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DNA Thermal Stability Depends on Solvent Viscosity Nancy C. Stellwagen, and Earle Stellwagen J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b01217 • Publication Date (Web): 01 Apr 2019 Downloaded from http://pubs.acs.org on April 8, 2019

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The Journal of Physical Chemistry

DNA Thermal Stability Depends on Solvent Viscosity

Nancy C. Stellwagen* and Earle Stellwagen Department of Biochemistry, University of Iowa, 51 Newton Road, Iowa City, IA 52242, USA. *Corresponding Author: Tel.: 319-335-7896; Fax: 319-335-9570; Email: [email protected]

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ABSTRACT: Capillary electrophoresis has been used to measure the thermal stability of small DNA hairpins in solutions containing 0.3 M cation, comparing the results observed in Na + and NH4+ with those observed in solutions containing various tetraalkylammonium ions. The midpoint melting temperatures of the hairpins decreased non-linearly with cation radius, but linearly with solvent viscosity, suggesting that the reversible melting transition involves DNA migration through the solvent to find stable base-pairing partners. The normalized melting temperatures increased linearly with the inverse viscosity of the solvent and agreed with values calculated from literature data for another small DNA hairpin, a small RNA duplex and sonicated calf thymus DNA in tetraalkylammonium ion solutions. The normalized melting temperatures calculated from literature data for poly(A)·poly(U) and two proteins, ribonuclease and lysozyme, in tetraalkylammonium ion solutions also increased linearly with inverse solvent viscosity. By contrast, the normalized melting temperatures calculated from literature data for DNA in solutions containing ethylene glycol or glycerol to modify the viscosity increased linearly with the logarithm of inverse solvent viscosity, not the first power of inverse solvent viscosity.


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INTRODUCTION Many studies of the thermal stability of DNA (and RNA) are carried out in dilute solution to eliminate solute-solute interactions during the measurements. By necessity, the viscosity of the solvent is then close to the viscosity of water. However, studies of the reversible denaturation/renaturation of double-stranded1,2 and single-stranded3 DNAs have shown that the rate of renaturation is inversely related to solvent viscosity. 1,2 Hence, the rate-limiting step in the reaction was proposed to be the diffusion of a critical number of DNA nucleotides into the correct configuration to nucleate stable base pair formation.1,2 Most studies of the viscosity dependence of DNA thermal stability have used cosolvents such as glycerol, ethylene glycol or polyethylene glycol to increase the viscosity of the solvent. 1-5 Other studies have used bulky cations, such as the tetraalkylammonium ions, to modify the viscosity.6-11 The midpoint melting temperatures observed for DNA molecules in solutions containing moderate concentrations of small monovalent cations such as Na +, K+, Li+, or NH4+ increase approximately linearly with the logarithm of cation concentration. 6,8,11 The melting temperatures also increase approximately linearly with the logarithm of cation concentration in solutions containing tetramethylammonium (TMA +) or tetraethylammonium (TEA+) ions, although the melting temperatures are lower than observed in solutions containing the same concentration of Na+ or NH4+ ions.6,8 Tetrapropylammonium (TPA+) ions can either increase7,8 or decrease6 DNA thermal stability with increasing cation concentration, suggesting that TPA + interacts equally with the duplex and coil conformational ensembles. By contrast, the DNA melting temperatures observed in solutions containing tetrabutylammonium (TBA +) or tetrapentylammonium (TPeA+) ions decrease linearly with the logarithm of increasing cation concentration.6,8,9 Why DNA and RNA thermal stability depends on the size of the alkyl side chain in tetraalkylammonium (TXA+) ion solutions is not well understood. The lower melting temperatures observed in solutions containing the bulkier TXA + ions have been attributed to hydrophobic interactions of the alkyl side chains with the nucleotide bases, pulling the helix ↔ coil equilibrium toward the coil conformation; 6,12 electrostatic interactions of bulky tetraalkylammonium ions with the relatively widely spaced phosphate residues in single-stranded DNAs, stabilizing the coil conformation; 7 decreased electrostatic interactions between nucleic acids and their counterions in solvents with lower dielectric constants; 9 and/or through-solvent effects of the TXA+ ions on water structure, affecting the hydration of the helix and coil differently.13 To better understand the effect of different monovalent cations on DNA and RNA thermal stability, we have been using free solution capillary electrophoresis (CE) to analyze the melting temperatures of small DNA hairpins in solutions containing various TXA + ions. Hairpins simplify the analysis of the helix/coil transition because the reaction is monomolecular in both the forward and reverse directions. Our previous study, using a well-characterized DNA hairpin with six base pairs in the stem and four thymine bases in the loop, showed that the melting temperature decreased approximately linearly with increasing cation radius. 8 We have now expanded this study to hairpins of the same size with different stem and loop sequences, using a higher TXA+ concentration to better characterize the effect. To determine the generality 3 ACS Paragon Plus Environment


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of the results, the normalized melting temperatures of the hairpins are compared with our previous studies as well with studies of DNA, RNA and proteins in the literature. MATERIALS AND METHODS DNA oligomers All DNA hairpins contained six base pairs in the stem and four nucleotides in the loop; two 16-residue single-stranded oligomers were used as single-stranded references. All oligomers were synthesized by IDT (Coralville, IA), purified by polyacrylamide gel electrophoresis, lyophilized, redissolved in 10 mM Tris-Cl buffer, pH 8.0, and stored at -20 ºC until needed. The sequences of the hairpins and references, along with their short names, are given in the first two columns of Table 1. The observed and predicted melting temperatures (see text) are given in columns 3-5. Table 1. Sequences and short names of hairpins and references, observed melting temperatures in 0.3 M TBA+, and predicted and observed melting temperatures in 0.3 M Na +. Short Namea G1 G1a G1b G2 G3 G3a G3b G3c G3d G3e G4 G5

Sequence Stem Loop Stem GAAAAA-CCCC-TTTTTC GATATA-CCCC-TATATC GATATA-TTTT-TATATC GATGTA-CTCC-TACATC GAGACA-TTCC-TGTCTC AGGACA-TTCC-TGTCCT GAGACA-TTCG-TGTCTC GAGACA-TTCT-TGTCTC GAGACA-TTTT-TGTCTC GAGACA-CCCT-TGTCTC GCAGAC-TTCT-GTCTGC GCAGGC-TTTT-GCCTGC

Tm in 0.3 M Na+ Observed Predictedb 48 48 43 39 45 44 50 59 61 62 60 64 60 68 82

Tm in 0.3 M TBA+ Observed 28 23 25 33 44 47 45 44 45 42 54 63

T16 TTTTTT TTTT TTTTTT R16c CGCAAC TTCT AACATT a The number following G in the hairpin short names corresponds to the number of GC or CG base pairs in the stem. Lower case letters following the number correspond to stem or loop sequence variants. b Calculated from DINAMELT.14 c Randomized sequence corresponding to hairpin G1. Buffers The background electrolytes (BGEs) used for the CE experiments contained diethylmalonic acid ([(CH3CH2)2C(COOH)2], Sigma-Aldrich, St. Louis, MO) as the buffering 4 ACS Paragon Plus Environment

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ion, titrated with the hydroxide of Na+, NH4+, TMA+, TEA+, TPA+ or TBA+ to pH 7.3, the pKa of the second carboxyl group. The tetrapentylammonium ion (NPe 4+) could not be used because it formed a coacervate with the ions in the BGE. Since diethylmalonate (DM) is an anion, the cation may be changed at will without altering the pH or buffering capacity of the BGE. Because the second carboxyl group is half-ionized at its pKa, the cation concentration was 1.5 times the diethylmalonic acid concentration; the ionic strength was twice the diethylmalonic acid concentration. For clarity in the text below, all BGEs are identified by their cation concentrations. The relative viscosities (η /ηo) of the various BGEs were calculated from eq 1, using the viscosity B-coefficients and known concentrations, ci, of the ions in each solution, assuming the viscosity A-coefficients to be negligibly small.15 η / ηo = 1 + ∑i Bi ci

(1)

The viscosity B-coefficients used in the calculations are given in Table 2. Most of the Bcoefficients correspond to ions in the BGEs used in the present study; others correspond to ions used in experiments taken from the literature (see text). The B-coefficients of most of the ions were taken from tables given by Marcus and coworkers.15,16 The B-coefficient of the tetraethanolammonium ion [(EtOH)4N+] was taken from Evans et al.17 However, the Bcoefficients of Tris+ and the mono- and divalent anions of diethylmalonic acid (DM -1 and DM-2) have not been reported. The B-coefficients of these ions were estimated from sums of the Bcoefficients of their functional groups, taken from tables given by Miki, et al. 18 The estimated Bcoefficients appear to be reasonable, since the B-coefficient calculated for DM -2 was 0.716 M-1, ~10% lower than the measured B-coefficient19 of the divalent anion of pimelic acid (0.775 M -1), a linear dicarboxylic acid with the same chemical formula as diethylmalonic acid. Table 2. Viscosity B-coefficientsa B-coefficient, B-coefficient, -1 Ion M Ion M-1 + -1 Na 0.085 DM 0.613 + -2 NH4 -0.008 DM 0.716 TMA+ 0.123 Br-1 -0.033 TEA+ 0.385 Cl-1 -0.005 TPA+ 0.916 OH-1 0.122 TBA+ 1.275 Tris+ 0.333 (EtOH)4N+ 0.32 a Taken from Jenkins and Marcus,15,16 except (EtOH)4N+, taken from Evans, et al.17 and Tris+, DM-1 and DM-2, estimated from tables of functional groups given by Miki, et al. 18 Capillary electrophoresis



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A Beckman Coulter (Fullerton, CA) MDQ capillary electrophoresis system was used to measure the free solution mobilities of the DNA oligomers. The instrument was run in the reverse polarity mode (anode on the detector side) with UV detection at 254 nm, using methods described elsewhere.20 The capillaries (Polymicro Technologies, Phoenix, AZ) were 31.4 ± 0.2 cm in length (20.9 ± 0.2 cm to the detector), with external diameters of 375 μm and internal diameters of 75 μm. The capillaries were coated internally with linear polyacrylamide to minimize the electroosmotic flow (EOF) of the solvent; previous studies have shown that this coating does not affect the observed mobilities.21 The applied electric field ranged from 1.2 to 5.8 kV/cm, depending on the concentration and identity of the ions in the BGE; the current was always 60 μA or less. Under these conditions, Joule heating effects are negligible and the observed mobilities are independent of the applied electric field. 21 DNA samples containing one of the hairpins and a reference, usually T16, were coinjected into the capillary using low pressure (0.5 psi, 0.0035 MPa) for 3 s. The injection volume was ~22 nL; the sample plug occupied ~2.6% of capillary length. The hairpin and reference were added in different molar ratios to enable easy identification of the peaks in the electropherograms; if necessary, the assignments were confirmed by running the oligomers separately. If the residual EOF in the capillary is negligible, the electrophoretic mobility, μ, of the hairpin can be calculated directly from the observed migration time using eq 2: μ = Ld / E t

(2)

where Ld is the distance from the capillary inlet to the detector (in cm), E is the electric field strength (in V/cm) and t is the time required for the sample to migrate from the inlet to the detector (in seconds). In practice, the residual EOF in the capillary varied somewhat on different days and in different capillaries because of small fluctuations in the capillary coating. To correct for this effect, the mobilities of the hairpin and T16 were measured in each experiment and the mobilities of T16 in a given BGE were averaged. The deviation of the observed mobility of T16 from the average, μ, [∆μ = μ(T16, observed) – μ(T16, average)] was used to correct the observed mobility of the hairpin using eq 3: μ(hairpin, corrected) = μ(hairpin, observed) ) + μ

(3)

For convenience, all mobilities are expressed in mobility units, m.u. (1 m.u. = 1 × 10 -4 cm2/V s). The corrected mobilities and mobility ratios of the hairpins were very reproducible. For example, the mobility ratio observed for hairpin G1 at 15ºC in 0.3 M TBA + was 1.279 ± 0.007 m.u.; for hairpin G3a, it was 1.332 ± 0.008 m.u. Hence, the error bars in most of the figures below are smaller than the sizes of the symbols. Thermal melting experiments In dilute solutions when the Debye-Hückel theory is valid, the mobility of a spherical polyion is directly proportional to its charge (Q) and inversely proportional to its radius (a) and the viscosity of the BGE (η), as shown in eq 4:


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μ=Q/6πηa

(4)

When a DNA duplex or hairpin is denatured either by heat or by chemical reagents, it becomes less compact, increasing the radius while the effective charge remains approximately constant. With increasing temperature (or denaturant concentration), the mobility of the hairpin or duplex gradually becomes equal to that of a random coil containing the same number of nucleotides. 8 Thermal melting studies were carried out by measuring the mobilities of the hairpins and a reference, usually T16, at temperatures between 15ºC and 60ºC, the range available on the CE instrument. The capillary was allowed to equilibrate at least 3 min at each temperature before the voltage was applied; previous studies have shown that a 3 min wait is sufficient to reach temperature equilibrium.22,23 The hairpins and T16 typically migrated as sharp, nearly Gaussianshaped peaks, as shown in Figure 1 for hairpin G1. No homodimers were present; homodimers migrate significantly faster than hairpins22 and would have added an additional peak to the electropherograms. The separation between the hairpin (left) and coil (right) peaks gradually decreased with increasing temperature and began to overlap at high temperatures, as the peak corresponding to the hairpin conformational ensemble began to include more and more slowlymigrating, coil-like conformations. The approximately Gaussian shapes of the hairpin and coil peaks indicate that the folded and unfolded hairpin conformations were in rapid exchange throughout the thermal transition. Similar electropherograms were observed for the other hairpins.

Figure 1. Electropherograms observed for hairpin G1 (left peak) and reference T16 (right peak) as a function of temperature in 0.3 M TBA+. The electropherograms correspond to temperatures of 15ºC, 30ºC, 40ºC, 50ºC, and 60ºC from bottom to top. The mobilities observed for hairpin G2 and references T16 and R16 in 0.3 M TBA + are plotted in Figure 2A as a function of temperature. The mobilities of the two references were essentially equal to each other in 0.3 M TBA+ (Figure 2A) and in 0.3 M Na+ (not shown), indicating that the mobilities were not sequence dependent. The mobilities of the references increased approximately linearly with increasing temperature because of the changes in the viscosity and dielectric constant of water with temperature. 24 The mobility of hairpin G2 was faster than that of references T16 and R16 at low temperatures but slowed and became equal to the mobility of the references at high temperatures, as shown in Figure 2A. To simplify the 7 ACS Paragon Plus Environment


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analysis of the thermal transitions, changes in the viscosity and dielectric constant of water with temperature were factored out by calculating mobility ratios, dividing the mobility of the hairpin at each temperature by the mobility of the reference, electrophoresed at the same time in the same solution.8,22,23 The mobility ratios observed for hairpin G2, calculated from the data in Figure 2A, are plotted as a function of temperature in Figure 2B. Such plots are called melting curves for brevity.

Figure 2. (A) Dependence of the mobilities of: (●), hairpin G2; (o), reference T16; and (Δ), reference R16 on temperature in 0.3 M TBA+. The solid line was drawn by linear regression. (B), Mobility ratios observed for hairpin G2 in 0.3 M TBA + as a function of temperature. The curved line was calculated from eq 5; the midpoint of the thermal transition was 33 ± 1 ºC. The melting curves were analyzed as two-state transitions and fitted to a four parameter sigmoid using eq 5, taken from SigmaPlot (Systat Software, Inc.):

𝑦=𝑦 +

(

(5)

)

Here, y is the mobility ratio observed at temperature T, yo is the limiting mobility ratio at high

temperatures, a is a constant that when added to yo is equal to the mobility ratio at low temperatures, x is the temperature of the measurement, xo is the temperature at the midpoint of the transition, called the melting temperature, Tm for brevity, and b describes the cooperativity of the transition. The limiting mobility ratios at high and low temperatures and the midpoints of the transitions were taken from the fits; replicate melting temperatures usually agreed within ±1ºC. For convenience in comparing the melting curves obtained in different BGEs, the fraction of hairpin present in the conformational ensemble at each temperature was plotted as a function of temperature. RESULTS AND DISCUSSION Dependence of thermal stability on the number of GC base pairs in the stem The normalized melting curves observed for hairpins G1 to G5 in 0.3 M TBA + are illustrated in Figure 3A, where the fractional hairpin concentration is plotted as a function of 8 ACS Paragon Plus Environment

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temperature. The midpoint melting temperatures, which ranged from 23ºC to 63ºC, are plotted in Figure 3B as a function of the number of GC base pairs in the stem (closed circles). The melting temperatures increased linearly with the number of GC base pairs in the stem, as observed in previous DNA studies using a variety of cations. 25-28 In 0.3 M Na+, the melting temperatures of all hairpins, except the G1 hairpins, were too high to be measured by CE; they were therefore predicted from the program DINAMELT. 14 The predicted melting temperatures of the G1 hairpins (open circles in Figure 3B) were close to the measured values (open triangles). Hence, DINAMELT provides a reasonable estimate of the melting temperatures that would have been observed for the hairpins in 0.3 M Na +. The predicted melting temperatures are plotted as a function of the number of GC base pairs in the stem in Figure 3B (open circles). As observed in 0.3 M TBA +, the predicted melting temperatures in 0.3 M Na+ increased linearly with the number of GC base pairs in the stem. However, the predicted melting temperatures in 0.3 M Na + were ~17ºC higher than observed for the same hairpins in 0.3 M TBA+. Since the differences in the melting temperatures were independent of the number of GC base pairs in the stem, the lower melting temperatures observed in TBA+ must be attributed to differences in the properties of the solvent, not to preferential interactions of the Na+ or TBA+ ions with the hairpin or coil conformations.

Figure 3. (A), Dependence of the fractional hairpin concentration of hairpins G1 to G5, (o), on temperature in 0.3 M TBA+. (B), Dependence of the melting temperatures, Tm, of the hairpins and their sequence variants on the number of GC base pairs in the stem. The symbols correspond to: (●), Tm of hairpins and sequence variants measured in 0.3 M TBA +; (Δ), Tm of hairpin G1 and its sequence variants measured in 0.3 M Na +; and (o), predicted Tm of various hairpins and sequence variants in 0.3 M Na+.14 The straight lines were drawn by linear regression; r2 = 0.980 for TBA+ and 0.972 for Na+. Although the melting temperatures of the various hairpins and their sequence variants depended primarily on the number of GC base pairs in the stem (Figure 3B), smaller sequencedependent effects were also noted. For example, the melting temperatures observed for hairpin G1, with an A5-tract in the stem, were somewhat higher than observed for hairpins G1a and G1b, with mixed AT and TA base pairs in the stem (see Table 1, columns 3-5). Hence, the presence of an A-tract stabilized the hairpin, both in 0.3 M Na+ and in 0.3 M TBA+. The relative 9 ACS Paragon Plus Environment


stiffness of AA·TT dimers29,30 and/or the cross-strand bifurcated hydrogen bonds characteristic of the A-tract conformation31-33 may have led to the increased the thermal stability of hairpin G1. Hairpins G3 and G3a, which differed only by the inversion of the last two base pairs in the stem, exhibited very similar melting temperatures in 0.3 M Na + and in 0.3 M TBA+ (Table 1). Hence, end-fraying was not observed for hairpin G3a, even though it has been observed for small DNA duplexes with terminal AT base pairs.34 By contrast, hairpin G1b, with a T4 loop, had a higher melting temperature in 0.3 M Na+ than hairpin G1a, with the same stem and a C4 loop. Similarly, hairpin G3d, with a T4 loop, was more stable in 0.3 M Na + than G3 hairpins with the same stem and mixed T and C residues in the loop. These results agree with previous studies showing that DNA hairpins with T4 loops are more stable than hairpins with the same stem and C4 loops.35 The loop-dependent differences in thermal stability observed in 0.3 M Na + were significantly reduced in 0.3 M TBA+ (compare columns 4 and 5 in Table 1), suggesting that TBA+ disrupts the intramolecular base stacking usually found in T4 loops. 36,37 Effect of cation size and solution viscosity on DNA thermal stability Melting curves were measured for hairpin G1 in BGEs containing 0.3 M Na +, NH4+, TMA+, TEA+, TPA+ or TBA+, with the results shown in Figure 4. The melting temperatures in the various BGEs decreased with increasing cation radius (Figure 4A), in agreement with previous studies.6-8,11 However, cation radius does not appear to be the variable determining DNA thermal stability, because the melting temperature of hairpin G1 decreased linearly with increasing solution viscosity, as shown in Figure 4B. Hence, the rate-controlling step in DNA denaturation/renaturation must involve diffusion of the DNA molecules through the solvent to find stable nucleation sites.1,2

Figure 4. (A), Dependence of the melting temperatures observed for hairpin G1 on the hydrated radius of the cation15,16 in solutions containing 0.3 M cation. The curved line is drawn to guide the eye. (B),

Dependence of the melting temperature of hairpin G1 on solvent viscosity, in centipoise (1 cP = 1 mPa·s). Solvent viscosity was calculated from eq 1 and the viscosity B-coefficients given in Table 2. The straight line was drawn by linear regression; r2 = 0.992. Cation release after DNA thermal melting 10 ACS Paragon Plus Environment

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The melting temperatures observed for hairpin G1 increased linearly with the logarithm of the Na+ ion concentration, over a 10-fold range of [Na+], as shown by the open circles in Figure 5A. Similar results have been observed for other DNA molecules in solutions containing Na+ ions.42 The solid line was calculated from the structure-prediction program DINAMELT;14,38 the predicted melting temperatures agree with the measured values. The slope of the line can be used to estimate the number of cations released per phosphate upon denaturation of a duplex or hairpin using eq 6: slope = dTm/d log[M+] = -2.3(αRTm2/∆Ho) ∆n

(6)

where [M+] is the cation concentration, R is the gas constant, α is a factor that accounts for changes in the activity coefficient of the cation with concentration and is often set equal to 0.9, 39 ∆Ho is the enthalpy of melting per nucleotide and ∆n is the number of cations released per nucleotide.8,39-44 Cation release occurs because the hairpin has a higher charge density than the random coil, so that greater numbers of counterions are condensed in the ion atmosphere and released upon melting.40,45 The factor in parentheses on the right-hand side of eq 6 is not significantly salt-and temperature-dependent and is often given the value of 50. 8,41,42 Using this value, the slope of the line suggests that 1.4 Na+ ions are released per hairpin, or 0.093 Na+ ions per phosphate. This value is within the range of values observed for small DNA and RNA oligomers in solutions containing less than 1M Na + ions.9,42-44,47-49

Figure 5. (A), Dependence of the melting temperatures observed for hairpin G1 on the logarithm of the cation concentration: (o), in Na+; and (♦), in TBA+. The straight lines were calculated by linear regression. (B), Comparison of the melting temperatures: (o), observed for hairpin G1 in Na+; and (♦), calculated for hairpin G1 from the viscosity-corrected melting temperatures in TBA+ (see text). The slope of the viscosity-corrected TBA+ line is equal to the slope of the line describing the melting temperatures in Na+. The dependence of the melting temperatures of hairpin G1 on the logarithm of TBA + concentration is illustrated by the closed diamonds in Figure 5A. The slope of the TBA + line is significantly lower than observed for Na+, suggesting that fewer TBA+ ions are released from the hairpin upon thermal melting. However, the Na+ and TBA+ melting temperatures cannot be compared directly because DNA melting temperatures depend on solution viscosity, as shown in Figure 4B. If the melting temperatures observed for hairpin G1 in TBA + are corrected for the 11 ACS Paragon Plus Environment


viscosity effect by multiplying the observed Tm by the ratio of the viscosities of the two solutions [η(TBA+)/η(Na+)], the viscosity-corrected melting temperatures in TBA + are indicated by the closed diamonds in Figure 5B. The slope of viscosity-corrected TBA + line is equal to the slope of the line observed in Na+, suggesting that equal numbers of Na+ and TBA+ ions are released upon thermal melting in the two BGEs. The observed results agree with previous studies showing that neither Li+ ions nor TBA+ ions form site-specific complexes with the DNA phosphates. 8 TBA+ lowers the melting temperature of hairpin G1, not because it binds preferentially to the phosphate groups in the random coil conformation, pulling the helix/coil equilibrium toward the coil, but because TBA+ increases the viscosity of the solution, a through-solvent effect that modifies the rates of hairpin opening and closing. Generality of the results Previous studies in the literature have noted that the thermal stability of DNA and RNA decreases with increasing solvent viscosity.6-9,50,51 Unfortunately, the melting temperatures obtained in different studies cannot be compared directly because they depend on the size and sequence of the DNA or RNA,25,26,52 the ionic strength of the solution,52-56 and the type of viscogen used.9,51 However, if the concentration and/or type of the viscogen is the only variable in a given series of measurements, different experiments in the literature can be compared by calculating the fractional decrease in the melting temperature as a function of the fractional change in the viscosity of the solvent. Figure 6 compares the fractional melting temperatures [T m(TXA+)/Tm(Na+ or NH4+)] observed for various nucleic acids in tetraalkylammonium ion solutions as a function of the normalized inverse viscosity of the solvent [η(Na+ or NH4+)/η(TXA+)]. For brevity, the fractional melting temperatures and normalized viscosities are called Tm ratios and viscosity ratios in the following figures and text. The viscosities of the various TXA+ solutions were calculated from eq 1, using the viscosity B-coefficients given in Table 2 and the known concentrations of the ions in each solution. The nucleic acids include: hairpin G1 (this study), a similar hairpin with the sequence 5’-ATCCTA-TTTT-TAGGAT,8 a 9 bp RNA duplex with the sequence CAACGCAAG/CUUGCGUUG,9 sonicated and/or sheared calf thymus DNA,6,7 and poly(A)·poly(U).51

Figure 6. Dependence of the Tm ratio [Tm(TXA+)/Tm(Na+)] observed for various nucleic acids on the viscosity ratio [η(Na+ or NH4+)/η(TXA+)]. The symbols correspond to: (●), hairpin G1; (o), a DNA 12 ACS Paragon Plus Environment

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hairpin of the same size with a different sequence; 8 (), 9 bp RNA duplex;9 (Δ), sonicated calf thymus DNA;6 (), sheared calf thymus DNA;7 and (□), poly(A)·poly(U).51 The straight lines were drawn by linear regression; r2 = 0.911 for the solid line and 0.935 for the dashed line. The fractional melting temperatures observed for small mixed-sequence DNAs and RNA in tetraalkylammonium ion solutions increased linearly with inverse solvent viscosity (increasing viscosity ratio), regardless of whether the nucleic acid was DNA or RNA, whether the conformation was a hairpin or duplex, and whether the sample was monodisperse or polydisperse. Hence, diffusion of the polynucleotide chains through the solvent appears to be involved in the rate-determining step of the denaturation/renaturation reaction. 4 The fractional melting temperature of poly(A)·poly(U) also increased linearly with inverse solvent viscosity (increasing viscosity ratio), as shown by the open squares in Figure 6. However, the slope of the line was not as steep as observed for the other nucleic acids, possibly because self-stacking of the adenine nucleotides in poly(A)57-59 affected the elastic properties59,60 and/or folding kinetics61 of the polynucleotide.1,50,62,63 Different results were observed when the viscosity of the solution was changed by adding viscogenic cosolvents to the buffer, as shown in Figure 7. The normalized melting temperatures observed for sheared bacteriophage T4 DNA1 and sheared salmon sperm DNA64 increased nonlinearly with increasing viscosity ratio (inverse solvent viscosity) when the viscosity was increased by adding ethylene glycol to the solution, as shown in Figure 7A. Similar results were observed for a DNA duplex with the sequence (ATCGCGAT) 2 when glycerol was added, as shown by the inverted triangles in Figure 7A. By contrast, the fractional melting temperatures of all three DNAs increased linearly with logarithm of the viscosity ratio (inverse viscosity), as shown in Figure 7B.

Figure 7. Dependence of normalized DNA melting temperatures on inverse viscosity in solutions containing added viscogenic cosolvents. (A), Dependence of the T m ratios on the viscosity ratio. (B), dependence of the Tm ratios on the logarithm of the viscosity ratio. The symbols correspond to: (●), sheared bacteriophage T4 DNA in ethylene glycol;1 (o), sheared salmon sperm DNA in ethylene glycol; 60 and (), 8 bp duplex DNA in glycerol.51 The curved line in (A) is meant to guide the eye; the straight line in (B) was calculated by linear regression; r2 = 0.859.



Proteins in tetraalkylammonium ion solutions The thermal stabilities of ribonuclease65 and lysozyme66 have been measured in solutions containing various tetraalkylammonium ions, with the results illustrated in Figure 8. The normalized melting temperatures increased linearly with increasing viscosity ratio (inverse solution viscosity), as observed for the nucleic acids. The slope of the line was approximately equal to that observed for poly(A)·poly(U) in TXA+ solutions, as shown by the open squares in Figure 8. The results suggest that large numbers of conformational states must be searched in denatured proteins and in polynucleotide homopolymers to find suitable renaturation sites, a process that would be diffusion-limited and, hence, depend on solvent viscosity. 1,4,63

Figure 8. Dependence of the fractional melting temperatures of: (●), lysozyme;66 (o), ribonuclease;65 and (□), poly(A)·poly(U)51 on the viscosity ratio of solutions containing tetraalkylammonium ions. The line was drawn by linear regression; r 2 = 0.853. Contrast between thermal stability in tetraalkylammonium ion solutions and in solutions containing viscogenic cosolvents The reason why the fractional decrease in the melting temperatures observed for nucleic acids in solutions containing tetraalkylammonium ions (Figures 6 and 8) differs from the results observed in solutions containing viscogenic cosolvents such as ethylene glycol or glycerol (Figure 7) is not clear. In TXA+ solutions, the number of components in each solution, as well as the volume of free water, is constant; the viscosity is determined primarily by cation size. In such solutions, interpenetration of the individual DNA chains to find and nucleate stable secondary structures appears to be independent of the size of the nucleic acid, whether the molecule is DNA or RNA, whether the native conformation is a hairpin or duplex, and whether the sample is monodisperse or polydisperse. However, sequence does factor into the thermal stability observed for nucleic acids in TXA , since the normalized melting temperatures observed for poly(A)·poly(U) increased less strongly with inverse viscosity than observed for mixed-sequence DNA and RNA oligomers (compare the dashed and solid lines in Figure 6). Interestingly, the normalized melting temperatures observed for poly(A)·poly(U) agreed with those measured for ribonuclease and +


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lysozyme in tetraalkylammonium ion solutions, as shown in Figure 8, suggesting a common influence of solvent viscosity on the search for stable nucleation sites in these two types of molecules. Importantly, as shown in Figure 7, the normalized melting temperatures observed for sheared bacteriophage T41 and salmon sperm64 DNAs in ethylene glycol-water solutions increased as the logarithm of inverse viscosity, not the first power of inverse viscosity as observed in TXA+ solutions. The semi-logarithmic dependence of the melting temperatures on inverse viscosity can probably be attributed to excluded volume 63,67 and/or steric hindrance1 effects. Increasing the concentration of ethylene glycol or glycerol would decrease the volume of free water available for the nucleic acids, effectively increasing the DNA concentration and making it more difficult for individual DNA chains to move through the solvent and find suitable nucleation partners. However, other factors must also affect the thermal stability of nucleic acids in solutions containing viscogenic cosolvents. For example, the normalized melting temperature observed for an 8 bp DNA duplex51 in glycerol-water solutions increased as the logarithm of inverse viscosity, as shown in Figure 7, while the normalized melting temperatures of small DNA hairpins and a 9 bp RNA duplex9 increased linearly with inverse viscosity in TXA+ solutions (Figure 6). Further studies will be needed to the clarify the factors determining DNA thermal stability in these two types of solvents. Biological relevance The thermal stability of DNA and RNA is variable and depends on the viscosity of the medium in which it is dissolved. Since the viscosities in various intracellular compartments can vary widely,68,69 DNA and RNA stability in various locations within the cell must also vary widely. The dynamic interchange between metastable and stable DNA and RNA conformations in various locales in the cell may therefore contribute to biological regulatory mechanisms. 70-72 CONCLUSIONS Using small DNA hairpins as a model system, we showed that the thermal stability observed in tetraalkylammonium ion solutions decreases linearly with the increasing viscosity of the solvent, not the radius of the cation or its hydrophobicity. The dependence of the normalized melting temperatures on inverse viscosity agrees with literature values of the normalized melting temperatures observed for another small DNA hairpin, a small RNA duplex, and sonicated calf thymus DNA in tetraalkylammonium ion solutions. The normalized melting temperatures of poly(A)·poly(U), lysozyme and ribonuclease in tetraalkylammonium ion solutions also depend linearly on inverse viscosity, although the dependence is not as strong as observed for mixedsequence DNAs and RNA. By contrast, the normalized melting temperatures observed for DNA in solutions containing ethylene glycol or glycerol to modify the viscosity depend on the logarithm of the inverse viscosity, possibly because of excluded volume effects. The decrease of the thermal stability of nucleic acids with increasing solvent viscosity has a number of important implications. In the first place, the stability of the same sequence can 15 ACS Paragon Plus Environment


vary at different compartments in the cell, if the compartments have different viscosities. Such differences may contribute to biological regulatory mechanisms. Secondly, sequence-dependent counterion condensation cannot be determined directly from the dependence of the melting temperature on cation concentration, if the viscosity of the solution changes significantly with cation concentration. Instead, the melting temperatures must first be corrected for the viscosity effect and then compared as a function of cation concentration. Hence, the viscosity of the solution is an important variable that must be taken into account when interpreting DNA results obtained in different solutions. Acknowledgment Partial financial support of this research (to N.C.S.) from NSF (Grant CHE0748271) and NIH (Grant GM061009) is gratefully acknowledged.

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