-estimated amounts of 6 ACS Paragon Plus Environment

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the buffer to achieve the desired molalities, m. The molar concentration, C, of urea was computed from its molal concentration, m, using C = [1/(mρb) + φV/1000]-1, where ρb is the density of urea-less phosphate buffer and φV = 44.1 cm3mol-1 is the apparent molar volume of urea 52-53. Urea solutions were subsequently used as solvents for the DNA solutions. The DNA was dissolved in and exhaustively dialyzed against a pH 7.0 phosphate buffer with NaCl and urea at desired concentrations. The dialysis was carried out in 1000 Da molecular weight cut-off Tube-O-Dialyzers from G Biosciences (St. Louis, MO, USA). To form the equilibrium structure, the DNA-containing solution was placed in a boiling water bath and allowed to cool to room temperature. The concentration of the DNA was determined from the absorbance at 260 nm measured at 25 °C using a molar extinction coefficient of 228,500 M-1 cm-1 for the unfolded conformation. The latter was calculated using an additive nearest neighbor procedure described by Owczarzy and colleagues 54. For circular dichroism (CD) measurements and temperature-dependent UV light absorption measurements, the DNA concentrations were ~30 and 3 µM, respectively. Circular Dichroism Spectroscopy. CD spectroscopy was used to probe the DNA conformation at each of the experimental conditions of this study. The CD spectra of Tel22 were recorded in a 1 mm path-length cuvette at 25 °C using an Aviv model 62 DS spectropolarimeter (Aviv Associates, Lakewood, NJ) or an Olis DSM 20 CD spectrophotometer (Olis, Inc., Bogart, GA). UV Melting Experiments. UV light absorption at 295 nm was measured as a function of temperature in a DNA sample contained in a 1 cm path-length cuvette.



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These measurements were performed using a Cary 300 Bio spectrophotometer (Varian Canada, Inc., Mississauga, Ontario, Canada) or a Lambda 800 spectrophotometer (Perkin Elmer, Inc., Waltham, MA). The temperature was changed at a rate of 1 °C per minute. The G-quadruplex-to-coil transition temperatures, TM, and the van't Hoff enthalpies, ∆HvH, were evaluated from the experimental UV melting profiles using standard procedures 55-59.

RESULTS Figure 1 presents the CD spectra of Tel22 at 25 °C in the absence of urea at NaCl concentration between 20 and 1000 mM NaCl. In the absence of urea, Tel22 exhibits a CD spectrum, characteristic of the antiparallel G-quadruplex conformation with a positive peak at 295 nm and a negative peak at 260 nm 5, 60-61. The CD spectrum does not change when the concentration of NaCl increases from 20 to 1000 mM. Figures 2a, b, c, d, e, f, g, and h show the CD spectra of Tel22 at urea concentrations between 0 and 8 M at 20, 50, 100, 200, 400, 600, 800, and 1000 mM NaCl, respectively. As is seen from Figure 2a, at 20 mM NaCl, Tel22 undergoes a urea-induced conformational transition as suggested by changes in the positions and the amplitudes of the original CD spectral peaks. However, an increase in salt leads to complete (at urea concentrations of up to 4 M) or partial (at 6 and 8 M urea) refolding of unfolded Tel22 (see Figures 2b to h). Figure 3 compares the CD spectra of the urea-induced unfolded state (at 20 mM NaCl and 8 M urea) and the temperature-induced (at 20 mM NaCl and 95 °C) unfolded states of Tel22. The two states are clearly distinct with the ureainduced unfolded state being more structured than the heat-induced unfolded state. 8 ACS Paragon Plus Environment

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Figure 4 shows a representative UV melting profile of Tel22 recorded at 295 nm in a solution containing 200 mM NaCl and 1 M urea. All UV melting profiles have been approximated by the two-state model of thermal denaturation: A = AN(T) + [AD(T) - AN(T)]/[1 + exp(∆HM(T-1 - TM-1)/R)]

(1)

where TM and ∆HM are the transition temperature and van’t Hoff enthalpy, respectively; and AN(T) and AD(T) are linear functions that approximate the pre- and postdenaturation baselines, respectively. Figure 5 plots TM as a function of urea at various NaCl concentrations. Inspection of Figures 5 reveals a steady decrease in TM as the concentration of urea increases, while TM increases with an increase in salt concentration. Figure 6 presents a compilation of van’t Hoff transition enthalpies, ∆HM, plotted against respective transition temperatures, TM. Linear regression analysis of the data plotted in Figure 6 yields a slope, ∆∆HM/∆TM, of 0.14 ± 0.04 kcal mol-1K-1 which may represent a change in heat capacity, ∆CP, associated with the heat-induced denaturation of Tel22. In fact, it has been reported that thermal unfolding of some Gquadruplexes is accompanied by an increase in heat capacity 62. On the other hand, our calorimetric measurements have not produced any significant changes in heat capacity accompanying thermal unfolding of the K+-stabilized Tel26 telomeric Gquadruplex in the hybrid-1 conformation 26.

DISCUSSION Urea-induced Unfolding of Tel22 is a Two-State Process. Inspection of Figures 2a to h reveals that, at each NaCl concentration employed in this study, an increase in urea 9 ACS Paragon Plus Environment


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leads to a conformational alteration of Tel22 as manifested in a decrease in the amplitude of the positive peak at 295 nm, a redshift and a significant decrease in the amplitude of the negative peak at 264 nm, and a redshift and a modest decrease in the amplitude of the positive peak at 246 nm. Comparative analysis of the CD spectra presented in Figures 2a to h reveals that the denaturing effect of urea weakens as the concentration of NaCl in solution increases. This observation clearly reflects an increase in G-quadruplex stability accompanying an increase in the concentration of stabilizing ions (in this case, Na+ ions) 5, 63-65. Thus, at increasing NaCl concentrations, increasingly higher concentrations of urea are required to unfold the Tel22 Gquadruplex. Further inspection of Figures 2a to h reveals the presence of nearly isoelliptic points at 252 and 275 nm. This observation is suggestive of a two-state nature of the ureainduced unfolding transition of the Tel22 G-quadruplex. In contrast to urea-induced unfolding of the Tel22 G-quadruplex, its heat-induced unfolding is not a two-state event but involves intermediates 21-24. Given the two-state nature of the urea-induced transition, it may be treated within the framework of the linear extrapolation model (LEM) in a manner similar to that applied to urea-induced denaturation of proteins 66-68. Figure 7 presents the urea dependence of the molar ellipticity at 295 nm of Tel22 at 20 mM NaCl. The experimental points were approximated by the LEM-based two-state analytical model in which the differential free energy, ∆G°, of the folded and unfolded states is given by the relationship: ∆G° = -RTlnK = ∆G0 – m[urea]

(2)


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where K = α/(1 - α) is the folded/unfolded equilibrium constant; α is the fraction unfolded; ∆G0 is the differential free energy of the folded and unfolded states in the absence of urea; and m is the so-called “m-value”, a proportionality coefficient. Fitting the data in Figure 7 with Eq. (2) yields the values of ∆G0 and m of 2.5±0.4 kcal mol-1 and 0.57±0.09 kcal mol-1M-1, respectively. A more conventional way of determining the differential free energy, ∆G0, is based on the thermodynamic parameters (the melting temperature, TM, and enthalpy, ∆HM) of the UV melting profile; ∆G0 = ∆HM(1 – T/TM). At 20 mM NaCl and in the absence of urea, TM and ∆HM are equal to 49.6 ± 0.1 °C and 38.1 ± 0.6 kcal mol-1, respectively. With these numbers, we calculate at 25 °C a ∆G0 of 2.9 ± 0.6 kcal mol-1 in good agreement with the ureaunfolding-based value of 2.5 ± 0.4 kcal mol-1. The agreement is significant and suggests that the LEM analysis of urea-induced unfolding profiles of G-quadruplexes provides an alternative way of characterizing G-quadruplex stability. Our determined m-value of 0.57 ± 0.09 kcal mol-1M-1 is within the range of similar values determined for other nucleic acid structures 37, 44-45. For example, the urea mvalues for the formation of a number of 12-meric DNA and RNA duplexes range between 0.58 and 0.98 kcal mol-1M-1 37. The Nature of the Urea-Induced Unfolded State. The CD spectrum of the ureainduced unfolded state of Tel22 at 20 mM NaCl and 8 M urea with a positive peak at 258 nm and a negative peak at 278 nm (see Figure 2a) is similar to that the unfolded state Tel22 adopts in the atmosphere of tetrabutylammonium ions in the absence of stabilizing Na+ ions 25. Figure 3 compares the CD spectra of the urea-induced unfolded state of Tel22 at 20 mM NaCl and 8 M urea and the heat-induced unfolded state at 20 11 ACS Paragon Plus Environment


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mM NaCl and 95 °C in the absence of urea. Inspection of Figure 3 reveals that the urea-induced unfolded state is more structured compared to the heat-induced state at 95 °C. The latter does not display the characteristic peaks at 258 and 278 nm. Significantly, an increase in temperature causes a gradual (non-cooperative) conversion of the CD spectrum of the urea-induced unfolded state of Tel22 into that of the heatinduced unfolded state. The inset in Figure 3 presents the temperature dependence of the molar ellipticity at 257 nm. Inspection of the inset reveals that the urea- and heatinduced unfolded states are not separated by a cooperative transition. Given these results, we conclude that the denaturing effect of urea on Tel22 is milder compared to that of temperature. At room temperature, the urea-induced unfolded state of Tel22 displays a significant amount of residual base stacking. This result is in agreement with the data from the Record lab who, based on a comparative analysis of the experimental and predicted m-values for duplex DNA and RNA, have concluded that the separated single strands are 60 to 90 % stacked 37. A qualitatively similar inference has been drawn from comparing the UV absorbance spectra of single stranded DNA and the respective mononucleotides 69. In agreement with our data, it has been suggested that the degree of single strand unstacking and the related exposure of previously buried solvent-accessible surface area is essentially independent of urea concentration 37. However, the stacking decreases as the temperature increases 37. Incomplete denaturation of DNA in the presence of urea contrasts the denaturing action of urea on proteins. As a general rule, urea-induced unfolded states of proteins are more fully disordered compared to the heat-induced denatured states which may retain significant amounts of secondary structural elements 38-39.


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Urea-Salt Phase Diagram. Figure 8 presents the three-dimensional plot of the combined effect of NaCl and urea on the transition temperature, TM. The TM-surface shown in Figure 8 separates the folded and unfolded conformations of Tel22. Inspection of Figure 8 along with Figure 5 reveals two interesting features. First, at all NaCl concentrations studied in this work, an increase in the concentration of urea brings about thermodynamic destabilization of the G-quadruplex as reflected in a nearly linear decrease in TM (see Figure 5). This observation is consistent with the reports of nonspecific detrimental effect of urea on the thermal stability of nucleic acid structures including an RNA pseudoknot and hairpin and polymeric DNA and RNA duplexes 43, 46, 48-49

. Second, at each experimental urea concentration, an increase in the

concentration of sodium ions leads to an increase in TM fully or partially offsetting the destabilizing influence of urea (see Figure 5). While a similar behavior has been reported for RNA structures 43, the molecular nature of the stabilizing action of cations in a G-quadruplex and other (canonical) nucleic acid structures is very different 63-65. In Gquadruplexes, the stabilizing action of cations originates predominantly from their specific binding inside the central cavity, while the nonspecific external binding (counterion condensation) may be very slightly stabilizing, insignificant, or even strongly destabilizing 63-65. In contrast to G-quadruplexes, canonical nucleic acid structures, e. g. duplexes, are stabilized by counterion condensation 70-73. As mentioned above, transition enthalpy data shown in Figure 6 exhibit a tendency to increase with temperature. The apparent slope, ∆∆HM/∆TM, of 0.14±0.04 kcal mol-1K1

may represent a change in heat capacity, ∆CP, associated with the heat-induced

denaturation of Tel22. An alternative explanation may reflect the fact that ∆CP is



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markedly influenced by a change in DNA solvation following the unfolding transition 7478

. An increase in urea leads to increasing replacement of water by urea within the

hydration shell of DNA with the resulting alteration of solute-solvent interactions. An alteration of the nature of solute-solvent interactions in the vicinity of the folded and unfolded states of Tel22 with an increase in urea concentration may result in the observed temperature dependence of the transition enthalpy, ∆HM, with an apparently positive ∆CP. Interactions of Urea with the Folded and Unfolded States of Tel22. The observed deleterious effect of urea on the stability of Tel22 is in line with the previous reports of urea destabilizing the secondary and tertiary structures of a wide range of DNA and RNA constructs as manifested, for example, in a decrease in their melting temperatures, TM 37, 43-45, 47-49. The destabilizing effect of urea on nucleic acid structures is due to its favorable interactions with the rings and functional groups of nucleic acid bases which become exposed to solvent in the unfolded state 37. Significantly, urea interacts favorably with all components of nucleic acids with a local/bulk microscopic partition coefficient, KP, ranging between 1.09 to 1.37 37. If normalized per solventaccessible surface area, urea interacts most favorably with the heterocyclic aromatic rings of all nucleic acid bases and with the methyl group of thymine 37. While this assessment is based on studies of small analogs of nucleic acids and has been applied to DNA and RNA duplex-to-single strand transitions 37, it is plausible to expect a similar molecular basis for the effect of urea on G-quadruplex stability. A thermodynamically rigorous way of quantifying accumulation or exclusion of a cosolvent around a macromolecule in question is given by preferential interaction


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parameter Γ23 = (∂m3/ ∂m2)T,P,µ3, where m2 and m3 are the molalities of the cosolvent and the macromolecule, respectively; while µ3 is the chemical potential of the cosolvent 40, 79

. The difference in preferential interaction parameter between the folded and

unfolded macromolecular (protein or DNA) states, ∆Γ23, is related to cosolvent-induced modulation of the folded/unfolded equilibrium constant, K, according to ∆Γ23 = (∂lnK/ ∂lna3)T,P,m2, where a3 is the activity of the cosolvent 79-82. The differential preferential interaction parameter, ∆Γ23, is related to the transition-induced changes in excess numbers of solvent, ∆n1, and cosolvent, ∆n3, around the solute as follows: ∆Γ23 = ∆n3 - (N3/N1)∆n1

(3)

where N1 and N3 are the numbers of mole of the principal solvent (water) and cosolvent in solution 40, 80, 83-86. Urea is very slightly excluded from the surface of double-stranded DNA but significantly accumulated at the surface exposed in DNA and RNA unfolding, in particular, the amide-like surfaces of guanine, cytosine, and, especially, thymine and uracil bases 47. There are no data on the preferential interactions of urea with Gquadruplexes. Nonetheless, it might be expected that, by analogy, urea is also very slightly excluded (or very slightly accumulated) from the surface of the G-quadruplex conformation while being strongly accumulated around the unfolded state. The m-value and differential interaction parameter, ∆Γ23, are related via the following relationship 36: m ≈ RT∆Γ23/[urea]

(4)

The urea m-value of a protein is proportional to a change in solvent-accessible surface area, ∆SA, accompanying the unfolding transition 87. Comparison of Eqs. (3) 15 ACS Paragon Plus Environment


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and (4) reveals that the ratio m/∆SA correlates with and reflects the net cosolvent excess or depletion around 1 Å2 of the newly exposed solute surface. Hence, m/∆SA reflects the complex of differential solute-water and solute-cosolvent interactions that determine the specific effect of a given cosolvent on the stability of the macromolecule in question. It is instructive to compare the ratio m/∆SA for the unfolding of the Tel22 Gquadruplex with that determined for other DNA and RNA denaturation events. A change in solvent accessible surface area, ∆SA, accompanying the unfolding of the Na+stabilized Tel22 G-quadruplex has been estimated to be 1230 Å2 25. With this estimate and our determined m-value of 0.57±0.09 kcal mol-1M-1, we calculate the ratio m/∆SA of 0.46 cal mol-1M-1Å-2. Survey of the literature reveals a large variation of the reported values of m/∆SA for DNA and RNA unfolding reactions. Shelton et al. 44 have estimated an average value of urea m/∆SA of 0.099 cal mol-1M-1Å-2 for five self-complementary RNA duplexes of varying lengths and tRNAPhe. While this estimate is close to that observed for protein unfolding 87, it is much smaller than 0.46 cal mol-1M-1Å-2, our estimate for the unfolding of the Tel22 G-quadruplex. On the other hand, Lambert and Draper 45 have determined, for a number of RNA structures including a hairpin, the tar-tar* RNA kissing loop complex, the tetraloop-receptor (TLR) structural motif, and the aptamer domain of the adenine riboswitch (A-riboswich), higher values of urea m/∆SA ranging between 0.48 (A-riboswitch) and 0.65 (hairpin) cal mol-1M-1Å-2 (close to our estimate for Tel22). Using the urea m-values determined for 12-meric DNA and RNA duplexes reported by Guinn et al. 37 and their calculated changes in solvent-accessible surface areas assuming 50 % stacking of the single stranded conformation, we evaluate the values of m/∆SA 16 ACS Paragon Plus Environment

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ranging between 0.24 and 0.40 cal mol-1M-1Å-2 (again, close to our estimate). Hence, the value of m/∆SA we determined for the Tel22 G-quadruplex is within the range of similar values determined by Lambert and Draper 45 and Guinn et al. 37, while being larger than those reported by Shelton et al. 44. As noted by Lambert and Draper 45, the smallness of the value of m/∆SA reported by Shelton et al. 44 may be due to an unrealistically large estimate of the change in solvent-accessible surface area, ∆SA, used by these authors in their analysis (the unfolded state has been modeled by the fully unstacked conformation). The use of more realistic (smaller) values of ∆SA would bring m/∆SA to higher values (similar to our estimate for Tel22). Given the similarity of urea m/∆SA ratios determined for the Tel22 G-quadruplex and other DNA and RNA structures 37, 45, we propose that the molecular basis of urea-induced destabilization of Tel22 is similar to that of other nucleic acid species.

CONCLUSION We presented the first systematic investigation of the combined effect of urea, salt, and temperature on the stability of a G-quadruplex. We derived the urea-salt-temperature phase-diagram of a telomeric DNA G-quadruplex which, in the presence of Na+ ions, adopts the antiparallel conformation. At 20 mM NaCl and room temperature, urea fully denatures the Tel22 G-quadruplex. An increase in salt mitigates the deleterious effect of urea on Tel22. The determined urea m-value of Tel22 normalized per change in solvent-accessible surface area, ∆SA, is similar to those for other DNA and RNA structures while being several-fold larger than that of proteins. Taken together, our



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results suggest that urea can be used as an analytical tool in thermodynamic characterizations of G-quadruplexes in a manner similar to the use of urea in protein studies. Studies involving a larger selection of G-quadruplexes varied in sequence, topology (parallel, antiparallel, hybrid), and molecularity (monomolecular, bimolecular, tetramolecular) to outline the advantages and the limits of the use of urea in Gquadruplex studies. In particular, a deeper understanding of the effect of solvent and cosolvents on the differential stability of the G-quadruplex and duplex conformations is a step towards elucidation of the modulating influence of different types of cosolvents on duplex-G-quadruplex molecular switches triggering genomic events.

Acknowledgements This work was supported by grant 203816-2012 from the Natural Sciences and Engineering Research Council of Canada (to T. V. C.) and grant 15T-1F048 from the State Committee of Sciences of the Republic of Armenia (to Y. B. D.).


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Figure 1 The CD spectra of Tel22 at 25 °C in the absence of urea at various NaCl concentrations. The NaCl concentration (in mM) for each spectrum is given in the inset. 2a 4000

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Figure 2 The CD spectra of Tel22 at 25 °C at various urea concentrations at 20 (panel a), 50 (panel b), 100 (panel c); 200 (panel d); 400 (panel e); 600 (panel f); 800 (panel g); and 1000 (panel h) mM NaCl. The urea concentrations (in M) for the spectra are given in the insets.



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Figure 3: The CD spectra of the urea-induced unfolded state at 20 mM NaCl and 8 M urea () and temperature-induced unfolded states at 20 mM NaCl and 95 °C in the absence of urea () of Tel22. The inset presents the temperature dependence of the molar ellipticity at 257 nm of the urea-induced unfolded state at 20 mM NaCl and 8 M urea.


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10

[urea], M



Figure 5: The G-quadruplex-to-coil transition temperature, TM, transition temperatures of Tel22 as a function of urea at various NaCl concentrations. The NaCl concentration (in mM) for each set of TM is given in the inset.

50

45

∆HvH, kcal mol-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

40

35

30

25 30

40

50

60

70

80

90

TM,°C

Figure 6: Compilation of transition enthalpies, ∆HM, of Tel22 measured at various combinations of NaCl and urea concentrations plotted against corresponding transition temperatures, TM.


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3000

-1

2000

-1

[θ ]295, deg M cm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1000

0

-1000 0

2

4

6

8

10

[urea], M

Figure 7: The dependence of the molar ellipticity of the Tel22 at 295 nm on the urea concentration at 20 mM NaCl. Experimental data were approximated with Eq. (2) (solid line).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 8: The three-dimensional dependence of the transition temperature, TM, on the concentrations of NaCl and urea.


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Effect of Urea on G-Quadruplex Stability - American Chemical Society

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