Sequence-specific interaction of pyrimidine oligonucleotides with double-stranded DNA at acidic pH complexes of different types

The interaction of pyrimidine oligonucleotides (OLN₁₅ and OLN₆) and their alkylating derivatives bearing 4-(3-amino)propyl-N-methyl and N-2-chloroethyl (RCl) aniline residues at the 5′-phosphate with a fragment of the human γ-interferon gene was studied. In the presence of 150 mM NaCI at pH 5.4, the yield of dsDNA alkylation was 60% for RCl-OLN₁₅ and 10% for RCl-OLN₆; at pH 4.0 in the presence of 150 mM NaCI and 10 mM MgCl₂, the yield of the dsDNA modification product was 100% for RCl-OLN₆ and 50% for RCl-OLN₁₅. It was shown by native electrophoresis that OLN₁₅ could form with the target dsDNA complexes of two types in the presence of magnesium ions at pH 4.0. One of the complexes was stable at pH 5.4 in the presence of magnesium ions, whereas the other was not. We found that only the complex stable in 10 mM Mg(OAc)₂, pH 5.4, was effectively alkylated.     

The interaction of magnesium ions with teichoic acid.

The binding of Mg2+ to the wall teichoic acid of Lactobacillus buchneri N.C.I.B. 8007 was measured by equilibrium dialysis at controlled ionic concentration and pH. In an aqueous solution containing 10mM-NaCl at pH 5.0 one Mg2+ ion was bound for every two phosphate groups of the teichoic acid, with an apparent association constant, Kassoc. = 2.7 x 10(3) M-1. On lowering the pH below the pKa of the phosphate groups the amount of bound Mg2+ decreased concomitantly with decreasing ionization of the phosphate groups. Both the amount of Mg2+ bound to the teichoic acid and the apparent association constants were similar in the presence of 10 mM concentrations of NaCl or KCl but decreased markedly in the presence of 10 mM-CaCl2 because of competition between Ca2+ and Mg2+ for the binding sites. A similar effect was found when the concentration of NaCl was increased from 0 to 50 mM. The results are discussed in relation to the function of teichoic acid in the walls of Gram-positive bacteria.     

The interaction of calcium and magnesium ions with deoxyribonucleic acid.

Conductance and equilibrium dialysis studies are reported for the aqueous systems (native calf thymus) DNA-CaCl₂ and DNA-MgCl₂ at various pH values and ionic strengths at 25 °C. Discontinuities occur in the conductance curves at mole ratios of Ca²⁺ and Mg²⁺ to nucleate phosphorus of 0.125, 0.30, and 0.50. The dialysis results show the formation of complexes of stoichiometry 0.50 and 1.00 mol Ca²⁺ or Mg²⁺/mol nucleate phosphorus (2:1 and 1:1 complexes), the latter only in neutral or alkaline solutions, in agreement with the conductance discontinuity at 0.50. The other discontinuities may be due to preferential binding in the formation of the 2:1 complex. Binding constants for the 2:1 complexes are evaluated. Absorption-temperature profiles have been determined for “native” and dialysed DNA in the presence of NaCl, CaCl₂, and MgCl₂. For dialysed DNA at 26 ° C and 260 nm the decrease of absorbance with increased salt concentration was halted for MgCl₂ and CaCl₂ at a concentration corresponding to the formation of the 2:1 complex. The absorbance of “native” DNA did not decrease. T_m and the reciprocal of the hypochromic rise ($\text{1}\text{h}$) increased linearly with log (salt concn). Values of T_m were the same at 230, 260, and 280 nm, but h was greater at 230 and 280 than at 260 nm, which may be due to the existence of alternating blocks of (A + T) and (G ? C) pairs. The entropy of transition was in the order Ca > Mg ? Na.     

High affinity interaction of HIV-1 integrase with specific and non-specific single-stranded short oligonucleotides.

Retroviral integrase (IN) catalyzes the integration of double-stranded viral DNA into the host cell genome. The reaction can be divided in two steps: 3'-end processing and DNA strand transfer. Here we studied the effect of short oligonucleotides (ODNs) on human immunodeficiency virus type 1 (HIV-1) IN. ODNs were either specific, with sequences representing the extreme termini of the viral long terminal repeats, or nonspecific. All ODNs were found to competitively inhibit the processing reaction with Ki values in the nM range for the best inhibitors. Our studies on the interaction of IN with ODNs also showed that: (i) besides the 3'-terminal GT, the interaction of IN with the remaining nucleotides of the 21-mer specific sequence was also important for an effective interaction of the enzyme with the substrate; (ii) in the presence of specific ODNs the activity of the enzyme was enhanced, a result which suggests an ODN-induced conformational change of HIV-1 IN.     

Modulation of the affinity of the vasopressin receptor by magnesium ions.

The binding of [3H]vasopressin (AVP) and the 125I-labelled vasopressin antagonist (VP-AT) d(CH2)5[Tyr2(Me),Tyr9(NH2)]AVP to rat liver membranes was examined with or without the addition of milimolar concentrations of divalent cations. The binding of vasopressin was enhanced by Mg2+ and Co2+ and markedly decreased by EGTA. The addition of EGTA and Mg2+ together restored the binding to a value similar to that of Mg2+ alone. On the contrary, the addition of Mg2+, Co2+, EGTA, and the combination of EGTA and Mg2+ decreased the binding of VP-AT to rat liver membranes. Kinetic analyses showed that Mg2+ increased the Kd twofold for VP-AT; that is from 0.13 nM to 0.28 nM. Moreover, it showed that the receptor with or without the addition of Mg2+ consists of a single population of binding sites, indicating that the receptor is switched from a high affinity to a low affinity state for VP-AT in the presence of 10 mM Mg2+. GTP gamma S was unable to block the effect of Mg2+ on the binding of VP-AT. These results suggest that this divalent cation interacts with receptor itself producing a conformational changes which thus modulates the affinity of the receptor.     

Formation constants for interaction of citrate with calcium and magnesium ions

Formation constants for the interaction of citrate ion with calcium and magnesium ions in solution at 37°C and a constant ionic strength of 0.15 were determined by potentiometric titration. Values for the formation of CaL^? and CaHL⁰ complexes were 1.88 × 10³ and 67, respectively. Corresponding constants for MgL^? and MgHL⁰ were 2.19 × 10³ and 42, respectively. The existence of other complexes was not confirmed. Protonation constants were also determined under the same conditions.     

The interaction of magnesium ions with the calcium pump of sarcoplasmic reticulum.

1. In the presence of Ca2+, ATP phosphorylates the Ca2+ pump of sarcoplasmic reticulum at the same site and to the same extent regardless of whether Mg2+ is added or not to the incubation media, the main effect of added Mg2+ being to increase the rate of phosphorylation. 2. When phosphoenzyme is made in Mg2+-containing media it dephosphorylates about 30-times faster than when it is made in the absence of added Mg2+. Addition of Mg2+ after phosphorylation is uneffective in accelerating the hydrolysis of phosphoenzyme even in solubilized enzyme, suggesting that phosphorylation of the Ca2+ pump results in occlusion of the site at which Mg2+ combines to accelerate the release of phosphate. 3. Occlusion of the site for Mg2+ can be partially reversed by trans-1,2-diaminocyclohexonetetraacetic acid (CDTA). Use was made of this property to demonstrate that for the rapid release of phosphate to occur Mg2+ has to be bound to the enzyme. 4. Results seem to indicate that Mg2+ combines with the Ca2+ pump prior to phosphorylation.     

Effect of magnesium ions on the structure of DNA thin films: an infrared spectroscopy study

Utilizing Fourier transform infrared spectroscopy we have investigated the vibrational spectrum of thin dsDNA films in order to track the structural changes upon addition of magnesium ions. In the range of low magnesium concentration ([magnesium]/[phosphate] = [Mg]/[P] < 0.5), both the red shift and the intensity of asymmetric PO₂ stretching band decrease, indicating an increase of magnesium-phosphate binding in the backbone region. Vibration characteristics of the A conformation of the dsDNA vanish, whereas those characterizing the B conformation become fully stabilized. In the crossover range with comparable Mg and intrinsic Na DNA ions ([Mg]/[P] ≈ 1) B conformation remains stable; vibrational spectra show moderate intensity changes and a prominent blue shift, indicating a reinforcement of the bonds and binding in both the phosphate and the base regions. The obtained results reflect the modified screening and local charge neutralization of the dsDNA backbone charge, thus consistently demonstrating that the added Mg ions interact with DNA via long-range electrostatic forces. At high Mg contents ([Mg]/[P] > 10), the vibrational spectra broaden and show a striking intensity rise, while the base stacking remains unaffected. We argue that at these extreme conditions, where a charge compensation by vicinal counterions reaches 92–94%, DNA may undergo a structural transition into a more compact form.     

CD studies on the conformation of oligonucleotides complexed with divalent metal ions: interaction of Zn2+ with guanine favours syn conformation.

The interaction of the divalent metal ions Mg2+, Mn2+, Zn2+ and Cu2+ with GpG and several other dinucleoside monophosphates were investigated by means of circular dichroism. The spectra of the complexes of GpG, GpU analogues and ApGpG caused in the presence of Zn2+ and other transition metals show a close similarity in the spectral CD shape to that previously reported in the literature for GpG and GpU at low pH and for m7GpG. From the results it may be concluded that transition metal ions-particularly considered for Zn2+/- tends to favour the degree of stacking with Guo in syn conformation in GpG or GpU due to the coordination of the metal ion at N-7 of the 3'-bound position while shielding of the phosphate site by Mg2+ does not influence the sugar-base torsional angle under comparable conditions. Stereochemical aspects and selectivity of the Zn2+ mediated conformation of the dinucleoside phosphates are discussed.     

Effect of magnesium ions on heat denaturation of DNA

The dependence of animal DNA denaturation on magnesium ion concentration has been studied in the range (10(-6)--10(-1) M with sodium ion content of 10(-3) and 10(-2) M. Special attention has been given to the effect of multivalent metallic impurities bound to DNA. An increase of DNA thermal stability has been shown to occur in the magnesium concentration rage of 10(-6)--10(-4) M. At concentrations exceeding 10(-3) M the T M begins to decrease. The dependence of the DNA melting range on magnesium ion concentration has a maximum at approximately 10(-5) M Mg2+. At low magnesium and sodium ion concentrations a strong asymmetry of the melting curves has been observed. This effect can be described in terms of the melting theory for DNA complexed with small molecules and is explained by magnesium ion redistribution from the denatured portions of DNA to native ones. The method for calculation of melting curves in the DNA-ligand system has been proposed. Studies of thermal denaturation parameters have been shown to be an effective method for the estimation of binding constants of ligands to native and denatured DNA.     

Effect of magnesium ions on red cell membrane properties

Summary Spectrin forms aggregates in solution when incubated at relatively high concentrations (several millimolar) of divalent cations. According to the evidence of electron microscopy, aggregates of globular appearance and, rather uniform size are cooperatively formed from spectrin dimers, no intermediate structures being seen. Inter-dimer chemical cross-linking of spectrin in intact red cell membranes is enhanced if magnesium ions at a concentration of 0.5mm or more are present. On the other hand, the elimination of magnesium from the interior of intact cells causes no significant change in shear elastic modulus, measured by micropipette assays, nor is there any dependence of membrane filtration rate on intracellular free magnesium concentration in the range 0–1mm. Magnesium-depleted cells are, however, converted into echinocytes within a short period, in which, control cells, exposed to ionophore and external magnesium ions, remain completely discoid. Magnesium-depleted cells also undergo structural, changes on heating below the temperature at which vesiculation sets in. These reveal themselves by the transformation of the cells to a unique characteristic shape, by grossly reduced filtrability, and by extensive agglutination of the cells when treated with a bifunctional reagent. Magnesium ions thus regulate the stability, but not to any measurable extent the gross elasticity, of the red cell membrane.     

Effect of pH on conjugal transfer at low temperatures.

The inhibitory effects of nonoptimal temperature and nonoptimal pH on F-type conjugal transfer were found to be synergistic. This finding is briefly discussed in an environmental context.     

Immobilization of oligonucleotides on a large pore support for plasmid purification by triplex affinity interaction

Triplex affinity interaction provides a new process for the purification of plasmid DNA, which is especially suited to meet the demands of a gene therapy use. We developed a method for the functionalization of a large pore affinity support suitable for this application. A 5-modified DNA oligonucleotide containing an aldehyde group was coupled to adipic acid hydrazide functionalized Sephacryl beads with a yield of 31% (over all immobilization yield 22.6% from starting oligonucleotide). The resulting selective and covalent immobilization of the ligand via a 16 atom, hydrophilic linker arm enables the oligonucleotide bases to freely bind to the target sequence. The proposed method provides affinity supports that might be used in large scale affinity purification of plasmid DNA.This revised version was published online in October 2005 with corrections to the Cover Date.     

SAXS study of crotapotin at low pH.

The structure of crotapotin, a protein extracted, from the venom of the Crotalus durissus terrificus, in solution at pH = 1.5, was studied by SAXS. The experimental results yield structural parameter values of the molecular radius of gyration Rg = 13.6 A, volume v = 16.2 x 10(3) A3 A3 and maximal dimension Dmax = 46 A. The distance distribution function deduced from the scattering measurements is consistent with an overall molecular shape of an oblate ellipsoid of revolution with asymmetry parameter v = 0.45.     

Effect of ions and nucleotides on the interactions of yeast Rad51 protein with single-stranded oligonucleotides

Rad51 protein is a eukaryotic homologue of RecA protein that is essential for homologous recombination. We developed a simple procedure for purifying yeast Rad51 protein, characterized its interaction with DNA, and compared it with those of RecA from Escherichia coli and Rad51 from higher eukaryotes. Fractionation of crude extract with 0.2% polyethylenimine eliminated contaminant proteins and nucleic acids, which can perturb the subsequent purification steps. Binding of Rad51 to single-stranded DNA was detected in solution by measuring the fluorescence anisotropy of a fluorescein probe attached to the 5' end of the oligonucleotides. The interaction was stabilized by ATP, as is that of RecA, but was neither stabilized by a non-hydrolysable analog of ATP, nor destabilized by ADP, unlike the interaction of RecA. This character was very similar to that of Xenopus XRad51.1, although the binding of yeast Rad51 to DNA was more sensitive to Mg(2+) ion in both the presence and absence of ATP, and was optimal at 5--10 mM Mg(2+). The dissociation of Rad51 protein from DNA is not, therefore, favored by the hydrolysis of ATP to ADP, in contrast to that of RecA. On the other hand, the high DNA-binding state of the Rad51-DNA complex promoted by ATP appeared to be short-lived. These features may be linked to the lower activity of Rad51 and the fact that Rad51 activity does not require the hydrolysis of ATP.     

Study of the interaction of polynucleotide chains with oligomers by means of chromatography. II. Effect of magnesium ions and noncomplementary bases on the stability of complexes

The interaction of the oligonucleotides ApA, ApApA, ApApC and ApApU with poly(U) and (Ip)₅I and (Ip)₆ with poly(C) has been studied by means of equilibrium gelfiltration through Sephadex.From sorption isotherms the free energies, energies and entropies of complexing have been computed for different concentrations of magnesium ions in the medium.The stoichiometric ratio of polymers to oligomers has been measured and found equal to 2 in the case of ApApA and ApApC. This shows that the cytidylic acid residue is included in the ternary complex. But in the case of ApApU the noncomplementary base is partly squeezed out of the complex.The stacking free energy of neighbouring oligomers has been found to be in the range 1000–3000 $\text{cal}\text{mole}$ depending on the conditions.The stoichimetric ratio has been found to be 1 in the case of poly(C): oligo(I), the stacking energy is equal to 1.2 $\text{kcal}\text{mole}$. The effect of magnesium is somewhat different in the case of double and triple helices and probably reflects the formation of coordination compounds with the nitrogen bases of nucleotides.