Thermal stability of PNA/DNA and DNA/DNA duplexes by differential scanning calorimetry

Thermodynamics of the thermal dissociation transitions of 10 bp PNA/DNA duplexes and their corresponding DNA/DNA duplexes in 10 mM sodium phosphate buffer (pH 7.0) were determined from differential scanning calorimetry (DSC) measurements. The PNA/DNA transition temperatures ranged from 329 to 343 K and the calorimetric transition enthalpies ranged from 209 +/- 6 to 283 +/- 37 kJ mol(-1). The corresponding DNA/DNA transition temperatures were 7-20 K lower and the transition enthalpies ranged from 72 +/- 29 to 236 +/- 24 kJ mol(-1). Agreement between the DSC and UV monitored melting (UVM) determined transition enthalpies validated analyzing the UVM transitions in terms of a two-state transition model. The transitions exhibited reversibility and were analyzed in terms of an AB = A + B two-state transition model which yielded van't Hoff enthalpies in agreement with the transition enthalpies. Extrapolation of the transition enthalpies and free energy changes to ambient temperatures yielded more negative values than those determined directly from isothermal titration calorimetry measurements on formation of the duplexes. This discrepancy was attributed to thermodynamic differences in the single-strand structures at ambient and at the transition temperatures, as indicated by UVM measurements on single DNA and PNA strands.     

Structural organization of double-stranded RNA from killer yeasts Saccharomyces cerevisiae by the thermal denaturation method

The thermal denaturation method for studying the structural organization of double-stranded RNA (dsRNA) from virus-like particles of killer yeasts Saccharomyces cerevisiae was used. High resolution derivative denaturation profiles of total dsRNA and its L- and M-types were obtained. Comparative analysis of these data with those on phage DNA denaturation demonstrated that the processes of denaturation of dsRNA and phage DNA were identical in quality. Increase of thermostability, interval of thermal denaturation and width of local helix-to-coil transitions in dsRNA as compared with phage DNA are caused by the differences of corresponding thermodynamic parameters. Derivative denaturation profiles of L- and M-types of yeasts dsRNA were shown to have certain identical local transitions. Low melting transition, consisting of three local thermalites, is due to the denaturation of AU-rich region (about 200 n.b.p.) in M-dsRNA.     

Human RAD52 protein has extreme thermal stability.

The human RAD52 protein plays an important role in the earliest stages of chromosomal double-strand break repair via the homologous recombination pathway. Individual subunits of RAD52 associate into seven-membered rings. These rings can form higher order complexes. RAD52 binds to DNA breaks, and recent studies suggest that the higher order self-association of the rings promotes DNA end joining. Monomers of the RAD52(1--192) deletion mutant also associate into ring structures but do not form higher order complexes. The thermal stability of wild-type and mutant RAD52 was studied by differential scanning calorimetry. Three thermal transitions (labeled A, B, and C) were observed with melting temperatures of 38.8, 73.1, and 115.2 degrees C. The RAD52(1--192) mutant had only two thermal transitions at 47.6 and 100.9 degrees C (labeled B and C). Transitions were labeled such that transition C corresponds to complete unfolding of the protein. The effect of temperature and protein concentration on RAD52 self-association was analyzed by dynamic light scattering. From these data a four-state hypothetical model was developed to explain the thermal denaturation profile of wild-type RAD52. The three thermal transitions in this model were assigned as follows. Transition A was attributed to the disruption of higher order assemblies of RAD52 rings, transition B to the disruption of rings to individual subunits, and transition C to complete unfolding. The ring-shaped quaternary structure of RAD52 and the formation of higher ordered complexes of rings appear to contribute to the extreme stability of RAD52. Higher ordered complexes of rings are stable at physiological temperatures in vitro.     

Salt dependence and thermodynamic interpretation of the thermal denaturation of small DNA restriction fragments.

The influence of cation concentration on the thermal denaturation of DNA restriction fragments from the E. coli lac regulatory region and from pVH51, ranging in size from 43- to 880- bp, is described. Upon increasing the ionic strength, the melting transitions broaden in a cooperative manner at salt concentrations characteristic for the specific fragment. For three fragments studied in detail, the salt concentration dependence at the midpoint varied between 0.03 and 0.19 M Na+. Along with the broadening, the melting transitions become more symmetrical. This result is discussed with respect to the irreversibility of melting transitions at low ionic strength. After a cooperative broadening, the shape of the melting curves remains constant up to salt concentrations of 0.5 M Na+. The dTM/dlog[Na+] values for three fragments fall between 15.7 and 16.7. An easily applicable approximation of the van't Hoff equation is used to evaluate the enthalpies of 13 transitions arising from the denaturation of 43 to 600 bp. The results of this analysis are compared to calculations of the expected enthalpies based on calorimetric measurements. The TMs of most transitions were directly related to the base composition, but several deviations from the predicted behavior were observed. The possible influences of fragment length and sequence on the thermal stability are discussed. The experimental and mathematical procedure to resolve a thermal denaturation transition with a width f 0.17 +/- 0.01 degrees and its distinction from another preceeding transition only approximately 0.15 degrees away in an 880-bp Hae III fragment from pVH51 is described. This transition is about half as wide as the smallest one reported to date.     

High-resolution thermal denaturation of DNA. I. Theoretical and practical considerations for the resolution of thermal subtransitions.

The fidelity achieved in first derivative profiles of DNA thermal denaturation is shown to depend on a number of factors including the thermal increment of data gathering, the precision of absorbance readings, and the manner in which data are smoothed prior to calculating the derivative of hyperchromicity. The closeness with which thermal denaturation data can be fitted by a cubic polynomial is carefully considered, and a derivation is presented for the estimated error in calculated values of the derivative of hyperchromicity with respect to temperature. After reviewing both theoretical and experimental evidence for the expected minimum width of a thermal transition in DNA, we conclude that thermal increments of 0.05°C or less are required for an adequate representation of transitions in naturally occurring DNA's. Data gathered under conditions meeting the requirements suggested here for quantitative recording of thermal denaturation profiles (Vizard and Ansevin, submitted for publication) show that virtually all of the high-resolution thermal denaturation profile of a simple, naturally occuring DNA may consist of small subtransitions, which we call thermalites. The finding of substransitions is consistent with current theories of DNA melting. A particularly well-resolved thermalite of λ bacteriophage DNA has a breadth of only 0.30°C (2σ width), and thus is narrower than previously reported thermal transitions for DNA. For this thermalite, the combination of width, shape, and position in the profile suggests that the substransitions observed in accurately recorded DNA thermal denaturation profiles are not described satisfactorily by existing theories. Knowledge of the requirements for the quantitative recording of thermal denaturation profiles should greatly favor the usefulness of denaturation experiments for physical genomic analysis.     

Differential scanning calorimetry of chromatin at different levels of condensation

The thermal denaturation of calf thymus total chromatin and of fractions enriched in heterochromatin or euchromatin, has been investigated by differential scanning calorimetry and compared to that of calf thymus DNA and DNA-histone complexes. In our experimental conditions, chromatin melts in three thermal transitions: the main one, assigned to separation of the DNA double helix, occurs at 83 °C, while the other two occur at 63 °C and 74 °C. The data show that: (a) the transition enthalpy for denaturation of DNA in the total chromatin and in DNA-histone complexes is nearly the same as that of DNA in solution; (b) the transition at 63 °C is present in the thermogram of the heterocromatin enriched fraction, while it is completely absent in that of the euchromatin enriched one. The results suggest that this transition can be attributed to the higher order structures of heterochromatin.     

Differential scanning calorimetry of chicken erythrocyte nuclei.

Investigation of structural features of native chromatin requires the use of intact nuclei, a turbid material which cannot be analyzed by optical methods. Differential scanning calorimetry does not require optically clear samples and has been proved by a number of authors to be a powerful tool in this field of study. By this technique, chicken erythrocyte nuclei were found to undergo at least four thermal transitions, centered at 59, 74, 88 and 98 degrees C. The highest temperature transition is strongly dependent on age and storage conditions of the nuclei. Adequate storage conditions overcame this problem and reproducible scans were obtained over a period of several months. This technical improvement has permitted the reconsideration of the occurrence of the fourth calorimetric transition, previously believed to be displayed only in replicating nuclei. Evidence gathered in the presence of perturbants and possible ligands allows the assignment of the four transitions to a nuclear protein scaffold, histones, nucleosomal DNA and a superstructured form of DNA. Moreover, it suggests that the higher-order structure is stabilized by fibronectin-like proteins.     

Ionic strength-dependent conformational transitions of chromatin. Circular dichroism and thermal denaturation studies

High-molecular-weight chicken erythrocyte chromatin was prepared by mild digestion of nuclei with micrococcal nuclease. Samples of chromatin containing both core (H3, H4, H2A, H2B) and lysine-rich (H1, H5) histone proteins (whole chromatin) or only core histone proteins (core chromatin) were examined by CD and thermal denaturation as a function of ionic strength between 0.75 and 7.0 × 10^?3M Na⁺. CD studies at 21°C revealed a conformational transition over this range of ionic strengths in core chromatin, which indicated a partial unfolding of a segment of the core particle DNA at the lowest ionic strength studied. This transition is prevented by the presence of the lysine-rich histones in whole chromatin. Thermal-denaturation profiles of both whole and core chromatins, recorded by hyperchromicity at 260 nm, reproducibly and systematically varied with the ionic strength of the medium. Both materials displayed three resolvable thermal transitions, which represented the total DNA hyperchromicity on denaturation. The fractions of the total DNA which melted in each of these transitions were extremely sensitive to ionic strength. These effects are considered to result from intra- and/or internucleosomal electrostatic repulsions in chromatin studied at very low ionic strengths. Comparison of the whole and core chromatin melting profiles indicated substantial stabilization of the core-particle DNA by binding sites between the H1/H5 histones and the 140-base-pair core particle.     

State transitions by molecules

In our previous paper, we described a method by which a state machine is implemented by a single-stranded DNA molecule whose 3'-end sequence encodes the current state of the machine. Successive state transitions are performed in such a way that the current state is annealed onto an appropriate portion of DNA encoding the transition table of the state machine and the next state is copied to the 3'-end by extension with polymerase. In this paper, we first show that combined with parallel overlap assembly, a single series of successive transitions can solve NP-complete problems. This means that the number of necessary laboratory steps is independent from the problem size. We then report the results of two experiments concerning the implementation of our method. One is on isothermal reactions which greatly increase the efficiency of state transitions compared with reactions controlled by thermal cycles. The other is on the use of unnatural bases for avoiding out-of-frame annealing. The latter result can also be applied to many DNA-based computing paradigms.     

Chromatin models. Thermal denaturation studies of (Lysx, Leuy)n-and (Lys)n(Leu)m-DNA complexes.

The structural transitions of (Lysx, Leuy)n-DNA and (Lysx)n(Leuy)m-DNA complexes have been studied by thermal denaturation utilizing simultaneous absorption and circular dichroism (CD) measurements [R. Mandel and G.D. Fasman (1974), Biochem. Biophys. Res. Commun. 59, 672]. These complexes are used as models for nucleohistones. At amino acid/nucleotide ratios r less than 1, the copolymers bind to DNA in a ratio of one amino acid residue per nucleotide, and such binding stabilizes the DNA double helix against thermal denaturation relative to the unbound regions. The leucine residues in the copolymers stabilize the bound portion of the complex against thermal denaturation but to a lesser degree than does poly(L-lysine). This study confirms the hypothesis that absorption melting profiles reflect only the change in secondary structure (helix-coil transition) of DNA. It was found that, in the absence of a higher ordered structure (condensed), the CD melting profile also reflects this same conformational transition, and the melting temperatures, Tm, in CD are equal to those in absorption. However, when a higher ordered structure (tertiary) exists in the complex, then the CD melting profile will be dominated by the structural transitions related to the melting of the higher ordered asymmetric structure in the condensed state, followed by the melting of the secondary structure. Under such circumstances, the Tm obtained from absorption may be slightly different from that of the CD, since only the secondary structural changes are being reflected in absorption. The relevance of these studies to the structure of chromatin is discussed.     

A scanning calorimetric study of natural DNA and antitumoral anthracycline antibiotic-DNA complexes

Thermal denaturation of natural DNA in the absence and presence of antitumor anthracycline antibiotics has been studied by adiabatic differential scanning calorimetry. The helix-coil transition is operationally irreversible as measured by DSC. Both the melting temperature and the overall molar transition enthalpy of the DNA samples was dependent on the percentage of GC base pairs. Calorimetric traces of anthracycline-DNA complexes have qualitatively similar features and the significance of this characteristic is discussed. The unsaturated drug-DNA complex melts through complex thermal transitions with one broad endotherm in the same temperature region as free DNA and the other at a higher temperature which is rf (mol ligand per mol DNA in base pairs) value dependent. Antibiotic binding at concentrations close to saturating conditions (rf = 0.2) reverts the melting range to a value near to its original one and increases the thermal stability of the duplex structure by around 30 degrees C. In addition, the calorimetric enthalpy is increased by between 64% and 150%, depending on which ligand was used.     

Equilibrium thermal transitions of collagen model peptides

The folding of collagen in vitro is very slow and presents difficulties in reaching equilibrium, a feature that may have implications for in vivo collagen function. Peptides serve as good model systems for examining equilibrium thermal transitions in the collagen triple helix. Investigations were carried out to ascertain whether a range of synthetic triple-helical peptides of varying sequences can reach equilibrium, and whether the triple helix to unfolded monomer transition approximates a two-state model. The thermal transitions for all peptides studied are fully reversible given sufficient time. Isothermal experiments were carried out to obtain relaxation times at different temperatures. The slowest relaxation times, on the order of 10-15 h, were observed at the beginning of transitions, and were shown to result from self-association limited by the low concentration of free monomers, rather than cis-trans isomerization. Although the fit of the CD equilibrium transition curves and the concentration dependence of T(m) values support a two-state model, the more rigorous comparison of the calorimetric enthalpy to the van't Hoff enthalpy indicates the two-state approximation is not ideal. Previous reports of melting curves of triple-helical host-guest peptides are shown to be a two-state kinetic transition, rather than an equilibrium transition.     

Changes in chromatin structure during the aging of cell cultures as revealed by differential scanning calorimetry

Nuclei from cultured human cells were examined by differential scanning calorimetry. Their melting profiles revealed four structural transitions at 60, 76, 88, and 105 degrees C (transitions I-IV, respectively). In immortalized (i.e., tumor) cell cultures and in normal cell cultures of low passage number, melting profiles were dominated by the 105 degrees C transition (transition IV), but in vitro aging of normal and Werner syndrome cells was associated with a marked decrease in transition IV followed by an increase in transition III at the expense of transition IV. At intermediate times in the aging process, much DNA melted at a temperature range (95-102 degrees C) intermediate between transitions III and IV, and this is consistent with the notion that aging of cell cultures is accompanied by an increase in single-strand character of the DNA. Calorimetric changes were observed in the melting profile of nuclei from UV-irradiated tumor cells that resembled the age-induced intermediate melting of chromatin. It is suggested that aging is accompanied by an increase in single-stranded character of the DNA in chromatin, which lowers its melting temperature, followed by strand breaks in the DNA that destroy its supercoiling potential.     

Caffeine enhancement of digestion of DNA by nuclease S1.

The activity of Aspergillus orzae nuclease S1 on DNA has been investigated under varying pH and metal ion conditions. Nuclease S1 was found to preferentially digest denatured DNA. With native DNA as substrate the enzyme could only digest the DNA when caffeine was added to the reaction mixture. The enzyme was more active in sodium acetate buffer (pH 4.5), than in either standard saline citrate (PH 7.0) or sodium phosphate buffer (pH 6.8). Caffeine was also found to affect the thermal stability of DNA, resulting in a melting profile characterized by two transitions. The first transition (poorly defined) was below the normal melting temperature of the DNA, while the next transition was at the normal melting temperature of the DNA, while the next transition was at the normal melting temperature of the DNA. The susceptibility of caffeine-treated DNA to nuclease digestion seems to be a result of the local unwinding that caffeine causes in the regions of DNA that melt in the first transition. This selective destabilization presumably sensitizes the unwound regions to nuclease hydrolysis. The hydrolysates of the DNA digested by nuclease S1 were subjected first to ion exchange chromatography followed by paper chromatography. The results from this partial characterization of the digestion products showed that they contain mononucleotides as well as oligonucleotides of varying lengths. The base composition of the mononucleotide digests suggests that caffeine has greater preference for interacting with A-T base-pairs in DNA.     

Simultaneous, coincident optical trapping and single-molecule fluorescence

We constructed a microscope-based instrument capable of simultaneous, spatially coincident optical trapping and single-molecule fluorescence. The capabilities of this apparatus were demonstrated by studying the force-induced strand separation of a dye-labeled, 15-base-pair region of double-stranded DNA (dsDNA), with force applied either parallel ('unzipping' mode) or perpendicular ('shearing' mode) to the long axis of the region. Mechanical transitions corresponding to DNA hybrid rupture occurred simultaneously with discontinuous changes in the fluorescence emission. The rupture force was strongly dependent on the direction of applied force, indicating the existence of distinct unbinding pathways for the two force-loading modes. From the rupture force histograms, we determined the distance to the thermodynamic transition state and the thermal off rates in the absence of load for both processes.     

Influence of lipid membranes on the conformational transitions of nucleic acids

The conformational transitions of nucleic acids which were enclosed in reverse phase evaporation vesicles (REV) were studied by thermal denaturation with optical recording. Cloned fragments of double-stranded DNA containing 179 base pairs and 187 base pairs, respectively, and polyA.polyU were enclosed in REV with a yield up to every vesicle containing 50 nucleic acid molecules. With the 179 base pairs DNA enclosed in the vesicle from egg lecithin two well resolved helix-coil transitions could be measured; one is very similar in the midpoint-temperature Tm and halfwidth delta T1/2 to the transition of the free nucleic acid, and the other transition occurs stabilized at a 3.5 degrees C higher Tm-value and with a broader delta T1/2, 2.7 degrees C instead of 0.6 degree C. Both transitions are from nucleic acids inside the vesicles. Varying the surface charge of the lipid membrane by adding the negatively charged phosphatidylserine or phosphatidylglycerol, an optimum in the yield of enclosure and a maximum in the increase in Tm (4.5 degrees C) and delta T1/2 (5.5 degrees C instead of 1.0 degrees C) was obtained at 20% phosphatidylserine or phosphatidylglycerol. In vesicles from pure negatively charged lipids no second population of nucleic acids was observed. Qualitatively, similar effects were observed with polyA.polyU. Stabilization and broadening of the second transition is higher for nucleic acids inside vesicles from lipids with unsaturated fatty acids, as dioleoyl-phosphatidylcholine, than with saturated fatty acids, dipalmitoyl-phosphatidylcholine. Stabilization and broadening decrease with increasing ionic strength, whereas the relative contributions of both transitions to the total hypochromicity remain unchanged; the second transition coincides with the first at 90 mM Na+. From the experimental results it was concluded that the interaction of nucleic acids and lipid membranes is mainly of electrostatic nature. The nucleic acids exist inside the vesicles in two populations, one behaving like nucleic acid free in solution and one influenced by the contact with the membrane. All results are in accordance with a model in which the interaction between the nucleic acid and the membrane is in competition with the dipole-dipole interaction inside the membrane surface.     

Differential scanning calorimetric investigation of pea chloroplast thylakoids and thylakoid fractions.

High sensitivity differential scanning calorimetry (DSC) was employed to study the thermal denaturation of components of pea chloroplast thylakoid membranes. In contrast to previous reports utilizing spinach thylakoids, several transitions are reversible, and deconvolution of the calorimetric curves indicates nine transitions in both first and second heating scans, but overlapping transitions obscure at least three transitions in the first heating scans of control thylakoids. Glutaraldehyde fixation increases the denaturation temperature of several transitions which is consistent with a reported increase in thermal stability of thylakoid function due to fixation. Acidic pH treatment has little effect on the DSC curves, although it has been reported to have a significant effect on membrane structure. Separation of grana from stroma thylakoids indicates that components responsible for transitions centered at approximately 56, 73, 77, and 91 degrees C are predominantly or exclusively associated with grana thylakoids, whereas components responsible for transitions centered at approximately 63 and 81 degrees C are predominantly associated with stroma thylakoids. A broad transition centered at 66 degrees C is associated with grana thylakoids, whereas a sharp transition at the same temperature is due to a component associated with stroma thylakoids. Evidence obtained by washing treatments suggests the latter transition originates from the denaturation of the thylakoid ATPase (CF1). Analysis of the calorimetric enthalpy values indicates most components of the grana thylakoids denature irreversibly at high temperature, whereas components associated with the stroma thylakoids have a considerable degree of thermal reversibility.     

Differences in thermal stability of frog and rabbit alpha alpha- and alpha beta-tropomyosins determined by optical rotatory dispersion

Frog and rabbit alpha alpha- and alpha beta-tropomyosins were purified, and their thermal stabilities determined by use of optical rotatory dispersion. The tropomyosins were found to be virtually completely helical at 5 degrees C. Regions of different thermal stabilities were seen for all tropomyosins. Rabbit and frog alpha alpha-tropomyosin show very similar thermal properties, with main transitions near 47-49 degrees C. The main transition for frog alpha beta-tropomyosin is at 32 degrees C. The results show that the alpha beta-tropomyosins are less stable than the alpha alpha-forms. Only thermal transitions of the alpha beta-forms appear to be correlated with the body temperatures of the animals.     

The influence of tertiary structural restraints on conformational transitions in superhelical DNA.

This paper examines theoretically the effects that restraints on the tertiary structure of a superhelical DNA domain exert on the energetics of linking and the onset of conformational transitions. The most important tertiary constraint arises from the nucleosomal winding of genomic DNA in vivo. Conformational transitions are shown to occur at equilibrium at less extreme superhelicities in DNA whose tertiary structure is restrained than in unrestrained molecules where the residual linking difference alpha res (that part of the superhelical deformation which is not absorbed by transitions) may be freely partitioned between twisting and bending. In the extreme case of a rigidly held tertiary structure, this analysis predicts that the B-Z transition will occur at roughly half the superhelix density needed to drive the same transition in solution, other factors remaining fixed. This suggests that superhelical transitions may occur at more moderate superhelical deformations in vivo than in solution. The influence on transition behavior of the tertiary structural restraints imposed by gel conditions also are discussed.