Interaction of nucleic acids. VI. Interaction of purine with nucleic acids

The interaction of purine with DNA, tRNA, poly A, poly C, and poly A. poly U complex was investigated. In the presence of purine, the nucleic acids in coil form (such as denatured DNA, poly A and poly C in neutral solutions, or tRNA) have lower optical rotations. In addition, hydrodynamic studies indicate that in purine solutions the denatured DNA has a higher viscosity and a decreased sedimentation coefficient. These findings indicate that through interaction with purine, the bases along the poly-nucleotide chain are unstacked and are separated farther from each other, resulting in increased assymmetry (and possibly volume) of the whole polymer. Thus, the de-naturation effect of purine reported previously can be explained by this preferential interaction of purine with the bases of nucleic acids in coil form through a hydrophobic-costacking mechanism. Results from studies on optical rotation and helix-coil transition show that the interaction of purine is greater with poly A than with poly C. The influence of temperature, Mg⁺⁺ concentration, ionic strength, and purine concentration on the effect of purine on nucleic acid conformation has also been investigated. In all these situations the unraveling of nucleic acid conformation occurs at much lower temperatures (20–40°C lower) in the presence of purine (0.2–0.6M).     

Unusual DNA Binding Exhibited by Synthetic Distamycin Analogues Lacking the N-terminal Amide Unit under High Salt Conditions

Abstract

The binding of three analogues of the minor-groove binding antiviral antibiotic distamycin (Dst) with double-stranded (ds)-DNA were monitored using ds-DNA melting temperature (T_m) measurements, ethidium bromide (EtBr) displacement assay, footprinting analysis and induced circular dichroism (ICD). These compounds contained 3–5 N-methyl-pyrrole-car- boxamide units and lacked the N-terminal formamide unit present in Dst. These experiments suggested that the present analogues did not compromise their AT-specificity despite the deletion of the N-terminal formamide unit. The binding affinities, however, were significantly affected. Interestingly, the analogue with three N-methyl-pyrrole-carboxamide units exhibited an initial decrease in ICD at >40 mM salt concentrations. This was followed by a pronounced recovery of ICD at > 1.6 M salt concentrations, a phenomenon hitherto not observed with any other DNA binding molecules. The pentapyrrole analogue exhibited the highest binding affinity with CT-DNA under normal (40 mM) salt conditions. However, it suffered maximum relative dissociation under high salt conditions and did not exhibit any recovery in ICD at higher NaCl concentrations. The analogues possessing four and five pyrrole rings exhibited intense ICD signals with poly d(GC) in the ligand absorption region in the presence of 40 mM NaCl, unlike the one with three pyrrole rings. These ICD signals were however, highly susceptible to changes in ionic strength. Thus subtle modifications in the ligand molecular structure can have dramatic effect on their DNA binding properties.     

Biphasic helix–coil transition of the ethidium bromide·poly(dA-dT) and the propidium diiodide·poly(dA-dT) Complexes. Stabilization of base-pair regions centered about the intercalation site

The nonexchangeable base and sugar proton nmr resonances and the 260 and 278-nm uv-absorbance bands of the nucleic acid were utilized to monitor the temperature-dependent duplex-to-strand transition of the alternating purine–pyrimidine deoxyribopolynucleotide poly(dA-dT) in the absence and presence of ethidium bromide (EB) at phosphate/drug = 50, 28, and 15 and propidium diiodide (PI) at P/D = 50, 25, 15, 10, and 5 in 0.1 M salt between 50° and 100°C. The nmr and optical methods monitor a biphasic duplex-to strand transition for the drug–poly(dA-dT) complexes. We have monitored the dissociation of the drug from the complex at the ethidium bromide phenanthridine ring and side-chain proton nmr resonances and the propidium diiodide 494 and 535-nm uv-absorbance bands and demonstrate that dissociation of the drug corresponds to the higher temperature transition in the biphasic nucleic acid melting curves. The lower temperature cooperative transition is assigned to the opening of drug-free AT base-pair regions in the drug–poly(dA-dT) complex and exhibits an increase in transition midpoint and a decrease in cooperativity with increasing drug concentration. The higher temperature cooperative transition is assigned to the opening of AT base-pair regions centered about the bound drug in the complex and exhibits an increase in the transition midpoint on raising the drug concentration. The large upfield shifts of the phenanthridine ring (but not side chain) protons of ethidium bromide on complex formation demonstrate intercalation of the drug between base pairs of the poly(dA-dT) duplex. The nucleic acid base and sugar resonances of poly(dA-dT) in 0.1 M phosphate undergo chemical shift changes between 0° and 50°C indicative of premelting conformational transition(s).     

On the theory of Ψ-condensation

We suggest a theory of Ψ-condensation, based on the assumption that a compact DNA particle is a globule, and specifically that a polymer solution is a strongly fluctuating system and that double-stranded DNA is a stiff homopolymer single-stranded chain. We show the DNA globule as it appears in a dilute poly(ethylene oxide) (PEO) solution. The corresponding phase transition is investigated in detail. Growth of the PEO concentration should lead to a decrease in the size of the compact particle and to an increase in its optical rotatory power. Conditions are defined at which drastic compaction of DNA takes place, accompanied by the loss of its optic rotatory power, in regions of high PEO concentrations.     

Spectrophotometric and NMR analysis of the interaction of fluorinated mono- and bifunctional intercalators with DNA, poly(dA-dT), and poly(dG-dC)

We have synthesized and investigated the DNA binding properties of three fluorinated acridine derivatives—a monomer (I), a short dimer (II) and a long dimer (III). Only III has a sufficiently long chain bridging the two acridine nuclei to permit binding by bisintercalation. Analysis of the equilibrium and kinetic binding properties of these compounds to poly(dA-dT) demonstrates that they behave very similarly to their unfluorinated parent compounds. Helix extension, as determined by viscosity measurements, shows that both compounds I and II bind by monointercalation while III binds by bisintercalation. These results are confirmed by ¹⁹F-nmr analysis, which indicates, in particular, that the two chromophores of III share the same molecular environment as that of I in the presence of either calf thymus DNA or poly(dA-dT). Negative nuclear Overhauser effects in the presence of DNA indicate tight binding such that the motion of the ligands is governed by the polynucleotide dynamics. Optical titrations establish that in 4M NaCl, both I and III bind to calf thymus DNA, but no binding was observed with poly(dG-dC). This result is in contrast to those for dimers of ethidium, which show substantial binding to polynucleotides under high salt conditions. Nuclear magnetic resonance experiments, however, carried out at considerably higher concentrations, show that compound I does indeed bind to poly(dG-dC) under these high salt conditions, albeit weakly, and leads to a conversion of the polynucleotide from a left-handed to a right-handed conformation.     

Thermal denaturation of chromatin and lysine copolymer-DNA complexes. Effects of ethylene glycol.

Chromatin was thermally denatured in the presence and absence of 1M ethylene glycol using a technique whereby both the hyperchromism and ellipticity are monitored simultaneously. Model complexes containing poly(L -Lys) or poly(L -Lys, L -Ala, Gly) and DNA were similarly melted in order to assist in interpreting the chromatin results. In both cases a general pattern emerged whereby ethylene glycol perturbed the resultant melting profile, showing increased hyperchromicity, ellipticity, and premelt slope. In addition, ethylene glycol destabilizes and reduces the high melting region of polypeptide-bound DNA and the extent of higher ordered structure in model complexes and chromatin. These results emphasize the importance of hydrophobic forces in polypeptide–polypeptide and polypeptide–DNA interactions.     

Poly(L–lysine)–DNA interactions in NaCl solutions: B → C and B → ψ Transitions

Thermal denaturation and circular dichroism (CD) properties of poly(L -lysine)–DNA complexes vary greatly when these complexes are prepared differently, that is, whether by NaCl-gradient dialysis starting from 2.0 M NaCl or by direct mixing at low salt. These differing properties were investigated in more detail by examining complexes, made by direct mixing in the presence of various concentrations of NaCl, both before and after the NaCl was dialyzed out of the complex solution. The precipitation curves of DNA due to polylysine binding indicate that such binding is noncooperative at zero salt; from 0.1 up to 1.0 M NaCl they exhibit varying degrees of cooperatively. Starting from zero salt, as the NaCl concentration used for complex formation is increased, both the CD and the melting properties of the complexes are shifted from those of directly mixed at zero salt to those of reconstitution: in the CD spectra there is a gradual shift from a B → C transition to a B → ψ transition; thermal denaturation results show a gradual increase in the melting temperatures of both free DNA (t_m) and polylysine-bound DNA (t′_m). The progressive shift from B → C to B → ψ suggests a close relationship between these two transitions. Large aggregates of the complexes do not warrant the appearance of ψ-type CD spectra: ψ-spectra have been obtained in the supernatants of polylysine–DNA complexes made and measured at 1.0 M NaCl while slightly perturbed CD spectra in B → C transition have been observed in turbid solutions of fully covered complexes made at very low salt. If the complexes are made at intermediate salts and dialyzed to a very low salt, although up to 60% of the DNA is still bound by polylysine, the CD spectra of the complexes are shifted back to the B-type CD characteristic of pure DNA.     

Competitive reactions in solutions of DNA and water-soluble interpolyelectrolyte complexes

The reaction of competitive binding of two polyanions—DNA and synthetic fluorescence-tagged poly(methacrylate) (PMA*)—with the polycation-quencher poly(N-ethyl-4-vinyl-pyridinium) (PEVP) was studied by fluorescence quenching technique. It was found that ability of DNA to displace PMA *from the water-soluble nonstoichiometric interpolyelectrolyte complex (NPEC) formed by PMA* and PEVP—NPEC(PMA*-PEVP)—and to form water-soluble NPEC(DNA-PEVP) can be determined by the parameter Ψ = P _PMA*/P _PEVP where P _PMA* and P _PEVP are the degrees of polymerization of PMA* and PEVP, respectively. In the case of Ψ < 1 the decrease of Ψ leads to the shift of the reaction equilibrium to the right, which can be explained by the gain of entropy due to the increase of the total number of polymeric particles in the solution. Introduction of alkali metal cations into the reaction mixture results in the shift of the reaction equilibrium, and according to their ability to shift the equilibrium to the right the cations can be arranged in the series Na⁺ > K⁺ > Li⁺. The substitution of native DNA by denatured DNA practically does not affect the reaction equilibrium in solutions of NaCl and KCl but considerably shifts it to the right in solutions of LiCl. The data obtained are in accordance with the differences in the selectivity of alkali cations binding with competitive polyanions. © 1995 John Wiley & Sons, Inc.     

Fluoroquinacrine binding to nucleic acids: Investigation of the 19F-nmr,optical, and fluorescence properties in the presence of DNA,poly(A), and tRNA

The interaction of fluoroquinacrine, 3-fluoro-7-chloro-9-(diethylamino-1-methylbutyl-amino)acridine, with poly(A), DNA, and tRNA has been investigated by monitoring changes in the ¹⁹F-nmr properties, the fluorescence, and the optical absorbance of the drug. The changes in the properties of fluoroquinacrine in the presence of nucleic acids are similar to those observed for quinacrine and suggest that the drugs bind in a similar fashion. The molecular dynamics of fluoroquinacrine bound to nucleic acids were determined by interpreting the data from a number of different nmr relaxation experiments with a two-correlation-time model. The two motions are the long-range bending motion of the drug-nucleic acid complex and the sliding of the drug between the base pairs. Both dipolar and chemical shift anisotropy contributions to the nmr relaxation parameters were taken into consideration. The binding of fluoroquinacrine to tRNA appears to be different from that observed for binding to DNA. Optical absorbance and ¹⁹F-nmr were also used to examine the helix-to-coil transitions of the drug–nucleic acid complexes. In the DNA complex, the ¹⁹F chemical shift changes parallel the absorption changes that occur during the transition. ¹⁹F-nmr and absorption show that the drug–tRNA complexes undergo a cooperative helix-to-coil transition, with the drug binding sites melting when the tRNA is 70% denatured.     

Metal-induced sequential transitions among DNA conformations

The action of [Co(NH₃)₆]Cl₃ on poly(dGdC) · poly(dGdC) can lead to a series of consecutive reactions, in which B-DNA is first converted to Z-DNA [M. Behe and G. Felsenfeld (1981) Proc. Natl. Acad. Sci USA 78 , 1619–1623], which in turn is transformed into an unidentified conformer that we tentatively call “U”, and finally the highly associated anisotropic ψ-DNA is produced. Conditions are given under which the sequence B ? Z ? “U” ψ(+) can be stopped at any point in the direction from left to right. The reverse processes, from right to left, occur when ψ(+) or “U”-DNA are treated with various amounts of salt concentrations and lowering temperature. Thus it is demonstrated that four conformers of poly(dGdC) · poly(dGdC) are readily interconvertible, and that Z-DNA and “U” conformers are intermediates in the reversible transformations of B- and ψ-DNA.     

Mutagen–nucleic acid complexes at the polynucleotide duplex level in solution: Intercalation of proflavine into poly(dA-dT) and the melting transition of the complex

The nmr chemical shifts and line widths of the nucleic acid base and sugar proton resonances and the proflavine ring protons can be monitored through the melting transition of the proflavine + poly(dA-dT) complex, phosphate/dye (P/D) ratio = 24 and 8 in 1M salt solution. The nucleic acid and mutagen protons in the complex are in fast exchange between duplex and strand states with the midpoint of the melting transition monitored at the nucleic acid resonances increasing from 72.6°C for poly(dA-dT) to 78.1°C for the P/D = 24 complex and 83.4°C for the P/D = 8 complex in 1M salt solution. The melting transition monitored by the proflavine resonances were 80.0°C for the P/D = 24 complex and 84.3°C for the P/D = 8 complex in 1M salt solution. Since the nucleic acid is in excess at high P/D ratios, the nucleic acid transitions are an average for the opening of mutagen-free and mutagen-bound base-pair regions, while the proflavine transitions monitor the melting of mutagen-bound base-pair regions. The observed 0.75 to 0.95 ppm unfield shift at all four proflavine protons on formation of the complex with poly(dA-dT) provides direct evidence for intercalation of the mutagen between base pairs of the nucleic acid duplex. We have deduced the approximate overlap geometry between the proflavine ring and nearest-neighbor base pairs at the intercalation site from a comparison between experimental proflavine complexation shifts and those calculated for various stacking orientations. The experimental chemical shift of the poly(dA-dT) adenine H-2 resonance in the duplex state in the absence and presence of proflavine suggests that intercalation occurs preferentially at dT-dA sites. The selective chemical shift changes at the sugar H-2′,2″ and H-3′ resonances of the poly(dA-dT) duplex on complex formation demonstrates changes in the sugar pucker and/or torsion angles of the sugar phosphate backbone at the intercalation site.     

The influence of water deficits on needle conductance, assimilation rate and abscisic acid concentration of seedlings of Pinus radiata D. Don

Abstract Seedlings of Pinus radiata, 10–20 weeks old and hitherto fully watered, responded rapidly when water was withheld. Wilting occurred 9d later, at which time soil matric water potential at dawn (Ψ_m) was –1.06MPa and shoot water potential (Ψ) was –1.9 MPa. Small reductions in Ψ_m elicited large responses in assimilation rate (A) and leaf conductance to water vapour (g). Seedlings appear to be more sensitive to small water deficits than are older Plants of P. radiata. After rewatering, significant increases of A and g occurred within one day, but neither regained the values measured prior to the imposition of a single drying cycle. This residual effect of drought on A, after one or six drying cycles, was partially caused by a decrease in photosynthetic capacity. In plants wilted for the first time, the concentration of abscisic acid (ABA) in the bulk foliage increased 3.4 times as Ψ decreased to –1.77 MPa. In comparison, pretreatment with six drying cycles significantly reduced Ψ to –2.13 MPa (indicating some osmotic adjustment) and induced only a doubling of ABA concentration. However, these differences in Ψ and ABA concentration did not Persist after the plants of all pretreatments had been watered for 7 d, although g of drought-pretreatment Plants remained approximately half that of continuously-watered plants.     

Altered nucleic acid partitioning during phenol extraction or silica adsorption by guanidinium and potassium salts

Nucleic acids were found to partition into the phenol phase during phenol extraction in the presence of guanidinium at certain concentrations under acidic conditions. The guanidinium-concentration-dependent nucleic acid partitioning patterns were analogous to those of the nucleic acid adsorption/partitioning onto silica mediated by guanidinium, which implied that phenol and silica interact with nucleic acids through similar mechanisms. A competition effect was observed in which the nucleic acids that had partitioned into the phenol phase or onto the silica solid phase could be recovered to the aqueous phases by potassium in a molecular weight–salt concentration-dependent manner (the higher molecular weight nucleic acids needed higher concentrations of potassium to be recovered, and vice versa). Methods were developed based on these findings to isolate total RNA from Escherichia coli. By controlling the concentrations of guanidinium and potassium salts used before phenol extraction or silica adsorption, we can selectively recover total RNA but not the high molecular weight genomic DNA in the aqueous phases. Genomic DNA-free total RNA obtained by our methods is suitable for RT-PCR or other purposes. The methods can also be adapted to isolate small RNAs or RNA in certain molecular weight ranges by changing the salt concentrations used.     

Germination sensitivities to water potential among co‐existing C3 and C4 grasses of cool semi‐arid prairie grasslands

An untested theory states that C₄ grass seeds could germinate under lower water potentials (Ψ) than C₃ grass seeds. We used hydrotime modelling to study seed water relations of C₄ and C₃ Canadian prairie grasses to address Ψ divergent sensitivities and germination strategies along a risk‐spreading continuum of responses to limited water. C₄ grasses were Bouteloua gracilis, Calamovilfa longifolia and Schizachyrium scoparium; C₃ grasses were Bromus carinatus, Elymus trachycaulus, Festuca hallii and Koeleria macrantha. Hydrotime parameters were obtained after incubation of non‐dormant seeds under different Ψ PEG 6000 solutions. A t‐test between C₃ and C₄ grasses did not find statistical differences in population mean base Ψ (Ψ_b(50)). We found idiosyncratic responses of C₄ grasses along the risk‐spreading continuum. B. gracilis showed a risk‐taker strategy of a species able to quickly germinate in a dry soil due to its low Ψ_b(50) and hydrotime (θ_H). The high Ψ_b(50) of S. scoparium indicates it follows the risk‐averse strategy so it can only germinate in wet soils. C. longifolia showed an intermediate strategy: the lowest Ψ_b(50) yet the highest θ_H. K. macrantha, a C₃ grass which thrives in dry habitats, had the highest Ψ_b(50), suggesting a risk‐averse strategy for a C₃ species. Other C₃ species showed intermediate germination patterns in response to Ψ relative to C₄ species. Our results indicate that grasses display germination sensitivities to Ψ across the risk‐spreading continuum of responses. Thus seed water relations may be poor predictors to explain differential recruitment and distribution of C₃ and C₄ grasses in the Canadian prairies.     

Viscosity study of DNA. II. The effect of simple salt concentration on the viscosity of high molecular weight DNA and application of viscometry to the study of DNA isolated from T4 and T5 bacteriophage mutants

Polyelectrolyte expansion effects on high molecular weight bacteriophage DNA have been studied by examining the influence of simple salt concentration upon the intrinsic viscosity, [η]. The viscosity–molecular weight exponent a in the expression [η] = KM^a diminishes from 0.8 in 0.005M simple salt to a limiting value of 0.6 for salt concentrations greater than 0.6M at 25°C. The ε parameter of the N^1+ε hydrodynamic representation thus varies from approximately 0.2–0.07 over this range of salt concentration. The intrinsic, viscosity of DNA decreases slightly with increasing temperature at low and moderate salt concentrations but becomes independent of temperature at high salt concentrations. The expansion of the DNA molecular domain is linear in the reciprocal square root of the simple salt concentration. Viscosity differences among DNA's isolated from several bacteriophage T5 mutants reflect small differences in molecular weight which are in agreement, with sine determination by other techniques. The DNA's isolated from various rII mutants of T4 bacteriophage including some very large deletion mutations were found to be identically the same size in accord with current genetic ideas. Details of the representation and extrapolation of viscosity data are discussed and the sensitivity of the technique is evaluated.     

Thermodynamics of the B to Z transition in poly(dGdC)

The thermodynamics of the B to Z transition in poly(dGdC) was examined by differential scanning calorimetry, temperature-dependent absorbance spectroscopy, and CD spectroscopy. In a buffer containing 1 mM Na cacodylate, 1 mM MgCl₂, pH 6.3, the B to Z transition is centered at 76.4°C, and is characterized by ΔH_cal = 2.02 kcal (mol base pair)^?1 and a cooperative unit of 150 base pairs (bp). The t_m of this transition is independent of both polynucleotide and Mg²⁺ concentrations. A second transition, with ΔH_cal = 2.90 cal (mol bp)^?1, follows the B to Z conversion, the t_m of which is dependent upon both the polynucleotide and the Mg²⁺ concentrations. Turbidity changes are concomitant with the second transition, indicative of DNA aggregation. CD spectra recorded at a temperature above the second transition are similar to those reported for ψ(–)-DNA. Both the B to Z transition and the aggregation reaction are fully and rapidly reversible in calorimetric experiments. The helix to coil transition under these solution conditions is centered at 126°C, and is characterized by ΔH_cal = 12.4 kcal (mol bp)^?1 and a cooperative unit of 290 bp. In 5 mM MgCl₂, a single transition is seen centered at 75.5°C, characterized by ΔH_cal = 2.82 kcal (mol bp)^?1 and a cooperative unit of 430 bp. This transition is not readily reversible in calorimetric experiments. Changes in turbidity are coincident with the transition, and CD spectra at a temperature just above the transition are characteristic of ψ(–)-DNA. A transition at 124.9°C is seen under these solution conditions, with ΔH_cal = 10.0 kcal (mol bp)^?1 and which requires a complex three-step reaction mechanism to approximate the experimental excess heat capacity curve. Our results provide a direct measure of the thermodynamics of the B to Z transition, and indicate that Z-DNA is an intermediate in the formation of the ψ-(–) aggregate under these solution conditions.     

Re-entrant cholesteric phase in DNA liquid-crystalline dispersion particles

In this research, we observe and rationalize theoretically the transition from hexagonal to cholesteric packing of double-stranded (ds) DNA in dispersion particles. The samples were obtained by phase exclusion of linear ds DNA molecules from water-salt solutions of poly(ethylene glycol)—PEG—with concentrations ranging from 120 mg ml^?1 to 300 mg ml^?1. In the range of PEG concentrations from 120 mg ml^?1 to 220 mg ml^?1 at room temperature, we find ds DNA molecule packing, typical of classical cholesterics. The corresponding parameters for dispersion particles obtained at concentrations greater than 220 mg ml^?1 indicate hexagonal packing of the ds DNA molecules. However, slightly counter-intuitively, the cholesteric-like packing reappears upon the heating of dispersions with hexagonal packing of ds DNA molecules. This transition occurs when the PEG concentration is larger than 220 mg ml^?1. The obtained new cholesteric structure differs from the classical cholesterics observed in the PEG concentration range 120–220 mg ml^?1 (hence, the term ‘re-entrant’). Our conclusions are based on the measurements of circular dichroism spectra, X-ray scattering curves and textures of liquid-crystalline phases. We propose a qualitative (similar to the Lindemann criterion for melting of conventional crystals) explanation of this phenomenon in terms of partial melting of so-called quasinematic layers formed by the DNA molecules. The quasinematic layers change their spatial orientation as a result of the competition between the osmotic pressure of the solvent (favoring dense, unidirectional alignment of ds DNA molecules) and twist Frank orientation energy of adjacent layers (favoring cholesteric-like molecular packing).     

Statistical mechanical deconvolution of thermal transitions in macromolecules. III. Application to double-stranded to single-stranded transitions of nucleic acids

The statistical mechanical deconvolution theory for macromolecular conformational transitions is extended to the case of nucleic acids transitions involving strand separation. It is demonstrated that the partition function, Q, as well as all the relevant thermodynamic quantities of the system, can be calculated from experimental scanning calorimetric data. In particular, it is shown that important thermodynamic parameters such as the size of the average cooperative unit during strand separation, the mean helical segment length, and the mean coil-segment length can be calculated from the average excess enthalpy function 〈ΔH〉. The theory is applied to the double-stranded to single-stranded transition of the system poly(A)·poly(U) at different salt concentrations. It is shown that the mean helical segment length is a monotonically decreasing function of the temperature well before strand separation occurs. On the other hand, the mean coil segment length remains practically constant until temperatures very close to T_m. Both experimental findings clearly indicate that the unfolding of poly(A)·poly(U) proceeds through the formation of many short helical sequences. The cooperative unit for the strand separation is calculated to be about 120 base pairs and essentially independent of the salt concentration. The existence of a minimum helical segment length of 10 ± 2 base pairs within the double-stranded form is calculated.