UU Base

Article Cite This: J. Phys. Chem. B XXXX, XXX, XXX−XXX

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Energetics, Ion, and Water Binding of the Unfolding of AA/UU Base Pair Stacks and UAU/UAU Base Triplet Stacks in RNA Carolyn E. Carr, Irine Khutsishvili,† and Luis A. Marky* Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, Nebraska 68198-6025, United States

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ABSTRACT: Triplex formation occurs via interaction of a third strand with the major groove of double-stranded nucleic acid, through Hoogsteen hydrogen bonding. In this work, we use a combination of temperature-dependent UV spectroscopy and differential scanning calorimetry to determine complete thermodynamic profiles for the unfolding of polyadenylic acid (poly(rA))·polyuridylic acid (poly(rU)) (duplex) and poly(rA)·2poly(rU) (triplex). Our thermodynamic results are in good agreement with the much earlier work of Krakauer and Sturtevant using only UV melting techniques. The folding of these two helices yielded an uptake of ions, ΔnNa+ = 0.15 mol Na+/mol base pair (duplex) and 0.30 mol Na+/mole base triplet (triplex), which are consistent with their polymer behavior and the higher charge density parameter of triple helices. The osmotic stress technique yielded a release of structural water, ΔnW = 2 mol H2O/mol base pair (duplex unfolding into single strands) and an uptake of structural water, ΔnW = 2 mol H2O/mole base pair (triplex unfolding into duplex and a single strand). However, an overall release of electrostricted waters is obtained for the unfolding of both complexes from pressure perturbation calorimetric experiments. In total, the ΔV values obtained for the unfolding of triplex into duplex and a single strand correspond to an immobilization of two structural waters and a release of three electrostricted waters. The ΔV values obtained for the unfolding of duplex into two single strands correspond to the release of two structural waters and the immobilization of four electrostricted water molecules.

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INTRODUCTION The overall physical and chemical properties of a nucleic acid molecule depend on base pairing, base stacking, ion binding, and hydration.1−4 Experimental and theoretical investigations have indicated that nucleic acid double helices are heavily hydrated.5−7 For instance, X-ray and NMR investigations have shown that water molecules create an ordered structure called “the spine of hydration” in the minor groove of A−T base pairs in B-DNA.8−12 Hydration of the major groove and other DNA conformations has also been reported.13−18 However, in spite of extensive investigations on the hydration of nucleic acids, the details of hydration as it relates to conformation, sequence, and nucleotide composition remain unknown. The reason for this is the presence of two distinct types of hydrating water molecules: structural (around polar and nonpolar groups) and electrostricted (around charges).19 These two types of water are difficult to detect and differentiate, complicating the measurement and analysis of their physical properties, especially their molar volume at the surface of a nucleic acid. Furthermore, the overall hydration of a nucleic acid molecule is closely associated with its number and type of hydrated counterions. For instance, in the interaction of divalent ions with nucleic acids, their close association involves the overlapping of their hydration shells, resulting in a release of water molecules; 20−22 the magnitude of the effect is © XXXX American Chemical Society

determined by the position of this cation relative to the surface of DNA. A larger dehydration effect takes place in the formation of inner-sphere counterion−DNA complexes relative to the formation of outer-sphere complexes because of changes in their molar volumes. Polyadenylic acid (poly(rA)) and polyuridylic acid (poly(rU)) form a double helix in an equimolar (1:1) mixture. Both temperature and salt affect the type of complex in solution, and depending on the experimental solution conditions the different types of species (single strand, duplex, and/or triplex) can be interconverted. The work of Krakauer and Sturtevant shows a phase diagram of the different helix-to-coil transitions or reactions with poly(rA) and poly(rU).23 There are four types of reactions: reaction I is the simple unfolding of a duplex poly(rA)·poly(rU) into single strands and can be achieved by an increase in temperature at lower salt conditions. Reaction II is the unfolding of the 1:2 triplex poly(rA)·2poly(rU), which is the melting of the triplex in a single step to form single strands and occurs at low temperature and high salt. Reaction III is the unfolding of the triplex into a duplex and a single strand poly(rA)·poly(rU) + poly(rU), which refers to the removal of the third or Hoogsteen strand and occurs at lower salt Received: June 11, 2018 Published: June 22, 2018 A

DOI: 10.1021/acs.jpcb.8b05575 J. Phys. Chem. B XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry B

ethylene glycol had their pH checked and adjusted back to 7.0 prior to use. While the dissociation of sodium cacodylate is essentially temperature-independent and therefore a better choice in that regard for melting experiments, the pKa of sodium phosphate is closer to 7.0 and has only a very minor temperature dependence.38 Temperature-Dependent UV Spectroscopy. Absorbance versus temperature profiles were measured at 260 nm using a thermoelectrically controlled Aviv model 14DS UV− Vis (Lakewood, NJ). The temperature was scanned at a heating rate of ∼0.6 °C/min from 1 to 100 °C. Analysis of the resulting melting curves yielded the temperature of the helix− coil transition, TM, and model-dependent van’t Hoff enthalpies, ΔHvH.39 The molecularity of each transition was obtained by monitoring the T M as a function of polynucleotide concentration. Intermolecular transitions show a dependence of TM on strand concentration, whereas the TM of an intramolecular transition is independent of strand concentration. However, because of the length of the polymeric duplex and triplex, they should display pseudo-intramolecular, concentration-independent behavior. Differential Scanning Calorimetry. The total heat required for the unfolding of each helical structure was measured using a VP-DSC differential scanning calorimeter from MicroCal (Northampton, MA). Two cells were heated from 0 to 80 °C at a rate of 0.6 °C/min. The sample cell contained 0.65 mL of polynucleotide solution, whereas the reference cell contained an equal volume of buffer solution. The thermograms were analyzed by buffer subtracting, normalizing by strand concentration, and deconvoluted using a non-two-state zero ΔCp model with the MicroCal software. The TMs and model-independent thermodynamic profiles of ΔHcal, TΔScal, and ΔG°(T) were obtained from a differential scanning calorimetry (DSC) experiment using the following relationships: ΔHcal = ∫ ΔCap dT and ΔScal = ∫ (ΔCap/T)dT, where ΔC p is the anomalous heat capacity of the oligonucleotides during the unfolding process and ΔHcal and ΔScal are the folding enthalpy and entropy, respectively, both assumed to be temperature-independent. The free energy change at any temperature, ΔG°(T), can be calculated with the Gibbs equation, ΔG°(T) = ΔHcal − TΔScal. Alternatively, ΔG°(T) can be calculated using the equation ΔG(T) ° = ΔHcal(1 − T/ TM). Additional DSC experiments were performed at several salt concentrations to indirectly obtain the associated heat capacity contributions, ΔCp, which were determined from the slopes of the lines of the ΔHcal versus TM plots. Determination of the Differential Binding of Counterions and Water. Additional UV melting curves were performed to determine both the differential binding of counterions, ΔnNa+, and differential binding of water molecules, ΔnW, between the helix and coil states. Application of the chain rule yielded the following relationships, respectively:40,41

conditions and lower temperatures. Reaction IV, called doublestrand disproportionation, involves one duplex acquiring the appropriate third strand from a second duplex to form a triplex and single strand.23 This type of reaction occurs at high temperatures and high salt.23 The classic work23 of Krakauer and Sturtevant was obtained 50 years ago using mainly spectroscopic measurements with only a few data points from calorimetric experiments. Our laboratory is recreating this work using modern instrumentation and experimental techniques to obtain complete thermodynamic profiles for the unfolding of these complexes, including the differential binding of ions and water and heat capacity effects. In addition, we wanted to understand the role of water on the unfolding of these complexes by correlating the osmotic stress measurements (differential binding of water) with the volume changes obtained from pressure perturbation calorimetry (PPC). Therefore, we provide a more complete picture of this system that is still currently utilized as a model system for drug binding24−29 and has been identified as a regulatory structure in numerous biological systems.30−32 The UAU triplex is one of two relevant RNA triplexes, as previous studies have shown that unlike DNA, RNA triplexes cannot be formed with a purine-rich third strand which binds antiparallel to the duplex through reverse Hoogsteen hydrogen bonds.33−35 As such, only UAU and C+GC triplexes can form using RNA. In this work, we used a combination of spectroscopy and calorimetric techniques to investigate the unfolding energetics, ion, and water binding, for the formation of a polymeric RNA duplex (duplex) and triplex (triplex). In both cases, the folding of each polymer helix was favorable as evidenced by the negative free energy terms. The thermodynamic results correlated well with those determined previously.23 Furthermore, the folding of these structures yielded an uptake of ions for both helices, immobilization of structural water (duplex) and release of structural water (triplex). Volume studies done in conjunction with osmotic stress experiments allowed us to determine that during unfolding of triplex into duplex and a single strand, there are two structural waters taken up and three electrostricted waters released. During unfolding of duplex into two single strands, those two structural waters are released and four structural waters are immobilized.

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MATERIALS AND METHODS Materials. The nucleic acids poly(rA)·poly(rU) and poly(rU) were purchased from Sigma-Aldrich. The average length of the duplex and triplex was 1075 base pairs. The poly(rU) was purchased as the potassium form and converted to the sodium form by dissolving it in 1 M NaCl, heating to 90 °C for 30 min, and cooling to room temperature. Both samples were then desalted by size exclusion chromatography using a G-10 Sephadex column. The solution concentration of these polynucleotides was determined using a PerkinElmer LAMBDA-10 spectrophotometer at 260 nm (single strand and duplex) or 257 nm (triplex) and 20 °C with the following molar extinction coefficients:36,37 9.35 mM−1 cm−1 (single strand), 8.98 mM−1 cm−1 (duplex), and 5.9 mM−1 cm−1 (triplex). Inorganic salts from Sigma-Aldrich were of reagent grade and used without further purification. All measurements were made in buffer solutions containing 10 mM sodium phosphate (NaPi) at pH 7.0 and adjusted to different salt concentrations using NaCl and different osmolyte concentrations using ethylene glycol. Buffers adjusted with NaCl and

ΔnNa+ = (∂ln K /∂TM)(∂TM /∂ln[Na +]) = 1.11[ΔHcal /RTM 2](∂TM /∂ln[Na +])

(1)

ΔnW = (∂ln K /∂TM)(∂TM /∂ln[aW ]) = [ΔHcal /RTM 2](∂TM /∂ln[aW ])

(2)

These ΔnNa+ and ΔnW terms represent the uptake/release of counterions and water, respectively, for the helix−coil transition of each complex. The terms in brackets are B


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Figure 1. (Left) Job plot for the formation of a triplex by titrating poly(rU) into poly(rA)·poly(rU). (Right) TM-dependence on total strand concentration at 260 nm in 10 mM NaPi, pH 7.0, for duplex (squares) and triplex (circles).

were measured with an Anton Paar (Graz, Austria) DMA densitometer in the differential mode, using two 602-M micro cells, each with a volume of ∼150 μL. The reference cell is filled with water, whereas the measuring cell is filled with solution or buffer. The density, ρ, is calculated from the oscillation period, T, of the cell using the following relationship: ρ = AT2 + B, where A and B are constants determined from calibrating densities (and periods) of water and air.36

determined directly from DSC experiments, whereas the terms in parentheses are the slopes of the plots of TM as a function of sodium concentration and water activity, respectively. The value of 1.11 in eq 1 is a constant used to convert the activity of Na+ into concentrations. The activity of water is varied by using different concentrations (0.5−2.5 M) of a cosolute, ethylene glycol, which does not interact specifically with nucleotides.42 The osmolality of the ethylene glycol solutions was measured with a model 830 UIC vapor pressure osmometer, which was calibrated with standardized NaCl solutions. The osmolalities were converted into water activity using the equation ln aW = −Osm/MW,43 where Osm is the measured solution osmolality and MW is the molality of pure water. Measurement of the differential binding of water using the osmotic stress method yields only the immobilization of structural, not electrostricted, water. Pressure Perturbation Calorimetry. A VP-DSC differential scanning calorimeter equipped with a pressure perturbation accessory was used to measure the heat (ΔQ) resulting from the application of an additional pressure change (ΔP) above the solutions of the calorimetric cells. A complete description of this PPC technique can be found elsewhere.44 Prior to the PPC scans, standard DSC curves were obtained for each helical polynucleotide, duplex and triplex, to determine the temperature range and temperature step to be used in the PPC experiment. A sample solution with a concentration of ∼50 OD was allowed to equilibrate against the same buffer solution (reference cell) at constant temperature and external pressure. The external pressure is then increased by ∼50 psi, causing heat to be absorbed differentially by the sample and reference cells. These heats, ΔQ, are obtained from integration of the compression and decompression peaks resulting from switching the external pressure on and off at particular temperatures determined from the DSC curves. The ΔQ values are used to measure the apparent coefficient of thermal expansion, α(T), from the integration of the relationship: (∂Q/ ∂P)T = −T(∂V/∂T)p = −Tα(T)V, yielding ΔQ = −TVα(T)ΔP, where V is the apparent molar volume of the solute. For a two-component system: α(T) = αo − [ΔQ/(TVΔP)], where αo is the thermal coefficient of the solvent. Integration of α(T) over the temperature range of the unfolding reaction, ∫ α(T) dT, yields the relative volume changes of the solute, ΔV/V, where ΔV is the unfolding volume of the macromolecule. The value of V is obtained from the equation: V = M/ρo − (ρo − ρ)/ρoC,45 where M is the molecular mass of the polynucleotide and ρo and ρ are the densities of the solvent and solution, respectively. The densities of these solutions

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RESULTS AND DISCUSSION UV Melting Curves. To determine if triplex (poly(rA)· 2poly(rU)) will form in a stoichiometric ratio of 1:1 from duplex (poly(rA)·poly(rU)) and single-strand (poly(rU)), a Job plot was obtained (Figure 1, left). Solutions of duplex and single strand were mixed at different molar ratios in 10 mM NaPi buffer containing 0.2 M NaCl at pH 7.0 to obtain the plot in Figure 1. The two lines intercept at a molar fraction of 0.5, which corresponds to the formation of a 1:2 triplex (poly (rA)·2poly(rU)). In future experiments, when forming a triplex, an excess of 5% of the single strand was added to promote complete formation of a duplex into a triplex. Although long polymers should display pseudo-intramolecular concentration-independent behavior, the molecularity of both duplex and triplex was determined. To obtain the molecularity of each complex, melting curves as a function of strand concentration were obtained, and their TM dependencies are shown in Figure 1 (right). Over a 10-fold range of strand concentration, the TM remains constant for duplex and triplex, which indicates that each polymer helix is unfolding through a pseudo-monomolecular transition, as expected for long polynucleotides. DSC Unfolding. DSC curves in 10 mM NaPi at pH 7.0 with salt concentrations ranging from 16 to 216 mM Na+ for duplex are shown in Figure 2 (top), the curves for triplex are shown in Figure 2 (bottom), and the thermodynamic parameters obtained are displayed in Table 1. At lower salt concentrations, the duplex unfolds in a single monophasic transition; at low salt, the only possible reactions that could occur are reaction I, duplex unfolding, or reaction III, unfolding of a triplex into a duplex and a single strand. Because this is the unfolding of the preformed duplex and triplex formation conditions have not been met, this peak is attributed to reaction I, or the melting of a duplex to form two single strands. However, upon increasing the salt concentration to 216 mM Na+, the transition becomes biphasic. At this salt concentration, an additional reaction could occur, either C


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namic profiles show that unfolding of duplex and triplex is unfavorable as evidenced by the positive ΔG(20) ° , indicating that folding is favorable. The enthalpy per base pair at low salt is lower than expected based on the nearest-neighbor enthalpy values.46 However, these N−N parameters are calculated at a 1 M salt concentration. Upon increasing the salt concentration, the enthalpy increases as expected, until it reaches a maximum at approximately 216 mM Na+ with a value of 8.1 kcal/mol, which is higher than the expected value for an AA/UU base pair stack calculated by Douglas Turner (6.8 kcal/mol).46 Formation of a triplex is favorable; at 91 mM Na+ the enthalpy doubles compared to 38 mM Na+, and the free energy is twice as large. The overall free energy contribution, determined at 20 °C, was favorable at all salt concentrations. It can be seen in Table 1 that the favorable free energy term, ΔG°(20), results from a favorable enthalpy term compensated by an unfavorable entropy term. The favorable enthalpy term is due to the formation of base pairs and base pair stacks, whereas the unfavorable entropy term involves the ordering of strands and the immobilization of ions and water. The enthalpy contributions were discussed previously. The entropy contributions, (TΔScal), for the unfolding of each molecule are shown in the sixth column of Table 1. The magnitude of these entropies follows the same order as those of the enthalpies. Heat Capacity Effects. The heat capacity of a molecule can provide information on the folded state; unfolding heat capacities are typically positive, indicating an exposure of hydrophobic groups to solvent upon unfolding.47 This is because hydration of nonpolar surfaces causes a positive effect on heat capacity, whereas hydration of polar surfaces has a negative effect on heat capacity;48−51 the magnitude of nonpolar hydration is greater than that of polar hydration. The folding of biomolecules secludes both nonpolar and polar surfaces, although the typically more nonpolar surface is buried. Upon unfolding, the nonpolar and polar surfaces are both exposed,3 but the magnitude of nonpolar exposure dominates the heat capacity term, leading to positive values. The heat capacity of a single base pair is ∼30−80 cal/K mol, based on previous research.37,52−54 Indirectly, the heat capacity for the unfolding of the duplex or triplex can be estimated from the slope of a plot of ΔHcal versus TM (Figure 3) obtained from DSC thermograms at different salt concentrations. The ΔHcal

Figure 2. DSC thermograms of duplex (top) and triplex (bottom) in 10 mM NaPi buffer at pH 7.0 with increasing salt concentrations.

reaction II, or the unfolding of the triplex into three single strands, or reaction IV, double-strand disproportionation, which involves one duplex taking a strand from another. The first peak at 62.5 °C is likely double-strand disproportionation, as outlined by Krakauer and Sturtevant, whereas the second peak at 64.7 °C is the melting of the remaining duplexes into single strands. The unfolding of the triplex is biphasic at a low salt concentration and becomes monophasic at high salt, the opposite trend of a duplex. At low salt concentrations, the only possible reactions are reaction I, or duplex unfolding into single strands, and reaction III, or triplex unfolding into a duplex and a single strand. Because triplex formation is more unstable than duplex formation, the first transition is likely attributed to reaction III, whereas the second transition is reaction I, or the unfolding of the remaining double-stranded oligonucleotides. At higher salt, both reactions are occurring simultaneously, as evidenced by the identical TMs compared to duplex dissociation (reaction I). Table 1 lists the thermodynamic profiles for the unfolding of duplex and triplex at all salt concentrations. The thermody-

Table 1. Thermodynamic Profiles for the Unfolding of Duplex and Triplexa [Na+] (mM) 16 38 91 216

16 38 91 216

transition

TM (°C)

ΔHcal (kcal/mol)

ΔG°(20) (kcal/mol)

1st 2nd

42.6 49.1 55.7 62.5 64.8

4.7 6.2 6.8 8.4 8.4

0.3 0.6 0.7 1.0 1.2

33.5 43.2 43.8 49.8 55.1 64.0

2.8 7.8 5.2 8.0 14.4 14.6

0.1 0.6 0.4 0.7 1.5 1.9

1st 2nd 1st 2nd

TΔScal (kcal/mol) Duplex 4.4 5.6 6.1 7.4 7.2 Triplex 2.7 7.2 4.8 7.3 12.9 12.7

ΔnNa+ (mol Na+/mol bp)

ΔnW (mol H2O/mol bp)

ΔCp (cal/mol K bp)

0.15

1.5

80

0.30

(−1.6) 2.2

190

Experimental errors are as follows: TM (±0.5 °C), ΔHcal (±5%), TΔS (±5%), ΔG(20) ° (±7%), ΔnNa+ (±12%), ΔnW (±12%), ΔCp (±20%). ΔnW values in parenthesis correspond to the unfolding of the triplex into duplex and a single strand. a

D


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study. As shown in both the UV and DSC melts, increasing the concentration of salt increases the TM in a linear fashion. For reactions I and IV, we obtained calorimetric results in excellent agreement with previously published data. However, for reaction III we obtained calorimetric results which differed from the classic work (solid vs dashed lines). Our value for the slope of reaction III was smaller (26.3 vs 34.3). Differential Binding of Counterions and Water Molecules. Equation 1 was used to calculate the differential binding of counterions, whereas eq 2 was used to calculate the differential binding of water. The slope of the TM-dependence on salt or osmolyte concentration was obtained from the UV melting curves as a function of NaCl or ethylene glycol concentration (Figures S1 and S2). The average ΔHcal/RTM2 term was obtained from the DSC thermograms at several salt and ethylene glycol concentrations. The folding of oligonucleotides is typically accompanied by an uptake of ions because of a shift in the helix−coil equilibrium toward the state with the higher charge density parameter.55 Folding is also accompanied by an uptake of water molecules toward the conformation with a higher hydration state.40,56 We obtained negative ΔnNa+ values for the folding of each complex, indicating that both duplex and triplex formation is accompanied by an uptake of Na+, which is indicative of the folded state having the higher charge density parameter as expected. The ΔnNa+ value of the −0.15 mol Na+/mol base pair is in excellent agreement with previously obtained values of −0.17 mol Na + /mol base pair obtained for polymer systems.1,2,57 Triplex uptake is larger, with a ΔnNa+ value of −0.30 mol Na+/mol base triplet, as expected because triplexes have a higher charge density parameter. Interestingly, the ion uptake is approximately twice as large despite the interaction of one additional strand. We used the osmotic stress technique to determine ΔnW values by increasing the amount of ethylene glycol in solution resulting in a change in the water activity. The osmotic stress method works by utilizing the difference in distribution of solute, which then changes the distribution of water.58 There are areas between, within, and around macromolecules where cosolutes are excluded, creating a negative pressure to draw water away from cosolute-depleted regions, in this case the oligonucleotides. This leads to an overall dehydration and a shifting of the helix−coil equilibrium toward lower temperatures. These values are listed in the eighth column of Table 1. We obtained a value of 1.5 mol of water per mole of base pair for the unfolding of the duplex into two single strands, indicating that during unfolding there is a release of structural water molecules. This is smaller than what is typically obtained for DNA (3−5 waters per base pair)40,59,60 and may be a feature of RNA hydration (i.e., that RNA is less hydrated or homogeneous A•U RNA tracts are less hydrated). The differential binding of water for the unfolding of the triplex is more complicated, as there are two transitions. The first transition, or the unfolding of the triplex into a duplex and a single strand, has a negative value, indicating that there is an uptake of 1−2 structural waters during unfolding, that is, removal of the third strand leads to an increase of 1−2 structural waters, most likely within the now vacant major groove. This can be compared to the heat capacity value obtained for the triplex; the RNA triplex exposes more hydrophobic groups, leading to an increase in structural water release upon unfolding. The second transition corresponds to the unfolding of the duplex into single strands and has a ΔnW =

Figure 3. Heat capacity plot of the duplex (squares) and triplex (circles).

increases with increasing TM for the unfolding of the duplex. This yielded a ΔCp value of 80 cal/K mol bp, in excellent agreement with previously determined per-base pair heat capacity values. The value for the triplex is higher, at 190 cal/K mol bp. Subtracting 80 cal/K mol for the duplex, insertion of the third strand yields a positive heat capacity value of 110 cal/ K mol. This is interesting, as a recent review from our lab indicated that most triplexes yield a negative heat capacity, because of the high sequestration of hydrophilic groups and the large hydrophobic surface when the triplex is fully folded.47 However, these previous studies were on DNA triplexes; RNA triplexes may have opposite heat capacity signs because of conformational differences. Nevertheless, the heat capacity value of the triplex indicates an exposure of more hydrophobic groups than hydrophilic groups upon unfolding compared to the duplex. Phase Diagram. Using the combined results from the UV and DSC melting curves, a phase diagram was constructed (Figure 4). A phase diagram can provide a visual guide to

Figure 4. Phase diagram for the potential unfolding reactions of the duplex and triplex. Calorimetric points are closed circles, and UV points are open circles. Dashed lines correspond to the work by Krakauer and Sturtevant, and solid lines correspond to the data obtained in this work. Reaction I: poly(rA)·poly(rU) → poly(rA) + poly(rU); reaction II: poly(rA)·2poly(rU) → poly(rA) + 2poly(rU); reaction III: poly(rA)·2poly(rU) → poly(rA)·poly(rU) + poly(rU), and reaction IV: 2(poly(rA)·poly(rU)) → poly(rA)·2poly(rU) + poly(rA).

determining what state the system will adopt under a given set of solution conditions. Figure 4 shows a phase diagram for the poly(rA)·poly(rU) system at pH 7, by plotting TMs as a function of salt concentration. There are four regions on this plot, and the four dotted lines correspond to the reactions of the work of Krakauer and Sturtevant.23 The solid lines in Figure 4 are based on the calorimetric data obtained in this E


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The Journal of Physical Chemistry B 2.2 water/mol base pair, indicating that duplex unfolding is releasing water molecules, that is, unfolding of the duplex into single strands releases those two waters in the major groove. This value is within the experimental error of the value obtained for the duplex, and it can be stated that the folding of a polymeric A•U tract RNA duplex immobilizes two water molecules per base pair. Volume Studies. We performed PPC to provide a more comprehensive picture of the hydration surrounding the polymeric complexes. ΔnW obtained through the osmotic stress method pertains only to structural water, while PPC measures the heat changes associated with changes in pressure and describes the overall hydration, including structural waters, electrostricted waters, and counterions forming inner sphere complexes. PPC follows the temperature dependence of the coefficient of thermal expansion, or α.44 The magnitude of α and its baselines provide information on the type of water involved in the unfolding process. The PPC data shown in Figure 5 start at

Table 2. Calorimetric and Volumetric Results for the Unfolding of the Duplex and Triplexa TM (°C)

ΔV/ΦV

ΦV (mL/mol)

45.9

−0.0088

328

ΔV (mL/mol)

ΔnW (mol H2O/mol)

Duplex −2.9

1.5

−0.3 −3.2

−1.6 2.2

Triplex 35.2 49.1

−0.0006 −0.0061

519

Experimental errors are as follows: TM (±0.5 °C), ΦV (±8%), ΔV (±15%), ΔnW (±12%).

a

first transition has a small volume contribution, ΔV, of −0.3 mL/mol bp and corresponds to the removal of the third strand, followed by a second transition with a ΔV of −3.2 mL/ mol bp, which corresponds to the unfolding of the duplex state to random coils. This is in excellent agreement with the value of the single transition obtained for the unfolding of the duplex, ΔV = −2.9 mL/mol (Table 2). The unfolding of the triplex (first transition) and the unfolding of the duplex (second transition) yielded a small contraction (negative value of ΔV) of the system. Overall, our unfolding volumes are in fair agreement with literature values obtained from different experimental techniques. For the helix−coil transition of poly(rA)·poly(rU), Chalikian et al.37 obtained a ΔV of 0 ± 2 mL/mol bp from ultrasonic velocity measurements, whereas Noguchi et al.62 obtained a ΔV value of 1.5 mL/mol base pair from dilatometry measurements. In addition, Noguchi et al. obtained a ΔV of 2.5 mL/mol base triplet for the unfolding of the poly(rA)·2poly(rU) triplex. Correlation of ΔV with ΔnW. Volume changes for a given process arise from differences in the molar volume of the immobilized (hydrating) water, structural and electrostricted. Using the assumption that voids contribute negligible packing effects in nucleic acids, we identified the net type of water involved in a given process by comparing the sign of ΔG°(20) (or enthalpy−entropy compensation) with the sign of ΔV. Similar signs indicate electrostricted hydration, whereas opposite signs suggest structural hydration.19,44,63 We obtained opposite signs for the unfolding of the duplex into single strands, ΔG(20) ° = 0.3 kcal/mol (enthalpy−entropy compensation = 4.4 kcal/mol) and ΔV = −2.9 mL/mol, indicating a net structural hydration. Opposite signs were also obtained for the unfolding of the triplex into duplex and a single strand, ΔG°(20) = 0.1 kcal/mol (enthalpy−entropy compensation = 2.7 kcal/ mol) and ΔV = −0.3 mL/mol, indicating a net structural hydration. On average, duplex unfolding yielded a net release of ∼2 structural water molecules (ΔnW = 1.5 mol water). Using a molar volume of 13.5 mL/mol44 for structural water gives a volume expansion of 6.7 mL/mol bp (ΔV = 1.5 × 4.5 mL/mol structural water, where 4.5 is the difference between bulk water and structural water molar volume). In the case of the unfolding of the triplex, the uptake of 1.6 structural waters yields a volume contraction of −7.2 mL/mol (ΔV = −1.6 × 4.5 mL/mol structural water); however, the release of electrostricted water contributes to a volume expansion of the system. The number of electrostricted water molecules in the unfolding of the duplex and triplex can be estimated by using our experimentally determined ΔV, which we will call ΔVu. ΔVu comprises the molar volume changes from structural water, ΔVSt, and electrostricted waters, ΔVEl. The molar

Figure 5. (Top) DSC melting curve of duplex and triplex taken before the PPC experiment. (Bottom) Volumetric measurements for duplex (circles) and triplex (squares). Oligonucleotides are indicated by closed symbols and baselines by open symbols. All measurements were obtained in 10 mM NaPi buffer, pH 7.0.

a positive value of α, and the negative slope indicates the exposure of hydrophilic groups. It can be seen from the starting point of the curve that the duplex exposes more hydrophilic groups than the triplex, which is seen in crystal structures of oligonucleotides; triplexes have more hydrophobic surface area than duplexes because insertion of the third strand hides the exposed hydrophilic areas of the base pairs. As the experiment continues and the polymer unfolds, the slope becomes positive, indicating exposure of hydrophobic groups, and the baseline moves closer to zero. In the end, the random coil state of each oligonucleotide complex is the same, indicating equal exposure of hydrophobic and hydrophilic groups. Analysis of the peaks61 yielded the ΔV values shown in Table 2. The triplex has two transitions. The F


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The Journal of Physical Chemistry B Present Address

volume changes due to structural water hydration were calculated previously (ΔVSt = 6.7 mL/mol for duplex and −7.2 mL/mol for triplex). In this calculation, we are not considering the binding of counterions, as the monovalent ions that are present in this study do not change their hydration state upon binding to a nucleic acid and so do not influence these calculations.1 In the case of the duplex, we obtained a ΔVEl of −9.6 mL/mol, and using a molar volume difference of 2.564 (difference in the molar volume between bulk and electostricted water), this yields an uptake of ∼4 electrostricted waters during unfolding. For the triplex, we obtained a ΔVEl of 6.9 mL/mol, which yielded a release of ∼3 electrostricted waters during unfolding. In summary, the volume change associated with the triplex unfolding into a duplex and a single strand results in the immobilization of two structural water molecules and the release of three electrostricted water molecules. Duplex unfolding into two single strands results in the immobilization of four electrostricted water molecules and release of the two structural water molecules.

†

Institute of Physics, Javakhishvili State University, Tbilisi, Georgia. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank Chris M. Olsen for preliminary data. This work was supported by grants MCB-0315746 and MCB1122029 from the National Science Foundation.

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CONCLUSIONS In this work, we have obtained complete thermodynamic profiles for the unfolding of two RNA polymers, poly(rA)· poly(rU) (duplex) and poly(rA)·2poly(rU) (triplex). We confirmed their pseudo-monomolecular melting behavior and triplex stoichiometry using a Job plot. We then determined their unfolding thermodynamic parameters (TM, ΔHcal, TΔScal, ΔG°(20), and ΔCp) using calorimetry. The favorable folding of both the duplex and triplex takes place through the characteristic compensation of a favorable enthalpy and unfavorable entropy contributions. The DSC unfolding at several salt concentrations allowed us to measure both heat capacity effects and ΔnNa+. The unfolding of the triplex yielded a higher heat capacity and a higher release of counterions. This means that the triplex is exposing more nonpolar groups to the solvent and has a higher charge density parameter than the duplex. Furthermore, we were able to confirm the phase diagram obtained earlier by Krakauer and Sturtevant. The correlation of the unfolding volumes, obtained from PPC, with the differential binding of water, obtained by the osmotic stress technique, shows the triplex unfolding into a duplex and a single strand, resulting in the immobilization of two structural waters and the release of three electrostricted water molecules, whereas the duplex unfolding into two single strands results in the release of two structural water molecules and the immobilization of four electrostricted water molecules.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.8b05575.

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REFERENCES

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TM dependence on salt and ethylene glycol concentration (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (402) 559-4628. Fax: (402) 559-9543. ORCID

Carolyn E. Carr: 0000-0002-9688-6015 Luis A. Marky: 0000-0001-5572-7707 G


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