Characterization of the synthetic compatible solute homoectoine as a potent PCR enhancer

Different substances such as dimethyl sulfoxide, tetramethylene sulfoxide, 2-pyrollidone, and the naturally occurring compatible solute betaine enhance PCR amplification of GC-rich DNA templates with high melting temperatures. In particular, cyclic compatible solutes outperform traditional PCR enhancers. We therefore investigated the effects that cyclic naturally occurring ectoine-type compatible solutes and their synthetic derivatives have on melting temperature of double-stranded DNA (dsDNA) and on PCR amplification of different templates. L-ectoine, betaine, and derivatives of L-ectoine decreased, whereas beta-hydroxyectoine increased, the melting temperature of dsDNA. The ability to decrease the melting temperature was greatest for homoectoine, a new synthetic derivative of l-ectoine. Furthermore, compatible solutes, especially homoectoine, enhanced PCR amplification of GC-rich DNA (72.6% GC content; effective range: 0.1-0.5M).     

Calculation of melting curves for DNA

A method is reported for calculating the melting curve of a DNA molecule of random base sequence, including in the formalism the dependence of the free energy of base pair formation on the size of a denatured section. Some explicit results are shown for a “typical” base sequence, in particular the probability of helix formation at individual base pairs in several different regions of the molecule and the amount of melting from the end of the chain. Particular attention is drawn to the variation of local melting behavior from one region of the molecule to another. It is found that sections rich in AT melt at relatively low temperatures with a fairly broad transition curve, whereas regions rich in GC pairs melt at higher temperatures (as expected) with a very abrupt, local transition curve. To account qualitatively for the results one may divide melting into two kinds of processes: (a) the nucleation and growth of denatured regions, and (b) the merging together of two denatured sections at the expense of the intervening helix. The first of these processes dominates in the first stages of melting, and leads to rather broad local melting curves, whereas the second process predominates in the later stages, and occurs, in a particular part of the molecule, over a very narrow temperature range. It is estimated that the average length of a helix plus adjacent coil section at the midpoint of the transition is approximately 600 base pairs. Since transition curves which measure the local melting behavior reflect local compositions fluctuations, these curves contain information about the broad outlines of base sequence in the molecule. Some suggestions are made concerning experiments by which this potential information source could be exploited. In particular, it is pointed out that one might hope to map AT or GC rich regions at particular genetic loci in a biologically active DNA molecule. Values of the relevant parameters found earlier for the transition of homopolymers produce melting curves for a DNA of random base sequence which are in good agreement with the experimental transition curve for T2 phage DNA. Hence the present theoretical picture of the melting of polynucleotides is at least internally self-consistent.     

Effect of ethylene glycol, urea, and N-methylated glycines on DNA thermal stability: the role of DNA base pair composition and hydration

The accumulation of the cosolutes ethylene glycol, urea, glycine, sarcosine, and glycine betaine at the single-stranded DNA surface exposed upon melting the double helix has been quantified for DNA samples of different guanine-cytosine (GC) content using the local-bulk partitioning model [Record, M. T., Jr., Zhang, W., and Anderson, C. F. (1998) Adv. Protein Chem. 51, 281-353]. Urea and ethylene glycol are both locally accumulated at single-stranded DNA relative to bulk solution. Urea exhibits a stronger affinity for adenine (A) and thymine (T) bases, leading to a greater net dehydration of these bases upon DNA melting; ethylene glycol local accumulation is practically independent of base composition. However, glycine, sarcosine, and glycine betaine are not necessarily locally accumulated at single strands after melting relative to bulk solution, although they are locally accumulated relative to double-stranded DNA. The local accumulation of glycine, sarcosine, and glycine betaine at single strands relative to double-stranded DNA decreases with bulk cosolute molality and increases with GC content for all N-methylated glycines, demonstrating a stronger affinity for G and C bases. Glycine also shows a minimum in melting temperature T(m) at 1-2 m for DNA samples of 50% GC content or less. Increasing ionic strength attenuates the local accumulation of urea, glycine, sarcosine, and glycine betaine and removes the minimum in T(m) with glycine. This attenuation in local accumulation results in counterion release during the melting transition that is dependent on water activity and, hence, cosolute molality.     

Enthalpies of DNA melting in the presence of osmolytes

The melting of DNA in the presence of osmolytes has been studied with the intention of obtaining information about how base pair stability is affected by changes in solution conditions. In previous investigations, the melting enthalpies were assumed to be constant as osmolalities change, but no systematic evaluation of whether this condition is true has been offered. This paper presents calorimetric data on the melting of two synthetic DNA samples in the presence of a number of common osmolytes. Poly(dAdT)*poly(dTdA) and poly(dGdC)*poly(dCdG) melting have been examined by differential scanning calorimetry in solutions containing ethylene glycol, glycerol, sucrose, urea, betaine, PEG 200 and PEG 1450 at increasing osmolalities. The results show small, but significant changes in the enthalpy of melting of the two polynucleotides that are different, depending on the structure of the cosolvent. The polyols, ethylene glycol, glycerol, PEG 200 and also urea all show decreases in melting enthalpy, while betaine and sucrose display increases with increasing concentration of cosolvent. The large stabilizing PEG 1450 shows no change within the experimental errors. Using concepts relating to preferential interactions of the cosolvents with the DNA base pairs, it is possible to interpret some of the observed changes in the thermodynamic properties of melting. The results indicate that there is strong entropy-enthalpy compensation upon melting base pairs, but entropy increases dominate to cause the decreases in stability with increased cosolvent concentration. Excess hydration parameters are evaluated and their magnitudes discussed in terms of changes in cosolvent interactions with the DNA base pairs.     

Melting profile and temperature dependent binding constant of an anticancer drug daunomycin-DNA complex

We calculate thermal fluctuational base pair opening probability and the drug binding constant of a daunomycin-bound Poly d(CGTA) · Poly d(TACG) at temperatures from room temperature to its melting temperature. For comparison we also carry out a calculation on a drug-free DNA with the same sequence. Our calculations are carried out by means of a statistical approach using microscopic structures and established force fields and with cooperative effects incorporated into the algorithm. Both hydrogen bond disruption probabilities and drug unstacking probability are determined self-consistently. These probabilities are then used to determine temperature dependent base pair opening probabilities and the drug binding constant. The calculated base pair opening probabilities and drug binding constant are found to be in fair agreement with experiments carried out at room temperature. Our calculation shows cooperative base pair disruption and drug dissociation at certain critical temperatures close to the observed melting temperatures for similar helices. We find that the temperature dependence of the drug binding constant fits well to the van't Hoff relation, in agreement with observations. Our calculation indicates the occurrence of a premelting transition in the drug-bound DNA helix. Some comments are made about this premelting transition.     

Sequence and temperature effect on hydrogen bond disruption in DNA determined by a statistical analysis

We use the modified self-consistent phonon approximation theory to calculate temperature dependent interbase hydrogen bond disruption profiles for a number of six base pair repeating sequence infinite B-DNA polymers with various guanine-cytosine/adenine-thymine ratios. For comparison we also include results we have obtained in our earlier work on several B-DNA homopolymers, copolymers and a four-base-pair repeating sequence polymer. Our theory gives a statistical estimate of thermal fluctuational disruption probability of individual hydrogen bonds in individual base pairs in DNA as a function of temperature. The calculated probabilities show no sequence dependence at premelting temperatures, in agreement with proton exchange measurements. These probabilities however become very sensitive to base sequence at temperatures close to the observed melting temperatures. Multi-phasic critical transitions are found in which a portion of base pairs are disrupted at temperatures below the final disruption temperature. These transitions include localized as well as non-localized base pair opening. The localized transitions involve disruption of a few base-pairs at every other location without large scale base unstacking, and they may not appear in the observed UV curves with current resolution. On the other hand the overall disruption behavior is consistent with observations. The midpoint transition temperatures are close to the observed melting temperatures and these temperatures show the observed linear dependence on guanine-cytosine content. Our calculations indicate that our theory can be used effectively to calculate H-bond disruption behavior of different DNA sequences. Received: 20 February 1996 / Accepted: 2 May 1996     

Dependence of the melting temperature of phage DNA on the GC pair content in a solvent with low ionic strength

Base ratio of DNA from 21 bacteriophage of Pseudomonas was determined by chemical hydrolysis and paper chromatography. Obtained values of the GC pair content were compared with melting temperature of DNA in 0,1 X SSC. The content of GC pairs correlates with melting temperature by equation %GC = 2,53 (Tm - 53,4). The content of GC pair for DNA from 30 bacteriophages of Pseudomonas was calculated. Some speculations concerning the distribution in DNA base ratio of bacteriophages of Pseudomonas are discussed.     

Base-pairing properties of the oxidized cytosine derivative, 5-hydroxy uracil

The most abundant base-substitution mutation resulting from oxidative damage to DNA is the GC to AT transition mutation. 5-hydroxyuracil (5-OHU), produced by the oxidative deamination of cystosine, has been established as the major chemical precursor for this most abundant transition mutation. Results from NMR spectroscopy and UV melting experiments show that 5-OHU would form the most stable pair with G, and the least stable pair with C. The hydroxyl group in the 5th position of the 5-OHU residue may play a role in increasing the stability of the 5-OHU:G pair over the normal Watson-Crick pair, the 5-OHU:A. The 5-OHU:C base pair would be least stable, and would destabilize the base-stacking in the duplex. Our results explain why certain DNA polymerases preferentially incorporate G opposite to 5-OHU over A and why C does not get incorporated against 5-OHU during DNA replication in vivo.     

Base- and sequence-dependent binding of aristololactam beta-D-glucoside to deoxyribonucleic acid

The dependence on base-pair composition and sequence specificity of the (aristololactam beta-D-glucoside)-DNA interaction was examined by spectrophotometric, spectrofluorometric, spectropolarimetric, thermal melting, thermodynamic, and viscometric studies. Binding of this alkaloid to various natural and synthetic DNAs was dependent upon the base composition and sequences of DNA. The binding parameters obtained from spectrophotometric analysis, according to an excluded-site model, indicated a relatively high affinity of the alkaloid binding to GC-rich DNA and alternating GC polymer. This affinity was further evidenced by the quenching of fluorescence intensity, decrease in quantum yield, and perturbations in circular dichroic spectrum. The alkaloid stabilized all DNAs against thermal denaturation. The temperature dependence of the binding constants was used to estimate the thermodynamic parameters involved in the complex formation of the alkaloid with various DNAs. The negative enthalpy and entropy change increased with increasing GC content of DNA and also compensated one another to produce a relatively small Gibbs free energy change. Viscometric studies showed that in the strong binding region the increase of contour length of DNA depended strongly on its base composition and sequence of bases, being larger for GC-rich DNA and alternating GC polymer. On the basis of these observations, it is concluded that the alkaloid binds to DNA by a mechanism of intercalation and exhibits considerable specificity toward alternating GC polymer.     

Agreement Between Deoxyribonucleic Acid Base Composition and Taxometric Classification of Gram-Positive Cocci.

Silvestri, L. G. (Università Statale, Milan, Italy), and L. R. Hill. Agreement between deoxyribonucleic acid base composition and taxometric classification of gram-positive cocci. J. Bacteriol. 90:136-140. 1965.-It had been previously proposed, from taxometric analyses, that gram-positive, catalase-positive cocci be divided into two subgroups. Thirteen strains, representative of both subgroups, were examined for deoxyribonucleic acid (DNA) base composition, determined from melting temperatures. Per cent GC (guanine + cytosine/total bases) values fell into two groups: 30.8 to 36.5% GC and 69 to 75% GC. Strains with low per cent GC values belonged to the Staphylococcus aureus-S. saprophyticus-S. lactis taxometric subgroups, and those with high per cent GC values belonged to the S. roseus-S. afermentans subgroup. The hypothetical nature of any classification is emphasized, and, in the present work, the hypothesis derived from taxometric analyses of division into two subgroups is confirmed by the study of DNA base ratios. The two subgroups correspond, respectively, to the genera Staphylococcus and Micrococcus.     

Salt effects on the denaturation of DNA

When DNA's of differing GC:AT base ratios, e.g. synthetic poly dAT, T4 DNA,calf thymus DNA, E. coli DNA, and M. lysodeikticus DNA, are heat-denatured at neutral pH in increasing concentrations of N(a)(2)SO(4) or C(s)(2)SO(4) as supporting electrolytes,the variation of melting temperature with average base composition, dT(m)/dX(G)(C), changes from 45°C (in 0.002M Na) to ll°C (in 4.5M Na) and from 42°C (in 0.002M Cs) to 3°C(in 4.5M Cs). The decrease of dT(m)/dX(G)(C) is a monotonic function of decreasing water activity in the salt solutions. We interpret this decreased composition dependence of the thermal stability of the various DNA's as being due to a destabilization of the GC base pairs relative to the AT base pairs by the concentrated salt media. A simple quantitative treatment shows that k = 8GC/SAT decreases from a value of 4.14 (in 0.01MN(a)) to 1.86 (in 3M Na) and from 4.18 (in 0.01M Cs) to 1.42 (in 3M Cs). SAT is the equilibrium constant for the formation of a hydrogen-bonded AT base pair from a pair of unbonded bases at the junction between a helical region and a denatured region and SGC is the like constant for the formation of a GC base pair. These results corroborate our previous findings of a strongly reduced composition dependence of the negative logarithm of the methylmercuric hydroxide concentration necessary to produce 50% denaturation when the helix-coil transition of DNA is studied in concentrated Cs(s)SO(4)(ultracentrifugation) instead of in dilute N(a)(2)SO(4) (ultraviolet spectrophotometry).     

The influence of intercalator binding on DNA triplex stability: correlation with effects on A-tract duplex structure

The triplex form of DNA is of interest because of a possible biological role as well as the potential therapeutic use of this structure. In this paper the stabilizing effects of two intercalating drugs, ethidium and the quinoxaline derivative 9-OH-B220, on DNA triplexes have been studied by thermal denaturation measurements. The corresponding duplex structures of the DNA triplex systems investigated are either A-tract or normal B-DNA. The largest increases in the triplex melting temperatures caused by the intercalators were found for sequences having A-tract duplex structures. Inserting a single base pair with an N2-amino group in the minor groove, e.g. a G-C pair, breaks up the A-tract duplex structure and also reduces the stabilizing effect of the drugs on the triplex melting temperatures. The large drug-induced increase in triplex melting temperature for complexes having an original duplex A-tract structure is correlated with a low initial melting point of the triplex, not with the triplex being unusually stable in the presence of the drug. Hence, we conclude that the large thermal stabilizing effect exhibited by ethidium and 9-OH-B220 on dTn.dAn-dTn triplexes is partly caused by the intercalators breaking up the intrinsic A-tract structure of the underlying duplex.     

Study of DNA melting in the region of the inversion of relative stability of AT and GC pairs]

Systematic data on the dependence of the melting curve parameters of DNA from different organisms on the concentration of salt (C2H5)5NBr have been obtained. The melting curves were studied by spectrophotometric as well as by microcalorimetric methods. The DNA melting range width is shown to pass through the minimum value delta0T = 0.6 +/- 0.1 degrees at the point of inversion of relative stability of AT and GC pairs that corresponds to the concentration of (C2H5)4NBr equal to 2.9 +/- 0.1 M. This concentration, as well as the value of delta0T, are the same for different DNA's of common chemical structure. The T2 and T4 DNA containing hydroxymethylated and glucosylated cytosine residues show an anomalous behaviour. The enthalpy of melting falls very slowly as the salt concentration increases. The possible causes of the observed value of delta0T are discussed. A conclusion is drawn that the main factor which governs the DNA melting process in the region of inversion of the relative stability of AT and GC pairs is the heterogeneity of stacking interaction between different base pairs.     

Enzyme stabilization using synthetic compensatory solutes

The protective effect of the synthetic compensatory solutes, dimethylthetin (CAS 4727-41-7) and homodeanol betaine (N, N-dimethyl-N-(2-hydroxyethyl)-N-(2 carboxyethyl) ammonium inner salt, CAS 6249-53-2), on two enzymes: lactate dehydrogenase (LDH from rabbit muscle) and a microbial lipase, was compared with that of glycine betaine, trehalose and sorbitol. When the enzyme plus 1 M solute were heated for 10 min at temperatures between 35-75°C, the temperature at which 50% of enzyme activity was lost increased most in the presence of trehalose (7.9° for LDH, 11.6° for lipase) and homodeanol betaine (10.7° for LDH, 11.0° for lipase). With both enzymes, more activity was retained at extreme temperatures in the presence of homodeanol betaine than with trehalose. Glycine betaine, dimethylthetin and sorbitol were less effective. Enzyme plus 1 M stabilizer solutions were frozen at -30°C and freeze-dried for 24 h. Trehalose was the most effective stabilizer of lactate dehydrogenase, and homodeanol betaine of lipase, during freeze-drying.     

Preferential interactions of glycine betaine and of urea with DNA: implications for DNA hydration and for effects of these solutes on DNA stability

Interactions of the solutes glycine betaine (GB) and urea with mononucleosomal calf thymus DNA in aqueous salt solutions are characterized by vapor pressure osmometry (VPO). Analysis of osmolality as a function of solute and DNA concentration yields the effect of the solute on the chemical potential, mu(2), of the DNA. Although both GB and urea generally are nucleic acid denaturants and therefore must interact favorably with the nucleic acid surface exposed upon melting, VPO demonstrates that neither interacts favorably with duplex DNA. Addition of GB greatly increases mu(2) of DNA, indicating that the average local concentration of GB in the vicinity of the double helix is much less than its bulk concentration. By contrast, addition of urea has almost no effect on mu(2) of duplex DNA, indicating that the average local concentration of urea in the vicinity of duplex DNA is almost the same as in bulk solution. Qualitatively, we conclude that the nonuniform distribution of GB occurs primarily because duplex DNA and GB prefer to interact with water rather than with each other. Comparison with thermodynamic data for the interaction of GB with various protein surfaces (Felitsky et al., Biochemistry, 43, 14732-14743) shows that GB is excluded primarily from anionic DNA surface and that the hydration of anionic DNA phosphate oxygen surface (>or approximately 17 H(2)O per nucleotide or >or approximately 0.22 H(2)O A(-)(2)) involves at least two layers of water. From analysis of literature data for effects of urea and of GB on DNA melting, we propose that urea is an effective nonspecific nucleic acid denaturant because of its favorable interactions with the polar amide-like surface of G, C, and especially T or U bases exposed in denaturation, whereas GB is a specific GC denaturant because of its favorable interaction with G and/or C surface in the single-stranded state.     

Proton nuclear magnetic resonance investigation of the conformation and dynamics in the synthetic deoxyribonucleic acid decamers d(ATATCGATAT) and d(ATATGCATAT)

A variety of one-dimensional proton NMR methods have been used to investigate the properties of two synthetic DNA decamers, d(ATATCGATAT) and d(ATATGCATAT). These results, in conjunction with the results of two-dimensional NMR experiments, permit complete assignment of the base proton resonances. Low-field resonances were assigned by sequential "melting" of the A . T base pairs and by comparison of the spectra of the two decamers. Below 20 degree C spin-lattice relaxation is dominated by through-space dipolar interactions. A substantial isotope effect on the G imino proton relaxation is observed in 75% D2O, confirming the importance of the exchangeable amino protons in the relaxation process. A somewhat smaller isotope effect is observed on the T imino proton relaxation. At elevated temperatures spin-lattice relaxation of the imino protons is due to proton exchange with solvent. Apparent activation energies for exchange vary from 36 kcal/base pair for base pairs (3,8) to 64 kcal/mol for the most interior base pairs (5,6), indicating that disruption of part, or all, of the double helix contributes significantly to the exchange of the imino protons in these decamers. By contrast, single base pair opening events are the major low-temperature pathways for exchange from A X T and G X C base pairs in the more stable higher molecular weight DNA examined in other studies. The temperature dependence of the chemical shifts and line widths of certain aromatic resonances indicates that the interconversion between the helix and coil states is not in fast exchange below the melting temperature, Tm. Within experimental error, no differential melting of base pairs was found in either molecule, and both exhibited melting points Tm = 50-52 degrees C. Spin-spin and spin-lattice relaxation rates of the nonexchangeable protons (TH6, AH8, and AH2) are consistent with values calculated by using an isotropic rotor model with a rotational correlation time of 6 ns and interproton distances appropriate for B-family DNA. The faster decay of AH8 compared with GH8 is attributed to an interaction between the thymine methyl protons and the AH8 protons in adjacent adenines (5'ApT3'). The base protons (AH8, GH8, and TH6) appear to be located close (1.9-2.3 A) to sugar H2',2" protons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Physicochemical properties of phosphorothioate oligodeoxynucleotides.

We have recently shown that phosphorothioate (PS) oligodeoxynucleotide (ODN) analogs, unlike their normal congeners, exhibit significant anti-HIV activity (Matsukura et al., (1987) Proc. Natl. Acad. Sci. USA 84, 7706-7710). We now report the syntheses, melting temperatures (Tm), and nuclease susceptibilities of a series of phosphorothioate ODN analogs. These include all-PS duplexes, duplexes with one normal chain and the other chain either all-PS, or end-capped with several PS groups at both 3' and 5' ends. The DNase susceptibilities of the S-ODNs are much less than the normal phosphodiesters, but by contrast duplexes of poly-rA with S-dT40 are much more susceptible to RNase H digestion. The Tm's for AT base pairs of S-ODNs are significantly depressed relative to normals, while GC base pairs show much less Tm depression. The Tm's of S-dT oligomers with poly-rA are reduced relative to the duplexes with normal dA oligomers. These results have significance for the biological properties of these analogs as anti-message inhibitors of gene expression, and provide a rational basis for the S-dC/G sequences as potential effective anti-AIDS agents.     

Fractionation of calf thymus DNA based on its interaction with homologeous f1 histone. Melting curves of the obtained fractions

Calf thymus DNA fractions were obtained by precipitation with the homologeous f₁ histone and their melting curves were investigated. An increase of the melting temperatures of DNA remaining in the supernatants was observed. Within the range of 5–50 % of the DNA precipitated the melting temperatures and the melting intervals of DNA in the sediment remained constant. The obtained values /38 mole % GC, ΔT_m = 7.0°C/ suggest that DNA found in the precipitates corresponds to the main calf thymus DNA. Despite its heterogeneity this group of DNA molecules does not undergo fractionation using f₁ histone. We assume that the molecules of the main DNA all contain specific areas to which the f₁ histone attaches in our experimental conditions. The main DNA molecules, regardless of their base composition, seem to contain these specific areas in amounts causing equal precipitation probability. They seem to differ in this respect from some GC rich fractions, possibly those of satellite DNA.