Simultaneous binding of adenosine and guanosine by polyuridylic acid: a further analysis

The dialysis data of Pitha, Huang, and Ts'o for the simultaneous binding of adenosine and guanosine to polyuridylic acid are analyzed here using a grand-partition function method described previously. The conclusion that the predominant mode of guanosine-binding cannot be a competition with adenosine for the primary hydrogen-bonding sites on the 2-polyuridylic acid complex emerges from this analysis. By setting a reasonable upper limit to the amount of competitive binding that might occur, it is found that the difference in standard free energies for the binding of guanosine and adenosine must be at least F _G ? F _A = 2400 cal/mole, provided the stacking energies for A ? A, A ? G, G ? G interactions are all equal. This difference in binding free energies implies a specificity of at least 80: 1 in favor of A on the primary sites at 5°C. Since this is a lower limit, the actual binding specificity may well be much greater. The desirability of achieving specificity through repulsion of incorrect bases, rather than via attraction of correct bases, is discussed.     

A proton magnetic resonance study of the interaction of adenylyl (3' is greater than 5') adenosine with polyuridylic acid

The interaction of adenylyl (3′ → 5′) adenosine (ApA) with polyuridylic acid in D₂O solution at neutral pD has been studied by high resolution proton magnetic, resonance spectroscopy. At temperatures above ～32°C, no evidence was obtained for the interaction of ApA with poly U. Below this temperature, a rigid triple-stranded complex involving a stoichiometry of 1 adenine to 2 uracil bases is formed, presumably via specific adenine–uracil base-pairing and cooperative base stacking of the adenine bases in a manner similar to that previously reported for the adenosine–poly U complex.     

The binding of deoxyguanosine to polycytidylic acid: an application of the thermodynamic model of monomer-polymer complex formation.

The thermodynamic model (equation 1) for formation of monomer-polymer complexes developed for better interpretation of the sigmoidal isotherms for the binding of adenosine to polyuridylic acid (2) and chemically modified polyuridylic acids (3) has been successfully applied to reproduce the isotherms for both the duplex binding of deoxyguanosine (d-G) to polycytidylic acid (at pH 6.8) and the triplex binding at pH 4.1. The value for the equilibrium constant, K, of the triplex complex (per unit of C-G-C) is ～2000 at the optimum value of n = 5 (n is the number of d-G units in the smallest complex that can form). The value of K for the duplex complex is 555 and the optimum value of n is 4.The value of ΔG for the triple helical complex is 4.15 kcal/mole, the value of w (the stacking free energy of the d-G units in the complex) is 2.05 kcal/mole. For the double helical complex at pH 6.8, ΔG° is 3.45 kcal/mole, w = 1.55 kcal/mole.It is also shown that equation (1) predicts that the shape and mid-point slope (i.e., w) of a binding isotherm depends only on the value of n; and thus the isotherms for rA-poly U (n = 5) and dG-poly C (n = 5) have the same mid-point slopes, and thus the same values of w. The difference between ΔG° and w is taken as a relative measure of the free energy of hydrogen bonding; values are calculated for the rA-poly U, the dG-poly C triple helix, and the dG-poly C double helical complexes.     

An analysis of the results for the binding of formaldehyde to polyuridylic acid

Spectrophotometric measurements were made on the extent of binding of formaldehyde to polyuridylic acid under conditions of varying temperature and formaldehyde concentration. The data is interpreted in terms of a temperature-dependent stacking of the bases in poly U at 20°, but not at 40°C. A theory of cooperative stacking is developed which considers the base residues to be either non-bonded, non-bonded and methylolated, or stacked. The results indicate essentially non-cooperative base stacking under these conditions with an equilibrium constant for base stacking of 0.92 at 20°C.     

Cooperative and thermodynamic parameters for oligoinosinate-polycytidylate complexes

The cooperative nature of the binding between polycytidylate and the oligoinosinates I(pI)_5–10 has been determined. Using the data of Tazawa, Tazawa, and Ts'o, it is shown that knowledge of the slope of the adsorption isothern allows one to determine the oligomer-polymer binidng constant, the oligomer–oligomer interaction constant, and the average degree of association (cooperative clustering) of the oligomers on the polymer. Knowledge of the above equilibrium constants as a function of temperature yields the respective thermodynamic parameters; no assumptions need to be made about the nature of the equilibrium constants or the thermodynamic parameters. For very long chains of polycytidylate, simple, explicit relations are given for the determination of the equilibrium constants involved. For finite chains of polycytidylate, the calculation of a single graph for each oligomer and polymer size allows the equilibrium constants to be determined for all experimental conditions of temperature and concentration. We find that the enthalpy and entropy of binding an oligomer n, bases to be δH_n = ±13.7 ? n(6.65) and δS_n = +32.5 ? n(18.8) given, respectively, in kcal/mole and e.u.; these parameters predict a melting temperature of 81°C for the poly(I)·poly(C) complex compared with the experimental value of 75°C. If the enthalpy is interpreted as arising from a sum of hydrogen bonding and stacking interactions, then the enthalpy of stacking is ?13.7 kcal/mole while the enthalpy of hydrogen bonding is +7 ± 4 kcal/mole; the positive enthalpy of hydrogen bonding presumably is a result of the fact that in the inosine-cytosine base pair, only two of the three sites on cytosine can hydrogen bond, the third being blocked from hydrogen bonding with water. The enthalpy of interaction between neighboring bound oligomers is found to be ?10.4 kcal/mole while the corresponding entropy is ?26.1 e.u. The binding is bound to be cooperative, though the extent of clustering varies markedly with temperature; the average number of oligomers in a cluster on the polymer is found to about five at a melting temperature of 25°C. The approach and equations given have generally applicability to oligomer-polymer associations.     

Volume changes accompanying the complex formation of polyadenylic and polyuridylic acids

The complex formation of polyadenylic acid (poly A) and polyuridylic acid (poly U) in 0.1M NaCl solution containing 0.01M sodium cacodylate was followed by dilatometric measurements at various mixing ratios of poly A and poly U. The volume changes, ΔV, accompanying the formation of poly A. poly U and poly A.2poly U were + l.5 and + 2.5 ml per mole of the nucleotide residue, respectively. This increase in volume was probably due to the increased counterion binding when the single-stranded polynucleotides were converted into the double- and triple-stranded helices, since depletion of charged species from the solvent proper would lessen the effect of electrostriction, thus resulting in a positive ΔV. The conversion of a single-stranded poly A to a double-stranded helix in acidic solution led to a ΔV of + 3.8 ml per mole of the nucleotide residue. This increase in volume was attributed to the charge neutralization as a result of protonation of the adenine bases.     

The binding of adenosine to polyuridylic acid in which uracil residues are chemically altered.

The binding of adenosine-¹⁴C to polyuridylic acid (poly(U)) and several modified poly(U)s has been studied by equilibrium dialysis. The poly(U) was modified by addition of appropriate reagents across the 5,6-double bond of the uracil ring to form the photohydrate, photodimer, dihydrouracil, the HOBr addition product and the HSO₃^? addition product. Modification of the uracil rings decreases the amount of adenosine which can be bound to the poly(U); the decrease in binding is a function of the fraction of uracil rings which have been changed. Using the expression S = S₀(1 ? αr)² to relate the fraction of uracil rings modified (r) to the number of binding “sites” remaining (S), it is found that α is about 1 for all the modifications except photodimer where it is about 2. These observations are taken to mean that the loss of binding capacity of the poly(U) resulting from modifications of the uracil ring is caused by loss of planarity of the uracil rings caused by the modifications, and consequent loss of double helix structure, but that for all modifications except photodimer there is no disruption of the poly(U) double helix on either side of the leison. There does appear to be local melting on either side of the photodimer lesion. The sigmoidal binding isotherms (A_b versus C_a) of modified and unmodified poly(U) can be approximated closely by the following equation: ((1)) (1) where A_b = bound A, C_a = free A, n = minimum number of adjacent A′s in complex, S = concentration of sites on poly(U), and K₁ = (K_m)^1/m for all m ≥ n. The stacking energy of adenosine (w) can be calculated accurately using the following equation, where dθ/d ln C_a is obtained from Eq. (1). ((2)) (2) For unmodified poly(U), w is ?2.0 kcal/mole and ΔG° (?;RT ln K₁) is ?3.2 kcal/mole. The difference (?1.2 kcal/mole) is attributed to hydrogen bonding. Heavily photohydrated poly(U) does not bind guanosine or guanosine-5′-phosphate.     

Stacking interaction and its role in kynurenic acid binding to glutamate ionotropic receptors

Stacking interaction is known to play an important role in protein folding, enzyme-substrate and ligand-receptor complex formation. It has been shown to make a contribution into the aromatic antagonists binding with glutamate ionotropic receptors (iGluRs), in particular, the complex of NMDA receptor NR1 subunit with the kynurenic acid (KYNA) derivatives. The specificity of KYNA binding to the glutamate receptors subtypes might partially result from the differences in stacking interaction. We have calculated the optimal geometry and binding energy of KYNA dimers with the four types of aromatic amino acid residues in Rattus and Drosophila ionotropic iGluR subunits. All ab initio quantum chemical calculations were performed taking into account electron correlations at MP2 and MP4 perturbation theory levels. We have also investigated the potential energy surfaces (PES) of stacking and hydrogen bonds (HBs) within the receptor binding site and calculated the free energy of the ligand-receptor complex formation. The energy of stacking interaction depends both on the size of aromatic moieties and the electrostatic effects. The distribution of charges was shown to determine the geometry of polar aromatic ring dimers. Presumably, stacking interaction is important at the first stage of ligand binding when HBs are weak. The freedom of ligand movements and rotation within receptor site provides the precise tuning of the HBs pattern, while the incorrect stacking binding prohibits the ligand-receptor complex formation.     

Proton magnetic resonance study of complex formation between N6-methyladenosine and polyuridylic acid

The interaction between N₆-methyladenosine and polyuridylic acid in D₂O solution at neutral pD has been studied as a function of temperature and N₆-methyladenosine concentration by proton magnetic resonance spectroscopy. A rigid double-stranded 1:1 complex is formed below ～10°C, involving hydrogen-bonded N₆-methyladenine:uracil base-pairing and stacking of the adenine bases. This complex is less stable than the 1:2 complex formed between adenosine and polyU, and involves a more rapid exchange of the monomer between free and polymer-bound environments.     

A calorimetric study of a polymer–monomer complex formed by polyuridylic acid 3′,5′-cyclic AMP

The existence of a soluble complex formed by polyuridylic acid (poly (U)) and 3′,5′-cyclic AMP (cAMP) is demonstrated by u.v. extinction vs. temperature curves, optical rotation, equilibrium dialysis, and reaction calorimetry. The complex hasthe stoichiometry of 2 poly (U)-cAMP and its formation is accompanied by an enthalpy change of ?13.0 kcal/mole of base triplet. The introuction of an empirical factor α in the equations given by Damle² and Crothers² leads to the evolution of a ΔH value of ?13.4 keal/mole. The parameter α is considered as a correction factor for the concentration dependence of the binding process. There is no relation between α and the reduction of monomer activity due to self-association of monomers. The study of the binding process at several temperatures showed that the cooperativity parameter, σ, is independent of temperature and its value of 6.5 × 10^?3 is in good agreement with σ = 5 × 10^?3 for the poly (U)·poly(A) system.³     

Three-stranded helix-coil equilibrium in polyuridylic acid-adenosine complex

Interaction of poly U (polyuridylic acid) and adenosine is studied by following the changes in ultraviolet absorbance in the wavelength region near the isochromic wave-length for the complex formation. The interaction is studied as a function of temperature, concentration of adenosine, and ionic strength, while the concentration of poly U was held constant. It is confirmed that only the three-stranded complex with the stoichiometry 1A to 2U is formed and that it dissociates directly into free poly U and adenosine. No discontinuity of any kind was apparent in the melting curves, and poly U was found to possess no ordered structure above 10°C under the conditions used. The results were, therefore, analyzed in terms of an exact helix–coil equilibrium theory using the mismatching model, i.e., assuming that either completely formed base triplet or completely free unbonded bases only exist, and that the two sections of the polymer chains forming closed loops need not contain the same number of unbonded bases. Self-association of free adenosine was taken into consideration. (Base triplet is analog of base pair for a three-stranded helical complex. It refers to a unit of three coplanar bases, in this case two uracils and one adenine, hydrogen bonded to one another to form a triplet. Such triplets may stack over one another along the helical axis, and when they are so stacked the bases of two triplets next to each other may have stacking interactions between them.) The values for enthalpy and entropy changes, both per mole of base triplets, were obtained for the following processes at neutral pH and moderate to high salt concentrations. (1) Growfh: Binding of one adenosine molecule to two uracil residues (one from each poly U strand) to form a base triplet next to an already formed base triplet with which it has stacking interactions, a process that involves both hydrogen bonding and base stacking interactions, ΔH_s, = ?19 ± 2 kcal, ΔS_s = ?55 ± 6 clausius; (2) Initiation: Binding of one adenosine molecule to two uracil residues (one from each poly U strand) to form an isolated base triplet, a process that involves only hydrogen bonding interactions, ΔH_b* = 4.5 ± 2 kcal, ΔS_b* = 6.6 ± 3 clausius; and (3)Interruption: Unstacking of two stacked base triplets initially next to each other by formation of an interruption (viz. a closed loop) between them, a process that involves only base stacking interactions, ΔH_b = 23.5 ± 3 kcal, ΔS_b = 61.6 ± 7 clausius, where the entropy changes include contributions other than the configurational entropy of closed loops. The discrepancy between our results and the calorimetric ΔH_s of ?13 kcal is attributed to (i) the possible effects of salt arid polymer on the self-association of free adenosine, (ii) the uncertainty in the value of the parameter for the probability of ring closure, and (iii) the contributions due to the partial molal enthalpy of the solvent and the unstacking of any poly U structure to the calorimetric enthalpy.     

Anti-cooperative interactions in single-strand oligomers of deoxyriboadenylic acid

Optical rotatory dispersion measurements were made on the deoxyribo nucleotides d(pA)₂, d(pA)₄, d(pA)₆ and poly(deoxyriboadenylic acid) at neutral pH over the temperature range 5–80°C. and were compared to similar data for the analogous oligoriboadenylic acids. The data were interpreted in terms of a temperature-dependent stacking of the bases in the single-strand deoxyribo oligomers. The thermal transition curves show an inverted chain-length dependence compared to the ribo oligomer curves. These results are explained by a theory of anti-cooperative interaction, where the nucleation parameter σ is >1. The theory, based on a one-dimensional Ising model involving both attractive nearest-neighbor and repulsive next-nearest-neighbor interactions, predicts the inverse chain length dependence and agrees rather well with the experimental data. At and above the transition temperature, the deoxyribo polymer is seen to consist of isolated stacked base pairs separated by at least one unit of random coil, there being only a very small probability for the existence of sequences of stacked residues longer than one. The partition function is seen to undergo an irregular behavior as a function of chain length because of the anti-cooperative phenomenon. It is necessary to use an enthalpy of stacking of ?5.0 kcal./mole in order to fit the experimental data with the theory. This value, 1.5 kcal./mole more positive than the ΔH found for the ribo oligomers, is reasonable, since the 2′ hydroxyl group would be expected to stabilize the stacking interaction in the ribo oligomers. Various kinds of distribution functions are calculated and plotted graphically for this theoretical model. A physical rationale is presented for the use of a repulsive next-nearest-neighbor term in this theory for the deoxyribo oligomers.     

Studies on the macroconidia of Microsporum canis

The characteristics of an in vitro polyuridylic acid dependent amino acid incorporating system prepared from germinating macroconidia of Microsporum canis are described. The incorporation of ¹⁴C-phenylalanine into polyphenylalanine is dependent on S-30 extract, adenosine triphosphate, magnesium ions and polyuridylic acid. Incorporation is slightly enhanced by yeast transfer ribonucleic acid and pyruvate kinase. The system is highly sensitive to ribonuclease, puromycin and miconazole (an antifungal agent), moderately sensitive to sodium fluoride and much less sensitive to phenethylalcohol, cycloheximide, chloramphenicol and deoxyribonuclease. Cell-free extract from ungerminated conidia has less capacity to synthesize the protein and during germination a marked increase in the protein synthetic activity is observed. The results from experiments wherein ribosomes and S-100 fraction from germinated and ungerminated spores are interchanged, revealed that the defect in the extract from the ungerminated spore is in the ribosomes.Abbreviations Poly(U) polyuridylic acid - tRNA transfer ribonucleic acid - ATP adenosine triphosphate - GTP guanosine triphosphate - BSA bovine serum albumin - RNase ribonuclease - DNase deoxyribonuclease - POPOP 1,4-bis-2(5-phenyl oxazolyl)benzene - PPO 2,5-diphenyl oxazole - TCA trichloracetic acid     

The interaction of collagen with the hydrophobic fluorescent probe-2-p-toluidinylnaphthalene-6-sulfonate.

Three kinds of fluorescence enhancement result from the interaction of 2-p-toluidinylnaphthalene-6-sulfonate and calf-skin collagen. They are negatively cooperative, independent, and highly cooperative fluorescence enhancement. In the independent region at pH 3.7, the binding number is about 36 moles of 2-p-toluidinylnaphthalene-6-sulfonate per mole of tropocollagen with a binding constant of 2.0 × 10⁴ M^?1; with ΔG = ?5.7 kcal/mole, ΔH = ?4.0 kcal/mole, and ΔS = 6 e.u. The pH dependence of fluorescence of native collagen shows that the deprotonated forms of the β and γ carboxyl groups of aspartic and glutamic acid decrease the intensity, possibly by charge repulsion of the negatively charged sulfonate group of 2-p-toluidinylnaphthalene-6-sulfonate. The positive charge of lysine is found to be unimportant in the interaction of 2-p-toluidinylnaphthalene-6-sulfonate with collagen. Fluorescence enhancement is caused mainly by the hydrophobic interactions of 2-p-toluidinylnaphthalene-6-sulfonate and collagen. Salt bridge formation between basic and acidic side chains in very low salt concentration may be detectable by 2-p-toluidinylnaphthalene-6-sulfonate fluorescence.     

Calorimetric determination of the heat capacity changes associated with the conformational transitions of polyriboadenylic acid and polyribouridylic acid

The heat capacities of the single-stranded and double-stranded forms of polyadenylic acid, polyuridylic acid, and poly(uridylic and adenylic acid) were determined with a drop heat capacity calorimeter. In addition, the temperature dependence of the apparent partial heat capacity (?Cp) was measured with a newly developed differential scanning calorimeter. The calculated ΔCp at 28°C for the transition poly(A)·poly(A) ? 2 poly(A) was found to be 165 ± 24 cal/Kmol-base pair, compared with a value of 140 ± 28 for the transition poly(A)·poly(U) ? poly(A) + poly(U). The temperature dependence of ?Cp of single-stranded poly(U) was consistent with the conclusion that it is totally unstacked at temperatures above 15°C. The temperature dependence of ?Cp of single-stranded poly(A) was used to determine the base-stacking parameters for poly(A). The experimental results are consistent with a stacking enthalpy change of ?8.5 ± 0.1 kcal/mol bases and a cooperativity parameter σ of 0.57 ± 0.03 for the stacking of adenine bases. These results demonstrate that the heat capacity of single-stranded polynucleotides is greater than that of the double-stranded forms. This increased heat capacity is mainly the result of the temperature dependence of the base-stacking interactions in the single-stranded form.     

A theory of complex multiple equilibria. The effect of poly-U concentration on the binding of deoxyadenosine

A grand-partition function method employed previously for the study of cooperative binding of nucleosides to polynucleotides is generalized to include the effect of changes in the polymer concentration, as well as the effect of changes in the nucleoside concentration. This method is applied to compute isotherms for the binding of deoxyadenosine by polyuridylic acid at various total concentrations of polyuridylic acid. It is found that these isotherms are extremely insensitive to the polymer concentration, as commonly assumed heretofore, and as demonstrated experimentally (to some degree) by Davies and Davidson. However, the conclusion inferred by Davies and Davidson that the complexes must be unimolecular in polyuridylic acid is now seen to be clearly unwarranted, though it remains still a viable possibility, until data over a much wider range of polyuridylic acid concentrations become available.     

Design and Synthesis of A3 Adenosine Receptor Ligands, 2′-Fluoro Analogues of Cl-IB-MECA

Abstract

Synthesis of 2′-deoxy-2′-fluoro-N ⁶-substituted adenosines as bioisosteres of Cl-IB-MECA and their binding affinities to A₃ adenosine receptor are described.     

Design and Synthesis of A3 Adenosine Receptor Ligands, 3′-Fluoro Analogues of Cl-IB-MECA

Abstract

Synthesis of 3′-deoxy-3′-fluoro-N ⁶-substituted adenosines as bioisosteres of Cl-IB-MECA and their binding affinities to A₃ adenosine receptor are described.     

Interaction of poly-5-bromouridylic acid. II. Thermodynamic analysis of its interaction with adenosine and 9-methyladenine

The interaction of poly-5-bromouridylic acid [poly(BU)] with adenosine and 9-methyladenine was studied by equilibrium dialysis, optical melting, and microcalorimetry. The stacking free energy, ω, was estimated as ?17.6 kJ/mol for adenosine·2poly(BU) and ?18.8 kJ/mol for 9-methyladenine·2poly(BU) from the binding isotherms constructed from equilibrium dialysis results. The binding isotherms constructed from a series of melting curves also gave ω values for adenosine·2poly(BU). The thermal stability of the complex depends on monomer concentration, and the partial molar enthalpies of the complex formation at the midpoint of the transition were evaluated from the T_m coefficients as a function of free monomer concentration. The values of ?92.0 and ?90.4 kJ/mol were obtained for adenosine·2poly(BU) and 9-methyladenine·2poly(BU) in 0.4M NaCl–0.02M Na-cacodylate–5 × 10^?4M EDTA (pH 7.0), respectively. Microcalorimetric measurements provided lower integral heats of reaction values for these complexes, i.e., ?73.2 kJ/mol for adenosine·2poly(BU) and ?71.5 kJ/mol for 9-methyladenine·2poly(BU). A comparison with a polyribouridylic acid system provided a quantitative understanding of a stabilization by bromination in terms of thermodynamic parameters.     

Thermodynamics of mucopolysaccharide–dye binding. II. Binding constant and cooperativity parameters of acridine orange–dermatan sulfate system

In the acridine orange–dermatan sulfate system, free and bound dye can be distinguished from each other spectroscopically. This permits the use of fluorometric methods to study the binding of acridine orange to the acid mucopolysaccharide dermatan sulfate. Experiments were conducted at 24°C in 10^?3 M citrate/phosphate buffer at pH = 7.0. The binding of the dye is highly cooperative, as evidenced by considerable interaction between adjacent bound dye molecules. Analysis of the data indicates that dermatan sulfate binds 2.3 ± 0.3 mol of acridine orange per dermatan sulfate uronic acid residue with a cooperative binding constant, K_q ranging from 4.9 to 6.0 × 10⁵ M^?1 which corresponds to a free energy of 7.74 ? ΔG° ? 7.86. The cooperativity parameter q apparently increases with increasing polymer-to-dye ratio.