DNA interaction with Mn2+ ions at elevated temperatures: VCD evidence of DNA aggregation

Interaction of natural calf thymus DNA with Mn(2+) ions was studied at room temperature and at elevated temperatures in the range from 23 degrees C to 94 degrees C by means of IR absorption and vibrational circular dichroism (VCD) spectroscopy. The Mn(2+) concentration was varied between 0 and 1.3M (0 and 10 [Mn]/[P]). The secondary structure of DNA remained in the frame of the B-form family in the whole ion concentration range at room temperature. No significant DNA denaturation was revealed at room temperature even at the highest concentration of metal ions studied. However at elevated temperatures, DNA denaturation and a significant decrease of the melting temperature of DNA connected with a decrease of the stability of DNA induced by Mn(2+) ions occurred. VCD demonstrated sensitivity to DNA condensation and aggregation as well as an ability to distinguish between these two processes. No condensation or aggregation of DNA was observed at room temperature at any of the metal ion concentrations studied. DNA condensation was revealed in a very narrow range of experimental conditions at around 2.4 [Mn]/[P] and about 55 degrees C. DNA aggregation was observed in the presence of Mn(2+) ions at elevated temperatures during or after denaturation. VCD spectroscopy turned out to be useful for studying DNA condensation and aggregation due to its ability to distinguish between these two processes, and for providing information about DNA secondary structure in a condensed or aggregated state.     

The denaturation transition of DNA in mixed solvents

The helix-to-coil denaturation transition in DNA has been investigated in mixed solvents at high concentration using ultraviolet light absorption spectroscopy and small-angle neutron scattering. Two solvents have been used: water and ethylene glycol. The "melting" transition temperature was found to be 94 degrees C for 4% mass fraction DNA/d-water and 38 degrees C for 4% mass fraction DNA/d-ethylene glycol. The DNA melting transition temperature was found to vary linearly with the solvent fraction in the mixed solvents case. Deuterated solvents (d-water and d-ethylene glycol) were used to enhance the small-angle neutron scattering signal and 0.1M NaCl (or 0.0058 g/g mass fraction) salt concentration was added to screen charge interactions in all cases. DNA structural information was obtained by small-angle neutron scattering, including a correlation length characteristic of the inter-distance between the hydrogen-containing (desoxyribose sugar-amine base) groups. This correlation length was found to increase from 8.5 to 12.3 A across the melting transition. Ethylene glycol and water mixed solvents were found to mix randomly in the solvation region in the helix phase, but nonideal solvent mixing was found in the melted coil phase. In the coil phase, solvent mixtures are more effective solvating agents than either of the individual solvents. Once melted, DNA coils behave like swollen water-soluble synthetic polymer chains.     

Rapid cycle DNA amplification: time and temperature optimization

Rapid temperature cycling with hot air allows rigorous optimization of the times and temperatures required for each stage of the polymerase chain reaction. A thermal cycler based on recirculating hot air was used for rapid temperature control of 10-microliters samples in thin glass capillary tubes with the sample temperature monitored by a miniature thermocouple probe. The temperatures and times of denaturation, annealing and elongation were individually optimized for the amplification of a 536-base pair beta-globin fragment from human genomic DNA. Optimal denaturation at 92 degrees-94 degrees C occurred in less than one second; yield decreased with denaturation times greater than 30 seconds. Annealing for one second or less at 54 degrees-56 degrees C gave the best product specificity and yield. Non-specific amplification was minimized with a rapid denaturation to annealing temperature transition (9 seconds) as compared to a longer transition (25 seconds). An elongation temperature of 75 degrees-79 degrees C gave the greatest yield and increased yields were obtained with longer elongation times. Product specificity was improved with rapid air cycling when compared to slower conventional heat block cycling. Rapid thermal control of the temperature-dependent reactions in DNA amplification can improve product specificity significantly while decreasing the required amplification time by an order of magnitude.     

Thermal analysis of CHL V79 cells using differential scanning calorimetry: implications for hyperthermic cell killing and the heat shock response

Differential scanning calorimetry (DSC) was used to assay thermal transitions that might be responsible for cell death and other responses to hyperthermia or heat shock, such as induction of heat shock proteins (HSP), in whole Chinese hamster lung V79 cells. Seven distinct peaks, six of which are irreversible, with transition temperatures from 49.5 degrees C to 98.9 degrees C are detectable. These primarily represent protein denaturation with minor contributions from DNA and RNA melting. The onset temperature of denaturation, 38.7 degrees C, is shifted to higher temperatures by prior heat shock at 43 degrees and 45 degrees C, indicative of irreversible denaturation occurring at these temperatures. Thus, using DSC it is possible to demonstrate significant denaturation in a mammalian cell line at temperatures and times of exposure sufficient to induce hyperthermic damage and HSP synthesis. A model was developed based on the assumption that the rate limiting step of hyperthermic cell killing is the denaturation of a critical target. A transition temperature of 46.3 degrees C is predicted for the critical target in V79 cells. No distinct transition is detectable by DSC at this temperature, implying that the critical target comprises a small fraction of total denaturable material. The short chain alcohols methanol, ethanol, isopropanol, and t-butanol are known hyperthermic sensitizers and ethanol is an inducer of HSP synthesis. These compounds non-specifically lower the denaturation temperature of cellular protein. Glycerol, a hyperthermic protector, non-specifically raises the denaturation temperature for proteins denaturing below 60 degrees C. Thus, there is a correlation between the effect of these compounds on protein denaturation in vivo and their effect on cellular sensitivity to hyperthermia.     

Effect of divalent copper ions on heat denaturation of DNA

Using the thermal denaturation method the effect of bivalent copper of (4-10(-6)-10(-3)) M concentrations on the helix-coil transition of DNA was studied in the solution of Na+ concentrations 10(-3)-10(-1) M. Unlike the previous studies, this paper makes allowance for the effect of impurity ions present in DNA and deionized water. It has been shown that in the region of low Cu2+ and Na+ concentrations, thermal stability increases, the melting range extends and the denaturation curves become asymmetric. At concentrations more than approximately 3-10(-5) M Cu2+, melting temperature starts to fall, and the range reduces to 1-1.5 degrees at [Cu2+] greater than or equal to 2-10(-4) M. As [Cu2+] reaches these values, the denaturation curve asymmetry and melting range increase again, which is due to the inversion of the relative stability of AT- and GC-pairs. Employing experimental and phase-transition-theory data for homopolymers, the constants of Cu2+ binding with phosphates and DNA bases were calculated. The concentration dependence of the DNA denaturation parameters was shown to be governed by the superposition of binding Cu2+ with phosphates and nucleic acid bases.     

Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide.

The thermal stabilities of RNA:DNA hybrids are substantially greater than those of DNA:DNA duplexes in aqueous electrolyte solutions containing high concentrations of formamide. Association rates to form DNA:DNA duplexes and DNA:RNA hybrids have been measured in these solvents. There is a temperature range in which DNA:DNA rates are negligible and RNA:DNA rates close to optimal.     

Denaturation of covalently closed circular DNA. Kinetics, comparison of several DNAs, mechanism and ionic effects

The rates of the alkaline denaturation of the covalently closed, circular DNAs (form I) of the replicative forms (RF) of phages G4, phi X174, and fd, and of plasmid pBR322 and phage PM2 have been measured at 0 degrees C and some at higher temperatures. These rates are orders of magnitude slower than the denaturation of linear DNA because of the increased stability of the helix to deprotonation that results from the accumulating positive superhelicity during denaturation. Denaturation reactions were initiated by rapid, infrasonic mixing (Camien, M.N., and Warner, R.C. (1984) Anal. Biochem. 138, 329-334), and their progress was measured by analytical ultracentrifugal analysis for the amounts of form I and denatured (Id) DNA after neutralization of the alkaline reaction. The comparative rates of the five DNAs varied over a wide range; the fastest, G4-RF, denatured at 500-fold the rate of the slowest, fd-RF. The differences are accounted for by the interaction of positive superhelicity with the sequence-dependent regions of relative helix stability in the various DNAs. Renaturation rates of Id DNAs varied similarly for Ids prepared at 0 degrees C, but only a few-fold for Ids prepared at 50 degrees C. The rate of denaturation of G4-RF was determined over a wide range of NaOH and NaCl concentrations at 0 degrees C, and the pHm was determined as a function of ionic strength and temperature. The effects of ionic strength have been analyzed in an application of the Manning ion condensation-screening theory (Manning, G.S. (1978) Q. Rev. Biophys. 11, 179-246) which is shown to account for the large destablizing effect of salts on the helix. The pH region of transition at 50 degrees C from renaturation to denaturation was examined, and it was found that the maximum rate of renaturation occurred at a pH about 0.05 units below the pHm.     

Renaturation of denatured, covalently closed circular DNA

The rate of renaturation of denatured, covalently closed, circular DNA (form Id DNA) of the phi X174 replicative form has been investigated as a function of pH, temperature, and ionic strength. The rate at a constant temperature is a sharply peaked function of pH in the range of pH 9 to 12. The position on the pH scale of the maximum rate decreases as the temperature is increased and as the ionic strength is increased. The kinetic course of renaturation is pseudo-first order: it is independent of DNA concentration, but falls off in rate from a first order relationship as the reaction proceeds. The rate of renaturation depends critically on the temperature at which the denaturation is carried out. Form Id, prepared at an alkaline pH at 0 degrees C, renatures from 5 to more than 100 times more rapidly than that similarly prepared at 50 degrees C. Both the heterogeneity in rate and the effect of the temperature of denaturation depend, in part, on the degree of supercoiling of the form I DNA from which the form Id is prepared. However, it is concluded that a much larger contribution to both arises from a configurational heterogeneity introduced in the denaturation reaction. The renaturation rate was determined by neutralization of the alkaline reaction and analytical ultracentrifugal analysis of the amounts of forms I and Id. The nature of the proximate renatured species at the temperature and alkaline pH of renaturation was investigated by spectrophotometric titration and analytical ultracentrifugation. It is concluded that the proximate species are the same as the intermediate species defined by an alkaline sedimentation titration of the kind first done by Vinograd et al. ((1965) Proc. Natl. Acad. Sci. U. S. A. 53, 1104-1111). Observations are included on the buoyant density of form Id and on depurination of DNA at alkaline pH values and high temperatures.     

Influence of hydrogen bonding in DNA and polynucleotides on reaction of nitrogens and oxygens toward ethylnitrosourea.

The reactivity of ethylnitrosourea toward hydrogen-bonded sites in double-stranded DNA or oly(rA).poly(rU) was compared with those sites in single-stranded DNA, RNA, or poly(rA). Alkylation of the N-1 of A in poly(rA).poly(rU) was almost suppressed at 5 degrees C but could be markedly increased by raining the reaction temperature to 25 degrees C, well below the Tm of 56 degrees C. In contrast, the N-7 and N-6 of A, which are not hydrogen bonded, reacted to the same extent at temperatures ranging from 5 to 65 degrees C. The extent of reaction at the N-3 of A varied inversely with the reactivity of the N-1 of A, indicating that of these two nitrogens the N-1 of A is the most reactive. The proportion of reaction at the various nitrogens in poly(rA) was not affected by temperature. Hydrogen-bonded oxygens in double-stranded DNA are the O-6 of G, the O-4 of T, and the O2 of C. All are equally reactive at 5, 25, and 51 degrees C. It is concluded that the observed temperature independence is due to these oxygens having an electron pair not involved in hydrogen bonding and, thus, available for reaction. In contrast, the electron pair of the N-1 of A (or the N-3 of C) is involved in hydrogen bonding, and the extent of their reactivity is dependent on thermal fluctuation providing transiently open base pairs at temperatures far below the Tm.     

N(4)C-alkyl-N(4)C cross-linked DNA: bending deformations in duplexes that contain a -CNG- interstrand cross-link

Short DNA duplexes containing a 1,3-N(4)C-alkyl-N(4)C interstrand cross-link that joins the two C residues of a -CNG- sequence were prepared using either a phosphoramidite or convertible nucleoside approach. The alkyl cross-link consists of 2, 4, or 7 methylene groups. The duplexes, which contain a seven-base-pair core and A(3)/T(3) complementary 3'-overhanging ends, were characterized by enzymatic digestion and MALDI-TOF mass spectrometry. Ultraviolet thermal denaturation studies showed that the duplexes denature in a cooperative manner and that the length of the cross-link affects the thermal stability. Thus, the transition temperature of the ethyl cross-linked duplex, 42 degrees C, is 16 degrees C higher than the melting temperature of the corresponding non-cross-linked control, whereas the transition temperatures of the butyl and heptyl cross-linked duplexes, 73 and 72 degrees C, respectively, are 46-47 degrees C higher. The reduced molecularity of denaturation of the cross-linked duplexes versus melting of the non-cross-linked duplex most likely accounts for these differences. Examination of molecular models suggests that the ethyl cross-link is too short to span the distance between the two C residues at the site of the cross-link in B-form DNA without causing distortion of the helix, whereas less and no distortion would be expected for the butyl and heptyl cross-links, respectively. The circular dichroism spectra, which show greatest deviation in the ethyl cross-linked duplex from B-form DNA, are consistent with this expectation. Anomalous mobilities on native polyacrylamide gels of multimers produced by self-ligation of each of the cross-linked duplexes suggest that the ethyl and butyl cross-linked duplexes undergo bending deformations, whereas multimers derived from the heptyl cross-linked duplex migrated normally. The bending angle was estimated to be 20 degrees, 13 degrees, and 0 degrees for the ethyl, butyl, and heptyl cross-linked duplexes, respectively. Thus, it appears that the degree of bending in these N(4)C-alkyl-N(4)C cross-linked duplexes is controlled by the length of the cross-link.     

Chromosomal DNA denaturation and reassociation in situ.

The degree of chromosomal DNA (cDNA) denaturation and renaturation on polytene chromosomes has been measured by UV microspectrophotometry. Also DNA losses occurring upon denaturation have been quantified by Feulgen, gallocyanin-chromalum and UV. It has been observed that denaturation in alkali (0.07 N NaOH at room temperature) and formamide (90% formamide; 0.1 SSC, pH 7.2) at 65 °C removes about 30% of the DNA. Low DNA loss occurs upon denaturation in HCl (0.24 M) at room temperature and 60% formamide: 2 × 10^?4 M EDTA (pH 8) at 55 °C. The presence of 4% formaldehyde in the denaturation buffer prevents DNA loss. After denaturation of chromosomes in 0.1 × SSC containing 4% formaldehyde at 100 °C for 30 sec, an hyperchromicity of 39 °C is observed. The denaturation efficiency varies with the denaturation treatment. The percentage reassociation was measured from the difference in the UV absorption of renatured chromosomes and that of denatured chromosomes from the same set. It seems that in our conditions DNA:DNA reassociation does not occur. The efficiency of hybridization is proportional to the denaturation extent of the DNA. However, the entire fraction of DNA which has been denatured is not available for hybridization.     

Effect of formaldehyde on the efficiency of hybridization of DNA immobilized on nitrocellulose filters.

We report in this study that under certain conditions formaldehyde interacts with DNA and makes it more efficient for hybridization on nitrocellulose filters. Hybridization signals of formaldehyde-treated DNA are stronger (up to 10 fold) as compared with that of the heat- or alkali-denatured DNA. Various parameters of the DNA-formaldehyde reaction are optimized as follows: (a) 6 x SSC, 10% formaldehyde, 60 degrees C, 20-30 min, reaction volume 10-200 microliters or (b) 6 x SSC, 5% formaldehyde, 98 degrees C, 15 min, reaction volume 10-200 microliters. Treatment of agarose gels after electrophoresis with formaldehyde improved both the transfer of DNA and the efficiency of hybridization. The following conditions are recommended for gel treatment: denaturation in 0.3 N NaOH, 1 M NaCl followed by neutralization with 0.5 M phosphate buffer, pH 7.0, containing 10% formaldehyde at 60 degrees C for 20 min.     

Synthesis and DNA nicking studies of a novel cyclic peptide: cyclo[Lys-Trp-Lys-Ahx-].

Two novel cyclic tetrapeptides: cyclo[Lys-Tyr-Lys-Ahx-] 7a and cyclo[Lys-Trp-Lys-Ahx-] 7b were synthesized by coupling protected amino acid in solution and the subsequent cyclization effected by the pentafluorophenyl ester method as described in previous papers. These cyclic peptides were designed and synthesized to study their interaction with DNA, based on previous reports that linear peptides Lys-Tyr-Lys and Lys-Trp-Lys could bind to various forms of DNA and cleaved supercoiled DNA at apurinic sites. Ethidium bromide displacement assay showed that the apparent DNA binding constant of linear Lys-Tyr-Lys and cyclic peptide 7a are far below 1 x 10(3) M(-1), whereas those of cyclic peptide 7b and linear Lys-Trp-Lys are 1.9 x 10(4) M(-1) and 9.5 x 10(3) M(-1), respectively. Kinetic studies using agarose gel electrophoresis showed that cyclic peptide 7b and Lys-Trp-Lys possessed DNA nicking activity on natural supercoiled phi X174 DNA with nicking rate of 50.7 and 75.6 pM min(-1) at 65 degrees C, respectively, whereas cyclic peptide 7a and linear Lys-Tyr-Lys were devoid of the corresponding activity. The DNA nicking rate increased significantly with increase in reaction temperature. At reaction temperatures lower than 65 degrees C, the DNA nicking rate of cyclic peptide 7b exceeded that of linear Lys-Trp-Lys. The addition of 1 microM ferrous ion did not give significant enhancement effect on the DNA nicking rate by the peptides. UV irradiation gave a marked rate enhancement on the DNA nicking rate of linear Lys-Trp-Lys and a moderate enhancement on the DNA nicking rate of cyclic peptide 7b.     

Circular dichroism studies of the interaction of a limited hydrolysate of T4 gene 32 protein with T4 DNA and poly[d(A-T)].poly[d(A-T)].

gp32 I is a protein with a molecular weight of 27 000. It is obtained by limited hydrolysis of T4 gene 32 coded protein, which is one of the DNA melting proteins. gp32 I itself appears to be also a melting protein. It denatures poly[d(A-T)].poly[d(A-T)] and T4 DNA at temperatures far (50-60 degrees C) below their regular melting temperatures. Under similar conditions gp32 I will denature poly[d(A-T).poly[d(A-T)] at temperatures approximately 12 degrees C lower than those measured for the intact gp32 denaturation. For T4 DNA gp32 shows no melting behavior while gp32 I shows considerable denaturation (i.e., hyperchromicity) even at 1 degree C. In this paper the denaturation of poly[d(A-T)].poly[d(A-T)] and T4 DNA by gp32 I is studied by means of circular dichroism. It appears that gp32 I forms a complex with poly[d(A-T)]. The conformation of the polynucleotide in the complex is equal to that of one strand of the double-stranded polymer in 6 M LiCl. In the gp32 I DNA complex formed upon denaturation of T4 DNA, the single-stranded DNA molecule has the same conformation as one strand of the double-strand T4 DNA molecule in the C-DNA conformation.     

Cytochemistry for bromodeoxyuridine/DNA analysis: stoichiometry and sensitivity

This report describes an improved immunochemical procedure for staining cells in suspension for amount of incorporated bromodeoxyuridine (BrdUrd) and total DNA. In this procedure, cellular DNA is partially denatured by extracting the cells with 0.1 M HCl and then heating them to 80 degrees C in a 50% formamide solution. The cells are then immunofluorescently stained using a monoclonal antibody against BrdUrd in single-strand DNA (ssDNA) and counterstained for DNA content with propidium iodide (PI), a dye that fluoresces preferentially when bound to double-strand DNA (dsDNA). We show that the relative amounts of immunofluorescently stained BrdUrd in ssDNA and PI in dsDNA can be altered reciprocally by changing the formamide concentration, denaturation time, and denaturation temperature. We show that this new immunochemical staining procedure allows more complete DNA denaturation so that fivefold lower levels of BrdUrd incorporation can be quantified. In addition, we show that the BrdUrd-linked immunofluorescence achieved using the new denaturation procedure is more linearly related to cellular BrdUrd content than that achieved after acid DNA denaturation. However, cell loss is sufficiently severe with the thermal denaturation procedure that it may not be applicable to all cell types.     

The helix-coil transition of closed and nicked DNAs in aqueous neutral trichloroacetate solutions.

The melting transition for closed, underwound DNAs and for nicked or linear DNAs was monitored by velocity sedimentation and by absorbance spectroscopy in aqueous NaCCl3CO2 (NaTCA) and RbTCA. The addition of neutral trichloroacetate lowers the midpoint of the helix-coil transition by 26% C/M for RbTCA and by 32% C/M for NaTCA, depressing the denaturation region to near room temperature at neutral pH. The melting of nicked DNA is cooperative, occurring over a temperature range of about 5.6 degrees C. The melting profile for closed DNA is broad and noncooperative with a transition breadth greater than 45 degrees. Closed DNAs undergo a structural alteration, as revealed by velocity sedimentation, resulting in a reduction in the number of superhelical turns at temperatures and salt concentrations substantially below the melting temperatures and salt concentrations substantially below the melting temperature of the nicked DNA. The reduction in the extent of supercoiling continues upon isothermal addition of salt up to the salt concentration at which all superhelical turns are removed. The salt concentration at the principal minimum in the sedimentation velocity profile (3.16 M NaTCA for PM-2 DNA) is approximately the same as that at the midpoint of the helix-coil transition for the nicked DNA.     

Oligonucleotide--minor groove binder 1:2 conjugates: side by side parallel minor groove binder motif in stabilization of DNA duplex

Synthetic polycarboxamides consisting of N-methylpyrrole (Py), N-methylimidazole (Im), N-methyl-3-hydroxypyrrole (Hp) and beta-alanine (beta) show strong and sequence-specific interaction with the DNA minor groove when they form hairpin structures with side-by-side antiparallel motifs. In the present paper, new conjugates containing two ligands linked to the same terminal phosphate of DNA strand were constructed. The paper describes optimized synthesis and properties of oligonucleotide-linked polyamide strands that insert into the minor groove of a duplex in a parallel or antiparallel orientation. Strong stabilization of DNA duplexes by two attached minor groove ligands is demonstrated by the thermal denaturation method. The unmodified duplex 5'-CGTTTATTp-3'/5'-AATAAACG-3' melts at 20 degrees C. When one tetra(Py) residue was attached to the first strand of this duplex, denaturation temperature was increased to 46 degrees C; attachment of the second tetra(Py) in a parallel orientation resulted in denaturation temperature of 60 degrees C. It is even higher than in case of "classic" octapyrrole hairpin ligand (Tm = 58 degrees C). Sequence-specific character of stabilization by two conjugated ligands was demonstrated for G:C-containing oligonucleotides attached to tetracarboxamide and octacarboxamide ligands constructed from Py, Im and beta units according to established recognition rules (deltaTm = 20 degrees C). The two-strand parallel minor groove binder constructions attached to addressing oligonucleotides could be considered as site-specific ligands recognizing single- and double-stranded DNA similarly to already described hairpin MGB structures with antiparallel orientation of carboxamide units.     

Large-scale isolation of plasmid DNA and purification of lambda phage DNA using hydroxylapatite chromatography

A rapid and relatively simple procedure for purifying large quantities of plasmid DNA is described. Plasmid thus purified contains no detectable chromosomal DNA and little RNA or protein. The procedure combines alkaline denaturation and hydroxylapatite chromatography and utilizes an improved method of separating DNA from RNA. It was observed that the phosphate concentrations at which previously bound DNA as well as RNA elute from hydroxylapatite changed markedly as a function of urea concentration. In the presence of urea concentrations higher than 4 M, the ranges of phosphate concentration over which DNA and RNA elute show no overlap. This permits efficient washing of hydroxylapatite-bound DNA under conditions which should remove all bound RNA. lambda Phage DNA is also easily eluted from hydroxylapatite under the conditions used.     

Thermodynamic dependence of DNA/DNA and DNA/RNA hybridization reactions on temperature and ionic strength

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, DeltaG degrees of the DNA hybridization reactions increased by about 6 kJ mol(-1) from 20 degrees C to 37 degrees 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 DeltaG degrees decreased non-linearly by 3.5 kJ mol(-1) at 25 degrees C and 6.0 kJ mol(-1) at 37 degrees 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 DeltaG degrees 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 degrees C from the melting transition at 56 degrees C.