Riboadenylic Acid Research Articles

Influence of position of hydroxyl group of flavonoids on their binding with single stranded polyriboadenylic acid: A spectroscopic evaluation.

Single stranded polyriboadenylic acid [poly (rA)] has been accepted widely as a suitable drug target owing to its vital role in the development of cancer since it controls gene expression during cell growth and differentiation. The biological properties of poly (rA) depend on its structural morphology. Pharmacologically active flavonoids can act as suitable binders to poly (rA) and significantly change its biophysical properties. Different factors favour flavonoid-poly (rA) binding. In our present work we have explored the role played by the position of hydroxyl groups in the flavonoids namely 3, 5, 6 and 7 hydroxyflavones in their course of interaction with poly (rA). A range of spectroscopic experiments reveal that 3HF binds best to poly (rA) among the four chosen flavonoids. This is probably due to the presence of a hydroxyl group in '3' position that enables it to exhibit ESIPT phenomenon which is missing for the other used flavonoids.

Drying dissipative structure of sodium salts of hyaluronic acid

Macroscopic and microscopic patterns during the course of dryness of aqueous solution in sodium salts of hyaluronic acid (NaHLA) were observed on a cover glass, a watch glass, and a Petri glass dish. Dendritic and rod-like microscopic patterns, which are similar to those of sodium salts of carboxymethyl cellulose, were observed for NaHLA especially on a cover glass and a watch glass. The microscopic patterns of NaHLA are supported to be originated from the hexose groups of polysaccharides, though the similar dendritic patterns are also observed for some of polynucleotides, sodium salts of deoxyribonucleic acid, and potassium salts of poly (riboadenylic acid), for example. Macroscopic broad ring size decreased substantially from the initial size of liquid and decreased slightly as polymer concentration decreased. These observations are consistent with existence of the rather strong interpolymer affinitive forces during the course of dryness. In the coexistence of sodium chloride, microscopic dendritic patterns grew large especially on a cover glass and a watch glass, which was so often observed for polysaccharides examined hitherto. Drying patterns are clarified to be formed by the successive and cooperative processes of evaporation, convection, sedimentation, and solidification.

Binding of the phenothiazinium dye methylene blue with single stranded polyriboadenylic acid

The binding of the phenothiazinium dye methylene blue to single stranded poly(riboadenylic acid) was investigated by spectroscopic and calorimetric techniques. The binding was cooperative and the affinity was of the order of 10 6 M −1 at 100 mM [Na +] as determined from absorbance, fluorescence and calorimetric studies. Ferrocyanide quenching studies showed intercalative binding of methylene blue to poly(riboadenylic acid). The binding perturbed the circular dichroism spectrum of poly(riboadenylic acid) with concomitant formation of prominent exciton split type of extrinsic CD bands in the 550–700 nm region. The interaction involved a single binding mode with a 1:2 binding stoichiometry. The binding affinity increased with [Na +] ion concentration in the range 10–200 mM [Na +]. Dye binding induced self-assembled duplex formation in poly(riboadenylic acid). The biological utility of the dye methylene blue in probing nucleic acid structure is revealed from these studies.

Dissipative crystallization of aqueous mixtures of potassium salts of poly(riboadenylic acid) and poly(ribouridylic acid)

Dissipative drying patterns of aqueous mixtures of potassium salts of poly(riboadenylic acid) (KPolyA) and poly(ribouridylic acid) (KPolyU) were studied on a cover glass, a watch glass and a glass dish at room temperature. Accumulation of the polymers forming the broad rings near the outside edge and the inner area of the dried film was observed. The fine multiple ring structures formed when the affinity of the polymer with the substrate is strong. Microscopic drying patterns changed drastically depending on the location in the dried film. Microscopic drying patterns were mainly dendritic long rods and sword (halberd)-like rods. They are assigned to the crystals of double-stranded and triple-stranded helices of the A:U and A:2U complexes, respectively. Cross-like drying patterns are also observed originated from the salt-polymer interaction.

Dissipative crystallization of potassium salt of poly(riboadenylic acid)

Dissipative patterns during the course of dryness of aqueous solution of potassium salt of poly(riboadenylic acid) (KPolyA) in the presence of potassium chloride were studied on a cover glass, a watch glass and a glass dish. Accumulation of KPolyA polymers forming the broad ring area and the drastic change in size and shape of the polymer single crystals depending on the location of the dried film, which are the typical effects of the dissipative crystallization, took place. Polymer crystals formed were spherulites, dendritic and rod-like assemblies, which are composed of the single or double helical chains depending on the pH-value of the initial solution.

The detrimental effect of orotic acid substitution in the peptide nucleic acid strand on the stability of PNA2:NA triple helices

We have investigated the incorporation of C6 derivatives of uracil into polypyrimidine peptide nucleic acid oligomers. Starting with uracil-6-carboxylic acid (orotic acid), a peptide nucleic acid monomer compatible with Fmoc-based synthesis was prepared. This monomer then served as a convertible nucleobase whereupon treatment of the resin-bound methyl orotate containing hexamers with hydroxide or amines cleanly converted the ester to an orotic acid or orotamide-containing peptide nucleic acid. Peptide nucleic acid hexamers containing the C6-modified nucleobase hybridized to both poly(riboadenylic acid) and poly(deoxyriboadenylic acid) via triplex formation. Complexes formed with poly(riboadenylic acid) were more stable than those formed with poly(dexoyriboadenylic acid), as measured by temperature-dependent UV spectroscopy. However, both of these complexes were destabilized relative to the complexes formed by an unmodified peptide nucleic acid oligomers. Internal or doubly substituted hexamers are destabilized more strongly than a terminally substituted one, and the type of substitution (carboxamide, ester, carboxylic acid) affects the overall triplex stability. These results clearly show that incorporation of a C6-substituted uracil into polypyrimidine PNA is detrimental to triplex formation. We have also extended this chemistry to incorporate uracil-5-methylcarboxylate into a peptide nucleic acid hexamer. After on-resin conversion of the C5 ester to the 3-(N,N-dimethylamino)propylamide, significant stabilization of the triplex formed with poly(riboadenylic acid) was observed, which illustrates the compatibility of C5 substitution with peptide nucleic acid directed triple helix formation. Key words: peptide nucleic acid, triple helix, orotic acid, orotamide, PNA.

A study on the effect of Cu2+ ions on the stability of triple helix poly[(riboadenylic acid):2 poly(riboinosinic acid)

AbstractThe effect of Cu2+ on the stability of the triple‐stranded helical structure of poly[(riboadenylic acid): 2 poly(riboinosinic acid)] poly(A : 21) was studied by means of circular dichroism spectroscopy and ultraviolet spectroscopy. The experimental results show that Cu2+ induces the hyperchromicity of poly(A : 2I) and causes the melting temperature Tm of poly(A : 2I) to decrease with increasing Cu2+ concentration. At the ratio of the concentration of Cu2+ to that of phosphate in poly(A : 2I), rt = 0, poly(A : 2I) is stable, is stable, at rt = 0.27, poly(A : 2I) decays in poly(A) and 2 poly(I), and at rt = 1.6, there is a racemate.

Effect of condensed tannins and related compounds on reverse transcriptase

AbstractInhibitory effects of 18 condensed tannins and related compounds on reverse transcriptase from an RNA tumour virus were examined in the presence of poly(riboadenylic acid) oligo(deoxythymidylic acid) ((rA)n(dT)12–18) or poly(ribocytidylic acid) oligo(deoxyguanylic acid) ((rC)n(dG)12–18) as a template‐primer. A potent inhibitory effect was found in galloylated monomers and oligomers as well as non‐galloylated tetramer, trimer and two of three dimers. The mode of the inhibition of one of them, procyanidin B‐2, was competitive with regard to the template‐primer.

Phosphorescence of adenosine and poly(riboadenylic acid). Evidence for dual emissions and n pi states.

Phosphorescence from the 9-adenylyl group in the form of microcrystalline powders of adenosine films of poly(riboadenylic acid) (poly(rA)) in hyaluronic acid has been studied at 77 K. For adenosine, clearly resolved vibronic structure consists of two progressions, A and B, with delta vA = 1363 cm-1 and delta vB = 1575 cm-1, correlated with in-plane C5-N7 and in-plane C4-C5 stretch, respectively. The relative strength of the progressions varies with excitation wavelength and this, together with the absence of a common origin, indicates the existence of two independent emitting states with 0-0' levels separated by either 300 or 1000 cm-1. Two different excitation spectra are observed lying below the normal (pi pi*) adsorption and one is assigned as a previously undetected 1(n pi*) transition. For poly(rA) films the emission band envelope is identical with that of adenosine but the vibronic structure is lost. Only one excitation peak is observed at 32.9 X 10(3) cm-1, identical with one of the adenosine spectra. The second adenosine excitation spectrum probably represents an intermolecular charge transfer transition. Comparison is made with the predictions of six semi-empirical MO calculations.

Proton, phosphorus and carbon nuclear magnetic relaxation studies on the interaction of poly(riboadenylic acid) with Cu2+

AbstractInteraction between poly(riboadenylic acid) (poly(A)) and Cu2+ in neutral aqueous (D2O) solution has been studied by 1H, 31P, and 13C nuclear magnetic resonance. electron‐nuclear hyperfine coupling constant and apparent electron‐nuclear distances were determined by measurement of T1 and T2 values as a function of temperature. The apparent distance from Cu2+ to H(2), H(8), H(1′), and phosphorus nuclei were estimated to be 4.1, 3.7, 5.1, and 3.1 Å from these results. Cu2+ was found to coordinate directly to the phosphate groups of poly(A) (Type I complex). Simultaneously there are bindings of Cu2+ directly to one of the nitrogen atoms of adenine ring, mainly to N(7) (Type II complex) and either N(1) or N(3) (Type III complex).

Calculation of induced circular dichroism of acridine orange-poly(riboadenylic acid) complexes and confirmation of their structure

Calculation of induced circular dichroism of acridine orange-poly(riboadenylic acid) complexes and confirmation of their structure

PPi analogs as inhibitors of human T-lymphotropic virus type III reverse transcriptase

Twenty-six PPi analogs were tested for inhibitory effects on human T-lymphotropic virus type III reverse transcriptase. The structural requirements for inhibition and mechanism of action of the most active inhibitors have been investigated. Foscarnet (phosphonoformic acid) was the most potent inhibitor of human T-lymphotropic virus type III reverse transcriptase with 50% inhibition at 0.5 microM. The mechanism was a noncompetitive type of inhibition of a (riboadenylic acid)n . (deoxythymidylic acid)12-18 [(rA)n(dT)12-18]-directed transcription at varied dTTP concentration. At constant substrate (dTTP) concentration and varied amounts of template, (rA)n(dT)12-18, the inhibitory action of foscarnet was of an uncompetitive type. The same pattern of inhibition was seen when the less active inhibitor carbonyldiphosphonate was studied under identical conditions. The structural requirements for inhibition of human T-lymphotropic virus type III reverse transcriptase by PPi analogs were similar to those shown by other reverse transcriptases.

Poly(riboadenylic acid) preferentially inhibits in vitro translation of cellular mRNAs compared with vaccinia virus mRNAs: possible role in vaccinia virus cytopathology.

Vaccinia virus-induced inhibition of host protein synthesis seems to be mediated by viral transcripts based on their differential inhibition of cellular mRNA translation in a rabbit reticulocyte lysate system. In this study, we demonstrated that the removal of poly(riboadenylic acid) [poly(A)] from the in vitro viral transcripts abolished this inhibition in the same cell-free system. This observation led us to the finding that less than 1 microM poly(A) completely inhibited HeLa cell mRNA translation in the reticulocyte lysate, whereas only 50% inhibition of vaccinia virus mRNA translation was observed at the same concentration. Similar results were also obtained in a wheat germ protein-synthesizing system. This inhibitory effect of poly(A) was totally abrogated by the addition of polydeoxythymidylate. This selective inhibition was highly specific for poly(A) since other homopolymers, including poly(G), poly(C), and poly(dA), were not capable of causing such an inhibition. Poly(U), however, had a moderate selective inhibitory effect. Among the several mRNAs tested, the translation of L-cell, encephalomyocarditis virus, and reovirus RNAs was also sensitive to poly(A). However, vesicular stomatitis virus mRNA translation was strikingly more resistant. These results suggest that poly(A), which is also synthesized by the virion-associated poly(A) polymerase may be involved in vaccinia virus-mediated host cell shutoff.

Isolation and characterization of cytoplasmic poly(A) polymerase from cryptobiotic gastrulae of Artemia salina.

Poly(A) polymerase has been purified to near homogeneity from the cytoplasm of Artemia salina cryptobiotic gastrulae by ion-exchange chromatography on DEAE-cellulose, DEAE-Sepharose CL-6B and phosphocellulose P11, gel filtration on CL-Sepharose 6B, affinity chromatography on poly(A)-Sepharose 4B and ATP-agarose. The enzyme is fully dependent on exogeneous oligo(riboadenylic acid) and is free of any nuclease or other enzyme activities. In standard assay conditions the enzyme preparation has a specific activity of 5.6 mumol AMP . h-1 . (mg protein)-1. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis reveals the presence of only two proteins with Mr 94 000 and 70 000. The Mr-70 000 protein has been identified as poly(A) polymerase. The enzyme is exclusively activated by Mn2+. Addition of Ca2+, Mg2+, Zn2+, NH4+, K+ or Na+ inhibits the enzymatic reaction. The activity is specific for ATP and competitive inhibition is observed in the presence of other ribonucleoside 5'-triphosphates. AMP incorporation is time-dependent and is increased non-linearly with protein and primer concentration.

Radiation-induced strand breakage in poly(riboadenylic acid). A pulse radiolysis study on the protective action of cysteamine under anoxic conditions.

Poly(riboadenylic acid) was irradiated in N2O-saturated dilute aqueous solution at pH 7.8 with single pulses (50-200 ns) of 16 MeV electrons. With the aid of Rayleigh light scattering measurements both the extent and rate of main-chain scission were measured in the absence and the presence of cysteamine (RSH). In both cases two modes of light scattering intensity (LSI) decrease were detected. From this finding it was concluded that two chemically different radical sites, giving rise to main-chain breakage, were produced by OH attack on the macromolecules. Cysteamine reduced the extent of LSI decrease and accelerated the rate of the slow mode of LSI decrease significantly. The fast mode of the LSI decrease is due to fragment diffusion and therefore the influence of cysteamine on the rate of decay of the short-lived radical could not be studied. The assessment of the relative importance of .OH scavenging (RSH + .OH----RS. + H2O) and of repair (PA. + RSH----PAH + RS.) with respect to protection yielded the following: in the case of the long-lived radical, repair contributes significantly at low RSH concentration (less than 10(-4) M); in the case of the short-lived radical, OH scavenging dominates and repair becomes noticeable only at [RSH] greater than 10(-4)M. The following data were evaluated in this work: 100 eV-yield of main-chain scission, G(sb) = 1.1; lifetimes of radicals: less than 50 microseconds (short-lived), 0.6 s (long-lived), kPA. + RSH = 3.4 X 10(6)M-1 S-1 (for long-lived radicals).

On the reactions of macroradicals in solution. A pulse radiolysis study

This paper summarizes work concerning oxidative and anoxic main-chain scission of single-stranded polynucleotides, e.g. poly(riboadenylic acid), and the oxidative main-chain scission of poly(ethylene oxide) and polyacrylamide. Degradation was initiated in aqueous solution by attack of OH radicals. The kinetics of the degradation process were followed by pulse radiolysis in conjunction with light scattering measurements. Intramolecular radical-radical reactions received special attention during these studies.

OH Radical‐induced chemical reactions of polynucleotide complexes

AbstractPoly(A + U), the 1:1 complex of poly(riboadenylic acid), poly A, and poly(ribouridylic acid), poly U, at pH 8 and self‐complexed poly A at pH 4 exist as double helices in dilute aqueous solution. These complexes exhibit a similar behavior as native calf thymus DNA upon irradiation with 16 MeV electron pulses. Thus time resolved Rayleight light scattering measurements showed that crosslinking and double strand breakage can be clearly separated, the former proceeding faster than the latter. The extent to which the two processes occur depends on the ionic strength of the solution. At ionic strenghts exceeding 10 −1 mol/l crosslinking is the dominant process indicating that hcrit, the critical length between two single strand breaks for the accomplishment of double strand breaks, is strongly reduced. The investigation of complexes of poly A and Mg2+ ions revealed that the destruction of salt bridges is the rate determining process for the decrease of the light scattering intensity due to mainchain scission. This implies that life‐times of salt bridges can be determined.

OH radical‐induced main chain scission of poly(riboadenylic acid)

OH radical‐induced main chain scission of poly(riboadenylic acid)

Interaction between polynucleotides in aqueous NaCl solution, 4. Poly(riboadenylic acid) — poly(2‐riboinosinic acid) system

AbstractSolution properties of poly(A) and poly(I) solution at a mole ratio of poly(A) to poly(I) of 1/2 were studied by means of calorimetry and a modified differential scanning calorimetry (DSC) with the help of circular dichroism (CD) and ultraviolet (UV) spectral methods. It was found that the heat of mixing, ΔHM, depends sharply on the ionic strength. At [NaCl] < 10−2 mol · dm−3 ΔHM is nearly zero. At higher ionic strength ΔHM decreases and reaches a minimum value at [NaCl] ≈︁ 2 · 10−2 mol − dm−3. Above this ionic strength ΔHM increases again. This behavior is explained on the basis of the formation of a triplex poly(A) · poly(2I) and the transition of poly(I) from a disordered to an ordered structure with increasing ionic strength. The heat of formation, ΔH, of the poly(A) · poly(21) triplex was estimated to be ca −41 kJ per mole of base triplet. Above an NaCl concentration of 2 · 10−1 mol · dm−3 the triplex formation may be represented by the following scheme:

Interaction between acridine orange and polyriboadenylic acid

The absorption spectra and circular dichroism of aqueous solutions of acridine orange mixed with polY(riboadenylic acid) [poly(rA)] have been measured for different mixing ratios at acid and neutral pH. The binding ratio of dye to poly(rA) has been determined by equilibrium dialysis. At acid pH where poly(rA) is in a double-stranded helix, monomeric dye molecules are intercalated between base pairs, first sparsely and then at neighbouring sites with mutual coupling, as the nucleotide-to-dye mixing ratio decreases. In the presence of excess dye, dimeric dye molecules of antiparallel type are bound to phosphate groups electrostatically and stack together to form linear sequences along a poly(rA) chain. At neutral pH where poly(rA) is single-stranded, isolated intercalation of monomeric dye molecules can occur in the helical parts. At intermediate mixing ratios, half-intercalated dimeric dye molecules are bound to adjacent sites and electronically coupled, inducing characteristic circular dichroism. In the presence of higher amounts of dye, external stacking of dimeric dye molecules of antiparallel type occurs along a poly(rA) chain. The binding of dye cations is suppressed to some degree at acid pH compared to that at neutral pH, owing to the repulsion exerted by protonated adenine bases.