doi:10.1016/j.bpc.2007.09.007 
Published by Elsevier B.V.
Thermodynamic dependence of DNA/DNA and DNA/RNA hybridization reactions on temperature and ionic strength
Brian E. Lang
, a, 
and Frederick P. Schwarza
aCenter for Advanced Research in Biotechnology/Biotechnology Division, National Institute of Standards and Technology, 9600 Gudelsky Drive, Rockville, MD 20850, USA
Received 15 August 2007;
revised 18 September 2007;
accepted 19 September 2007.
Available online 26 September 2007.
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Abstract
The thermodynamics of 5′-ATGCTGATGC-3′ binding to its complementary DNA and RNA strands was determined in sodium phosphate buffer under varying conditions of temperature and salt concentration from isothermal titration calorimetry (ITC). The Gibbs free energy change, ΔG° of the DNA hybridization reactions increased by about 6 kJ mol− 1 from 20 °C to 37 °C and exhibited heat capacity changes of − 1.42 ±0.09 kJ mol− 1 K− 1 for DNA/DNA and − 0.87 ± 0.05 kJ mol− 1 K− 1 for DNA/RNA. Values of ΔG° decreased non-linearly by 3.5 kJ mol− 1 at 25 °C and 6.0 kJ mol− 1 at 37 °C with increase in the log of the sodium chloride concentration from 0.10 M to 1.0 M. A near-linear relationship was observed, however, between ΔG° and the activity coefficient of the water component of the salt solutions. The thermodynamic parameters of the hybridization reaction along with the heat capacity changes were combined with thermodynamic contributions from the stacking to unstacking transitions of the single-stranded oligonucleotides from differential scanning calorimetry (DSC) measurements, resulting in good agreement with extrapolation of the free energy changes to 37 °C from the melting transition at 56 °C.
Keywords: DNA/DNA binding; DNA/RNA hybridization; Thermodynamics
Fig. 1. (top) the ITC scan of 10 μl aliquots of 177 μM DNA(TG) into 15.8 μM DNA'(CA) in pH = 7.0 PBS buffer at 25 °C. (bottom) The binding isotherm for this titration.
Fig. 2. van't Hoff plot of DNA/DNA duplex at pH = 6.0 (diamond), pH = 7.0 (square) and pH = 8.0 (circle) in 100 mM NaCl solution.
Fig. 3. DSC plot of the DNA duplex melting (black line) with scaled plots of the plots of the single-stranded DNA unstacking transition for DNA(TG) (dotted line) and DNA'(CA) (dashed line).
Fig. 4. Variation of ΔG as a function of Na+ concentration at 25 °C (circle) and 37 °C (square), along with the fits using Eq. (6).
Fig. 5. Plot of − ΔH vs. − TΔS for the DNA/DNA interaction from all data sets collected. The plot represents data from 25 °C to 37 C, pH conditions ranging between pH = 6 to pH = 8, and sodium chloride concentrations from 0.1 M to 1.0 M.
Fig. 6. Plot of ΔG as a function of Na+ concentration at 39 °C from Owczarzy et al. [11] with fit using Eq. (6) and coefficients generated from the functions in Table 7.
Table 1.
Thermodynamic values for the DNA(TG)/DNA'(CA) binding reaction from ITC measurements as a function of temperature
Given are the average stoichiometric ratios, N, from the fit of the ITC data, the observed apparent binding constant, Kb, and the concentration range of the final DNA duplex.
Table 2.
Thermodynamic quantities for the DNA(TG)/RNA'(CA) binding reaction from ITC measurements as a function of temperature
Given are the average stoichiometric ratios, N, from the fit of the ITC data, and the observed apparent binding constant, Kb. (Final duplex concentration ≈ 16 μM).
Table 3.
Thermodynamic quantities for the DNA(TG)/DNA'(CA) binding reaction from ITC measurements as a function of temperature and pH
Given are the average stoichiometric ratios, N, from the fit of the ITC data, and the observed apparent binding constant, Kb. (Final duplex concentration ≈ 13.6 μM).
Table 4.
Thermodynamic quantities for the DNA(TG)/DNA'(CA) binding reaction from ITC measurements as a function of temperature and sodium ion concentration
Given are the average stoichiometric ratios, N, from the fit of the ITC data, and the observed apparent binding constant, Kb. (Final duplex concentration ≈ 15 μM).
Table 5.
Thermodynamic quantities from DSC measurements on the melting of the DNA(TG)/DNA('CA) duplex
The van't Hoff enthalpy, ΔH°vH, is calculated from the fit of the two-state transition model, and the calorimetric enthalpy, ΔH°cal, is the is calculated from the integration of the specific heat of the transition.
Table 6.
Thermodynamic properties of the single-strand nucleic acid unstacking transitions
Data is given for the total unstacking enthalpy and entropy of transitions and melting temperatures, Tm, based on the DSC data, ΔuH° and ΔuS°, respectively. Also given are the Gibbs free energies at 37 °C, ΔG°u,37, the molar quantity of nucleotide in the unstacked conformation at 37 °C, n, and the contributions of stacking free energy to the binding reactions at 37 °C, nΔuG°.
Table 7.
Temperature dependent coefficients for Eq. (6)
ΔbG°max = ΔbG°[Na+] = 1 + k, along with temperature dependent functions for the coefficients (T in °C).
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