Raman spectral studies of nucleic acids. XVII. Conformational structures of polyinosinic acid.

Laser-Raman spectra of poly(rI) show the formation of an ordered complex in aqueous solutions of high ionic strength. This structure exhibits the A-helix geometry, contains stacked bases and is apparently stabilized by specific hydrogen bonding involving hypoxanthine C6=0 groups. Thermal dissociation of the poly(rI) complex (Tm=45 degrees C) yields single-stranded and disordered poly (RI) chains. A disordered structure also occurs for poly (rI) in aqueous solutions of low ionic strength. In oriented films, poly (rI) forms an ordered structure probably the same as that which occurs in solutions of high ionic strength. Raman intensities measured at 815 and 1100 cm-1 in spectra of poly (rI) and poly (rU)-poly (rA)-poly(rU) indicate that the correlation previously established for single- and double-stranded ribopolymer structures is valid also for these multi-stranded structures. X-ray diffraction and model-building studies confirm the A-helix structure.     

Protonated polynucleotide structures. IX. Disproportionation of poly (G)-poly (C) in acid medium

The interaction between poly (G) and poly (C) was investigated in neutral and acid medium by optical methods. Three main points arise from this investigation. (1) The formation of poly (G)·poly (C) was complete only above an ionic strength of about 0.6M [Na⁺]. Lowering the ionic strength increased the amounts of free poly (G) and free poly (C) that could be detected. (2) When titrating towards acid pH values a transition took place which was characterized by potentiometry, mixing curves, and circular dichroism: a three-stranded poly (G)·poly (C)·poly (C⁺) complex was formed analogous to the transition observed for the acid titration of poly (I)·poly (C). (3) Even when the poly (G)·poly (C) complex was incompletely formed (at low ionic strength) in neutral medium all poly (C) entered the triple-stranded complex.     

Protonated polynucleotides. VII. Thermal transitions between different complexes of polyinosinic acid and polycytidylic acid in an acid medium

The interaction between poly (I) and poly (C) in acid medium has been studied by potentiometric titration, mixing curves and thermal denaturation. Phase diagramms as a function of ionic strength, pH, and temperature have been established. From these data it is shown that the acid titration of the complex poly (I) · poly (C) passes through a triple-stranded intermediate poly (I) · poly (C) · poly (C⁺) to yield finally the protonated double-helical complex poly (I) · poly (C⁺). The mixing curves indicate the sole presence of the three-stranded complex in the intermediate zone. On the basis of the pK's the coexistence between the three-stranded complex with the neighboring double-stranded structure is demonstrated in a narrow rang of pH and ionic strength. The geometry of the base arrangements, their conformation and the sense of the strands are discussed in the light of the data presented. A Hoogsteen-type pairing between the bases for poly (I) · poly (C⁺) is favored, although the reverse Hoogsteen pair cannot be excluded.     

A basic isozyme of yeast glyceraldehyde-3-phosphate dehydrogenase with nucleic acid helix-destabilizing activity

A nucleic acid helix-destabilizing protein has been purified from Saccharomyces cerevisiae using affinity chromatographic techniques. Crude protein extracts at low ionic strength (approx. 0.05 M) were applied sequentially to tandem columns of native DNA-cellulose, aminophenyl-phosphoryl-UMP-agarose, poly(I . C)-agarose, poly(U)-cellulose and denatured DNA-cellulose. The 2 M NaCl eluant of the poly(U)-cellulose column was dialyzed to low ionic strength and recycled through native DNA-cellulose, poly(I . C)-agarose and poly(U)-cellulose. Purified helix-destabilizing protein eluted from the poly(U)-cellulose between 0.1 and 0.5 M NaCl. On the basis of enzymatic activity, immunological cross-reactivity, mobility on SDS gels, amino acid analysis and preliminary peptide mapping experiments, this material was identified as an isozymic fraction of glyceraldehyde-3-phosphate dehydrogenase. The major crystallizable isozyme of this enzyme from yeast is, however, considerably more acidic than the helix-destabilizing protein, and displays significantly lower helix-destabilizing activity. Stoichiometric levels of the isolated protein at low (approx. 0.01) ionic strength depress the Tm of poly(A-U) and poly [d(A-T)] by as much as 28 and 22 degrees C, respectively. Longer double helices, poly(A . U) and Clostridium perfringens DNA are also denatured by the helix-destabilizing protein, but at relatively slow rates. The binding of this protein to [3H]-poly(U) on nitrocellulose filters in [Na+]-dependent, with a 50% reduction at 0.09 M NaCl. Based on its effect on the circular dichroism spectrum of poly(A), the protein was shown to distort the conformation of the polynucleotide chain. An analogous protein from mammalian cells, P8, was also shown to depress poly(A-U) Tm.     

Ionic strength dependence of the hysteresis in the polyriboadenylate-polyribouridylate system.

The hysteresis observed in cyclic acid-base titrations of the three-standed polyribonucleotide helix poly (A)-2 POLY (U) strongly depends on ionic strength. For NaCl and at 25 degrees C, hysteresis occurs in the limited concentration range between 0.03 M and 1.0 M(NaCl). The transition points associated with the cyclic conversions between the triple helix and the poly (A)-poly (A) double helix and (free) poly (U) constitute a (pH ionic strength) phase diagram covering the ranges of stability and metastability of the hysteresis system. Variations with NaCl concentration of some hysteresis parameters can be quantitatively described in terms of polyelectrolyte theories based on the cylinder-cell model for rodlike polyions. The results of this analysis suggest that the metastability is predominantly due to dlectrostatic energy barriers preventing the equilibrium transition of the partially protonated triple helix above a critical pH value. Ultraviolet absorbance and potentiometric titration data of poly (A)in the acidic pH range can be analyzed in terms of two types of double-helical structures. Spectrophotometric titrations reveal isosbestic wavelengths for structural transitions of poly (A). "Time effects" commonly observed in poly (A) titrations are suggested to reflect helix transitions between the two acidic structures.     

Studies on the bacteriophage MS2. XXII. Conformation of MS2 RNA in acid medium

MS2 RNA, which sediments at 27S in a neutral buffer, can be converted to a compact 57S conformation at pH 3.8. Requirements for this conversion, besides protonation, are small concentrations of Mg⁺⁺ ions and a low ionic strength. On the other hand, after heating in the presence of EDTA and at low ionic strength, the RNA can be unfolded to an 11.7S form at pH 6.8 and to 10.5S at pH 3.8. The compact 57S form has lost at least 50% of its secondary structure, as determined by its hypochromicity. It corresponds to a monomer species, as will be shown in a following paper (XXIV). Comparative studies with the homopolymers poly A and poly C and with the heteropolymers poly A,U, poly A,C, and poly A,G indicate that the interactions involved in the acid RNA conformation are not simply explainable by the known interactions of the A–A⁺, C–C⁺, and/or A–C⁺ type.     

The binding of echinomycin to deoxyribonucleic acid.

Echinomycin is a peptide antibiotic which binds strongly to double-helical DNA up to a limit of approximately one molecule per five base-pairs. There is no detectable interaction with rRNA and only extremely feeble non-specific interaction with poly(rA)-poly(rU). Heat denaturation of DNA greatly decreases the binding, and similarly limited interaction is observed with naturally occurring single-stranded DNA. Association constants for binding to nine double-helical DNA species from different sources are presented; they vary by a factor of approximately 10, but are not simply related to the gross base composition. The interaction with DNA is ionic-strength-dependent, the binding constant falling by a factor of 4 when the ionic strength is raised from 0.01 to 0.10mol/litre. From the effect of temperature on the association constant for calf thymus DNA, the enthalpy of interaction is calculated to be about -13kJ/mol (-3kcal/mol). Binding of echinomycin persists in CsCl gradients and the buoyant density of nicked bacteriophage PM2 DNA is decreased by 25 mg/ml. Echinomycin interacts strongly with certain synthetic poly-deoxynucleotides, the binding constant decreasing in the order poly(dG)-poly(dC) greater than poly(dG-dC) greater than poly(dA-dT). For the latter two polymers the number of base-pairs occluded per bound antibiotic molecule is calculated to be three, whereas for poly(dG)-poly(dC) it is estimated to be four to five. Poly(dA)-poly(dT) and poly(dI)-poly(dC) interact only very weakly with the antibiotic. Poly(dI-dC) interacts to a slightly greater extent, but the binding curve is quite unlike that seen with the three strongly binding synthetic polynucleotides. Echinomycin affects the supercoiling of closed circular duplex bacteriophage PM2 DNA in the characteristic fashion of intercalating drugs. At low ionic strength the unwinding angle is almost twice that of ethidium. Likewise the extension of the helix, determined from changes in the viscosity of rod-like sonicated DNA fragments, is nearly double that expected for a simple (monofunctional) intercalation process. On this basis the interaction process is characterized as bifunctional intercalation. At higher ionic strength the unwinding angle relative to that of ethidium and the helix extension per bound echinomycin molecule fall, indicating a smooth progression towards more nearly monofunctional intercalation. Two simpler compounds which act as analogues of the quinoxaline chromophores of echinomycin, quinoxaline-2-carboxamide and the trypanocidal drug Bayer 7602, interact with DNA very much more weakly than does echinomycin, showing that the peptide portion of the antibiotic plays an essential role in determining the strength and specificity of the interaction.     

Immunological and spectroscopic studies of poly(dG-dC).poly(dG-dC) modified by cis-diamminedichloroplatinum(II)

The conformational changes induced by the binding of cis-diamminedichloroplatinum(II) to poly(dG-dC).poly(dG-dC) have been studied by reaction with specific antibodies, by circular dichroism and 31P nuclear magnetic resonance. Polyclonal and monoclonal antibodies to Z-DNA bind to platinated poly(dG-dC).poly(dG-dC) at low and high ionic strength. Antibodies elicited in rabbits immunized with the platinated polynucleotide bind to double stranded polynucleotides known to adopt the Z-conformation. At low and high ionic strength the circular dichroism spectrum of platinated poly(dG-dC).poly(dG- dC) does not resemble that of poly(dG-dC).poly(dG-dC) (B or Z conformation). At low ionic strength, the characteristic 31P nuclear magnetic resonance spectrum of the Z-form is not detected. It appears only at high ionic strength, as a component of a more complex spectrum.     

The structures and properties of poly-5-methylcytidylic acid

The optical rotatory dispersion properties of poly 5MeC, poly diMeC, and 5MeCMP-(5′) in 0.1M Na+ have been studied at various pH values and temperatures. Poly 5MeC and poly diMeC have optical properties which are similar to those for poly C; however, poly 5MeC has a biphasic melting profile in the pH range from 3.8 to 5.4 similar to that observed for poly 51C. Using titration, ionic strength, and pH dependence measuements, the data for poly 5MeC are interpreted in terms of the following scheme at pH 4.0 and 0.1 ionic strength: triple-strand helix ^37°C double-strand helix ^79°C single-strand coil. Support for this scheme is discussed. The effect of the methyl group is discussed in terms of similar structural possibilities for other polymers of cytidylic acid.     

Temperature dependence of the hydrogen ion equilibria in poly(riboadenylic acid)

Hydrogen ion titration curves have been obtained for poly (riboadenylic acid) (poly A) at temperatures of 0–40°C, and ionic strengths of 0.001, 0.01, and 0.15. Where comparable, the data are in general agreement with those previously reported by other investigators. Correlations between the titration data and thermal denaturation curves have been obtained. Formation of a two-stranded helix is catalyzed by the uptake of protons by the adenine base. Partial protonation of the base is required for formation of the two-stranded helix, but under appropriate conditions it is stable at degrees of ionization less than 0.2. The degree of ionization required for formation of the two-stranded helix increases with temperature and decreases with ionic strength.     

Relation between differential pulse voltammetric oxidation of polyriboinosinic acid and its structure

Electrooxidation of poly (I) at a paraffin wax-impregnated spectroscopic graphite electrode was studied by means of differential pulse voltammetry. It was found that the transition of single-stranded poly (I) to its multistranded form, induced by increasing the ionic strength of neutral medium, is accompanied by a lowering of the oxidation current of poly (I). The marked lowering of the oxidation current is also observable as a consequence of the formation of double-stranded complex of poly (I). poly (C). The voltammetry at carbon electrodes provides for the study of poly(I) structure in principle identical information as optical methods.     

Interaction of the alternating double stranded copolymer poly(dA-dT) x poly(dA-dT) with NiCl(2) and CdCl(2): solution behavior

The thermal denaturation of the synthetic high molecular weight double stranded polynucleotide poly(dA-dT) x poly(dA-dT) has been studied in aqueous buffered solution (Tris 1.0 mM; pH 7.8+/-0.2) in the presence of increasing concentrations of either Ni(2+) (borderline cation) or Cd(2+) (soft cation) at four different constant ionic strength values (NaCl), making use of UV and circular dichroism (CD) spectroscopies. The experimental results show that the B-type double helix of the polymer is stabilized against thermal denaturation in the presence of both cations at low concentrations, relative to the systems where only NaCl is present, in the same conditions of ionic strength and pH. The effect is more pronounced for Ni(2+) than for Cd(2+). At higher concentrations, both cations start to destabilize the double helix, with Cd cations inducing larger variations of T(m). In many cases, when denaturation starts, interstrand cross-linking occurs with formation of aggregates that precipitate.     

Protein dissociation from DNA in model systems and chromatin.

Salt induced dissociation of protamine, poly(L-lysine) and poly(L-arginine) from DNA was measured by relative light scattering at theta = 90 degrees and/or centrifugation. Dissociation of histones from DNA was studied using relative light scattering and intrinsic tyrosine fluorescence. Protamine was dissociated from DNA at 0.15 M MgCl2 (ionic strength mu = 0.45) or 0.53 M NaCl (mu = 0.53) based on light scattering data and at approximately 0.2 M MgCl2 (mu = 0.6) or 0.6 M NaCl based on centrifugation data. NaCl induced dissociation of poly(Lys) or poly(Arg) from natural DNAs measured by light scattering did not depend on the guanine plus cytosine content. To dissociate poly(Arg) from DNA higher ionic strength using NaCl, MgCl2, or CaCl2, similar ionic strength using NaClo4, and lower ionic strength using Na2SO4 was needed then to dissociated poly(Lys). Both the decrease in light scattering and the enhancement of tyrosine fluorescence of chromatin occurred between 0.5 and 1.5 M NaCl when histones were dissociated.     

Mechanical stability of single DNA molecules

Using a modified atomic force microscope (AFM), individual double-stranded (ds) DNA molecules attached to an AFM tip and a gold surface were overstretched, and the mechanical stability of the DNA double helix was investigated. In lambda-phage DNA the previously reported B-S transition at 65 piconewtons (pN) is followed by a second conformational transition, during which the DNA double helix melts into two single strands. Unlike the B-S transition, the melting transition exhibits a pronounced force-loading-rate dependence and a marked hysteresis, characteristic of a nonequilibrium conformational transition. The kinetics of force-induced melting of the double helix, its reannealing kinetics, as well as the influence of ionic strength, temperature, and DNA sequence on the mechanical stability of the double helix were investigated. As expected, the DNA double helix is considerably destabilized under low salt buffer conditions (相似文献   

Interaction of metal ions with polynucleotides and related compounds. IX. Unwinding and rewinding of poly(A + U) and poly(I + C) by copper(II) ions

It is demonstrated that, poly(A + U) and poly(I + C) are both formed under low ionic strength conditions. Continuous variation studies indicate the formation of copper(II) complexes of poly A, poly C, and poly I, but not of poly U. Copper(II) in a 1:1 ratio to polynucleotide prevents the formation of poly(A + U) and brings about the dissociation of the poly (A + U) complex produced in the absence of the metal. Poly (I + C) is similarly dissociated by copper(II) ions. The addition of sufficient electrolyte reverses the copper(II) induced dissociation of poly(I + C). The effect of copper(II) on ordered synthetic polynucleotides is thus very similar to its effect on DNA.     

Adduct formation between the carcinogen N-acetoxy-2-acetylaminofluorene and synthetic polydeoxyribonucleotides.

The chemical carcinogen N-acetoxy-2-acetylaminofluorene (NA-AAF) was reacted with poly(dG-dC) - poly(dG-dC); poly dG - poly dC; poly(dA-dT) - poly (dA-dT); and poly dA - poly dT under a variety of conditions. Poly (dG-homo GC polymer and 10--20 more reactive the A + T polymers. Lowering the ionic strength increased the extent of reaction, while pH change (8.9 vs. 5.5) had only a small effect. If ionic strength was adjusted so that the two guanine-containing polymers showed equal thermal stability (as judged by Tm) then the alternating copolymer was 7 times as reactive as the homopolymer. In aggreement with previous investigators, the major product was found to be 8-(N-2-fluorenylacetamido) deoxyguanosine.     

On the application of polyelectrolyte limiting laws to the helix–coil transition of DNA. V. Ionic effects on renaturation kinetics

The bimolecular rate constant k₂ for the association of complementary polynucleotide strands has been observed to increase strongly with increasing ionic strength—in fact, proportional to its third or fourth power. This effect is here interpreted quantitatively by means of polyelectrolyte theory starting with the Wetmur–Davidson postulate of a pre-equilibrium between separated strands and aligned segments close to one another but unbonded. The correct form, a power dependence of k₂ on ionic strength, is predicted. Comparison of the theoretical exponent with data allows the conclusion that each of the two single-stranded segments in the aligned but unbonded configuration consists of about 13–16 nucletides (not to be confused with the much smaller number of bonded base pairs in the nucleus), and that this number, denoted by Q, is possibly correlated either with a minimum length for duplex stability or with the persistence length of a single polynucleotide strand. It is suggested that experimental determination of the dependence of Q on (G+C)-content may distinguish between these possibilities. It is also suggested that addition of sufficient amounts of divalent metal ions such as Mg²⁺, Ca²⁺, or Co²⁺ may reverse the dependence of k₂ on ionic strength; under these conditions, k₂ is predicted to decrease with about the first power of ionic strength. At fixed ionic strength, k₂ should increase with increasing concentration of divalent metal ion, and, in fact, the published observation that the formation of poly(A)·2 poly(U) from poly(A)·poly(U) and poly(U) is second order in Mg²⁺ concentration is here correctly predicted from a priori molecular considerations. Finally, published association rate data for oligonucleotides are discussed in the present theoretical context.     

Influence of protein net charge on the nucleic acid helix-destabilizing activity of various pancreatic ribonucleases

Summary Helix-destabilization of double-stranded poly[d(A-T)] induced by various homologous pancreatic ribonucleases which differ in their net charges has been studied under different ionic strength conditions.The response of the destabilizing activity of the various proteins to ionic strength is represented by bell-shaped curves, whose maxima are shifted to higher ionic strength values the higher the number of positive charges of the RNAase involved in the nucleic acid-protein complex.This observation is discussed, and a model proposed, that could explain the experimental results presented.     

Rupture of base pairing in double-stranded poly(riboadenylic acid)-poly(ribouridylic acid) by formaldehyde: medium chain lenghts.

By assuming that the opening of hydrogen bonds due to thermal fluctuations is a very fast step and that the reaction of formaldehyde with the imino or amino group is a slow step, we have constructed a model for the unwinding process of poly(A-U) induced by formaldehyde. The denaturation equation derived from the model is essentially the same as that of the zipper model for moderately long chain lengths. The model predicts the following phenomena which are in agreement with our experimental findings. The rate of unwinding is approximately first order for unfractionated polynucleotides and zero order for fractionated samples. This means that formaldehyde ruptures helical residues sequentially starting from the ends and working toward the center. Our model further predicts that the denaturation rate is linearly dependent on -log[Na+] and pH at low ionic strength and is almost independent of [Na+] and pH at high ionic strength. Spectrophotometric measurements on poly(A-U) were done to confirm our theoretical findings.     

Importance of glycerol and fatty acid residues on the ionic properties of phosphatidylglycerols at the air-water interface.

Ionic properties of didodecanoylphosphatidylglycerol (C12PG), didodecanolyphosphatidyl-l'-propanol (C12PP), di-(12-methyl, 13-methyl)-pentadecanoylphosphatidylglycerols (C15PG) and dihexadecanoylphosphatidylglycerol (C16PG) have been studied at the air-water interface using titration experiments at constant ionic strength and film expansion experiments at constant pH, with Li+, Na+, K+ and Cs+ in the subphase. For each lipid, the apparent pK in the surface is strongly dependent on the subphase salt concentration and differs from expected intrinsic pK in the bulk. Discrimination between alkaline cations is observed. These results can be accounted for by strong surface potentials, which are satisfactorily calculated by using the Gouy and Chapman theory of the diffuse double layer. The comparison of C12PP and PG expansion data shows the importance of the glycerol residue of PG ionic properties, favouring penetration of cations in the films. Lipids in the liquid-crystalline state, such as C12-and C15PG, do not interact with alkaline cations as does C16PG in the gel phase. In particular, film condensations bring about a clear-cut discrimination between Na+ and K+. Results are discussed with regard to cation penetration and the structure of water at the interface. The importance on membrane functions of these strong surface potentials generated by PG monolayers is suggested.