Analysis of 2',5'-oligoadenylates in cells and tissues

Complex mixtures of 2',5'-oligoadenylates are formed in cells and tissues under several different circumstances, and methods for analyzing such mixtures are reviewed. Separation is achieved by high-performance liquid chromatography and quantitation by competition-binding assays, using three different types of antibodies or a specific binding protein, or by functional assay, using preparations of an endonuclease specifically activated by some of the 2',5'-oligoadenylates. Representative results from three different biological systems are presented. The function of 2',5'-oligoadenylates as activators of intracellular RNA degradation is discussed, along with the possibility that these compounds may serve as signals for other intracellular regulatory processes.     

Sensitive radioimmuno assay for 2',5'-oligoadenylates using a novel 125I-labeled derivative of 2',5'-triadenylate 5'-triphosphate

A novel 125I-labeled derivative of 2',5'-triadenylate 5'-triphosphate, pppA2'p5'A2'p5'A, with high specific radioactivity was synthesized by coupling of periodate-oxidized pA2'p5'A2'p5'A with beta-alanyltyrosine methyl ester followed by 5'-triphosphorylation and iodination with 125I. Antisera toward 2',5'-oligoadenylate 5'-triphosphate were produced in rabbits by immunization with the conjugate of pppA2'p5'A2'p5'A2'p5'A with bovine serum albumin, and an antiserum with high specificity and high sensitivity for 2',5'-oligoadenylates was selected and tested extensively. Radioimmuno assaying of 2',5'-oligoadenylates was carried out by a competitive double antibody method in which the amount of the antibody bound to the 125I-labeled probe was measured after precipitation with goat anti-rabbit IgG. The concentration of pppA2'p5'A2'p5'A required for 50% inhibition of the binding between the antiserum and the probe was 0.6 nM. The cross reactivity of the antiserum with the 3',5'-triadenylate was more than 10,000 times weaker compared to in the case of 2',5'-oligoadenylates. Very low or no cross reaction was observed with ATP, AMP, and adenosine. The radioimmuno assay using the 125I-labeled compound and the antiserum allows the direct analysis of 2',5'-oligoadenylates in the range of 4 fmol to 1 pmol (0.04-10 nM in a 100 microliter sample). This assay was applied to the measurement of the activity of 2',5'-oligoadenylate synthetase in cells stimulated by interferon. The properties of the 125I-labeled derivative of pppA2'p5'A2'p5'A are described.     

Chemical synthesis and biological activities of analogues of 2',5'-oligoadenylates containing 8-substituted adenosine derivatives.

The synthesis of sequence-specific 2'-5'-oligonucleotides and analogues of 2'-5' linked oligoadenylates containing 8-substituted adenosine derivatives [8-hydroxypropyladenosine (AHPr) and 8-hydroxyadenosine (AOH)] is reported. The reaction of 5'-phosphoroimidazolidate of 8-substituted adenosines under conditions of lead ion catalyst did not give the corresponding 2'-5' oligoadenylates containing pAHPr and pAOH. When these reactions were carried out in the presence of uranyl ion (UO2(2+] in place of lead ion as a catalyst, the desired 2'-5' oligoadenylates were obtained. The p5'AHPr2'p5'AHPr2'p5'AHPr and p5'AOH2'p5'AOH2'p5'AOH, p5'A2'p5'A2'pAOH were slightly resistant to snake venom phosphodiesterase. The both circular dichroism and 1H-NMR spectra studies were used to characterize the modified 2'-5' oligoadenylates. Further, the biological activity evaluations of 8-substituted analogues of 2-5A are also described.     

Studies towards the large scale chemical synthesis of the precursors of ribonucleosides-3',4',5',5'-2H4 and -2',3',4',5',5'-2H5

A summary delineating the large scale synthetic studies to prepare labeled precursors of ribonucleosides-3',4',5',5'-2H4 and -2',3',4',5',5'-2H5 from D-glucose is presented. The recycling of deuterium-labeled by-products has been devised to give a high overall yield of the intermediates and an expedient protocol has been elaborated for the conversion of 3-O-benzyl-alpha,beta-D-allofuranose-3,4-d2 6 to 1-O-methyl-3-O-benzyl-2-O-t-butyldimethylsilyl-alpha,beta-D-ribofuranose-3,4,5,5'-d4 16 (precursor of ribonucleosides-3',4',5',5'-2H4) or to 1-O-methyl-3,5-di-O-benzyl-alpha,beta-D-ribofuranose-3,4,5,5'-d4 18 (precursor of ribonucleosides-3',4',5',5'-2H4).     

2-Methyladenosine-Substituted 2',5'-oligoadenylates: conformations, 2-5A binding and catalytic activities with human ribonuclease L

2-Methyladenosine-substituted analogues of 2-5A, p5'A2'p5'A2'p5'(me2A), p5'(me2A)2'p5'A2'p5'A, and p5'(me2A) 2'p5'(me2A)2'pS'(me2A), were prepared via a modification of a lead ion-catalyzed ligation reaction. These 5'-monophosphates were subsequently converted into the corresponding 5'-triphosphates. Both binding and activation of human recombinant RNase L by various 2-methyladenosine-substituted 2-5A analogues were examined. Among the 2-5A analogues, p5'A2'p5'A2'p5'(me2A) showed the strongest binding affinity and was as effective as 2-5A itself as an activator of RNase L. The CD spectra of both p5'(me2A)2'p5'A2'p5'A and p5'A2'p5'A2'p5'(me2A) were superimposable on that of p5'A2'p5'A2'p5'A, indicative of an anti orientation about the base-glycoside bonds as in naturally occurring 2-5A.     

Regulation of poly(ADP-ribose) transferase activity by 2',5'-oligoadenylates

pppA2'pA2'pA appears to be a potent natural noncompetitive inhibitor of poly (ADP-ribose) transferase activity in the histone dependent reaction of ADP-ribosylation with Ki=5 microM. Moreover, it is a noncompetitive inhibitor of the Mg2+ dependent reaction of autoADPRT-ribosylation with Ki=20 microM. The activity of ADPRT falls down abruptly both in the cytoplasm and nuclei of mouse L-cells treated with interferon. In contrast, the activities of 2',5'-oligo (A) polymerase and 2'-phosphodiesterase remain virtually unchanged after the treatment with ADPRT preparation. The regulation of ADPRT activity and active form of ADPRT by 2',5-oligoadenylates is presumed to be one of the factors responsible for inducing the antiviral and/or antiproliferative effects of interferon.     

Antibody-nucleic acid interactions. Monoclonal antibodies define different antigenic domains in 2',5'-oligoadenylates

To define the epitopes involved in binding anti-oligonucleotide antibodies, several hybridomas producing monoclonal antibodies directed against 2',5'-oligoadenylate were established. A solid-phase enzyme-linked immunoassay that employed microtiter wells coated with Ficoll-2',5'-oligoadenylate conjugates proved useful in screening and characterizing hybridoma supernatants. Control experiments demonstrated that the conjugates were irreversibly adsorbed to polystyrene wells under the conditions employed in the assay. Reactivity of monoclonal antibodies with numerous analogues of 2',5'-oligoadenylate was measured by using a competition assay. Several monoclonal antibodies originating from different mice immunized with the same or different immunogens possessed distinctive fine specificities. At least one 2',5'-phosphodiester bond was important in forming each epitope, suggesting that the ribose phosphate backbone is a critical element in defining an antigenic domain of an oligonucleotide. The purine bases were also important, and modification of the bases had varied effects on the extent of antibody recognition. The length of the oligonucleotide and the nature of the termini were also of some importance. In several instances the modification created by linkage of 2',5'-oligoadenylate to carrier protein also contributed to the determinant. The monoclonal antibody most specific for 2',5'-oligoadenylates was relatively insensitive to ionic strength. In contrast, a monoclonal antibody with a 2',5'-oligopurine specificity appeared to bind 2',5'-oligoadenylate through one ion pair, whereas the binding of a monoclonal antibody with a low degree of base specificity appeared to bind through two ion pairs. The results demonstrated that 2',5'-linked oligoadenylate-protein complexes possess at least three distinct oligonucleotide-related antigenic surfaces that can be recognized with high apparent affinity by monoclonal antibodies. A model for the three epitopes is presented.     

Purine 8-substitution modulates the ribonuclease L binding and activation abilities of 2',5'-oligoadenylates

Analogues of the 2',5'-linked adenylate trimers monophosphate (p5'A2'p5'A2'p5'A) containing 8-hydroxypropyladenosine, 8-bromoadenosine, and 8-hydroxyadenosine in the first, second, and third nucleotide positions were tested for their ability to bind to and activate RNase L of mouse L cells. p5'AHPr2'p5'AHPr2'p5'AHPr (pAHPr3) (1b) and p5'ABr2'p5'ABr2'p5'ABr (pABr3) (1d) were markedly decreased in ability to bind to the 2-5A dependent endonuclease. On the other hand, analogue of the 2',5'-linked adenylate trimer monophosphate substituted by 8-hydroxyadenosine in the first, second, and third nucleotide position was bound about as well as parent 2-5A [pppA(2'p5'A)2] (p3A3) (1e) to RNase L. Additionally, p5'AOH2'p5'AOH2'p5'AOH (pAOH3) (1c) was as active as parent 2-5A in the rRNA cleavage assay, while pAHPr3 (1b) and pABr3 (1d) were devoid of activity. The 8-substituted analogues of 2-5A were more resistant to the degradation by the (2',5') phosphodiesterase. Finally of particular interest was monophosphate, pAOH3 (1c) which possessed nearly 100% of the translation inhibitory activity of 2-5A triphosphate itself. These results suggest that changes in the base-sugar torsion angles of 2-5A may modulate both binding to and activation of mouse L cell RNase L.     

Uranyl ion-catalyzed synthesis of several 8-substituted 2'-5'-oligoadenylates, and their properties

We have synthesized 2'-5' linked oligomers from 8-substituted adenosine-5'-phosphorimidazolides using uranyl ion catalyst. 8-amino derivative, as highly susceptible to hydrolysis, gave short chained oligomers in a low yield, while the rest of 8-substituted or unsubstituted derivatives gave the corresponding oligomers in high yields. Properties of 8-substituted 2'-5' oligomers were studied applying spectrometer and through enzymatic digestion.     

Poly(ADP-ribose) polymerase activity is inhibited by 2',5'-oligoadenylates in mouse L-cells

Down regulation of poly(ADP-ribose)polymerase (ADPRP) activity was observed in mouse LW-cells after treatment with 2'-5'oligoadenylates or with fibroblast interferon and poly(rI) poly(rC). The poly(rI) poly(rC)-induced inhibition of the enzymatic activity correlates with the observed increase of endogenous 2',5'-oligoadenylate cores which were reported to be potent inhibitors of ADPRP in vitro.     

Synthesis of 2'-end-modified 2'-5'-oligoadenylates

Analogs of 2'5'-oligiadenylates (2-5As) have been prepared on the basis of the recently developed internucleotide-linkage formation via selective hydroxyl activation of N-unprotected nucleosides. The analogs synthesized include the trimeric cores having 2'-deoxyadenosine, cordycepin (3'-deoxyadenosine), and 2',3'-bisdeoxyadenosine at the 2' termini, and the tetramer with 2'-end 2'-deoxyadenosine.     

Effects of K+ concentration on inhibition of protein synthesis by trimer and tetramer triphosphates of 2',5'-oligoadenylates in rabbit reticulocyte lysates

The inhibition of protein synthesis by trimer or tetramer triphosphates of 2',5'-oligoadenylates (2-5A) in rabbit reticulocyte lysates is dependent on the concentration of K+ in the assay mixture. Based on studies using mRNA-dependent rabbit reticulocyte lysates and the degradation of [3H] polyadenylated mRNA, the greater inhibitory effects of trimer or tetramer triphosphates at the higher K+ is shown to be due to a more active 2-5A-dependent endoribonuclease (RNase L).     

The adnosine 3',5'-monophosphate and guanosine 3',5'-monophosphate phosphodiesterases from human lung tissue.

Human lung tissue contains phosphodiesterase enzymes capable of hydrolyzing both adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP). The cyclic AMP enzyme exhibits three distinct binding affinities for its substrate (apparent Km = 0.4μM, 3μM, and 40μM) while the cyclic GMP enzyme reveals only two affinities (Km = 5μM and 40μM). The pH optima for the cyclic AMP and cyclic GMP phosphodiesterase are similar (pH 7.6–7.8). Both are inhibited by known inhibitors of phosphodiesterase activity (aminophylline, caffeine, and 3-isobutyl-1-methylxanthine). The divalent cations Mg²⁺ and Mn²⁺ stimulate cyclic AMP phosphodiesterase activity (in the absence of Mg²⁺) while Ca²⁺, Ni²⁺, and Cu²⁺ inhibit the enzyme. Histamine and imidazole slightly stimulate cyclic AMP hydrolytic activity. Thus, human lung tissue does contain multiple forms of both the cyclic AMP and cyclic GMP phosphodiesterase which are influenced by a variety of effectors.     

3',5' Cyclic nucleotide phosphodiesterases class III: members, structure, and catalytic mechanism

3',5' Cyclic nucleotide phosphodiesterases (PDEs) comprise a superfamily of enzymes that were previously divided by their primary structure into two major classes: PDE class I and II. The 3',5' cyclic AMP phosphodiesterase from Escherichia coli encoded by the cpdA gene does not show any homology to either PDE class I or class II enzymes and, therefore, represents a new, third class of PDEs. Previously, information about essential structural elements, substrate and cofactor binding sites, and the mechanism of catalysis was unknown for this enzyme. The present study shows by computational analysis that the enzyme encoded by the E. coli cpdA gene belongs to a family of phosphodiesterases that closely resembles the catalytic machinery known from purple acid phosphatases and several other dimetallophosphoesterases. They share both the conserved sequence motif, D-(X)(n) GD-(X)(n)-GNH[E/D]-(X)(n)-H-(X)(n)-GHXH, which contains the invariant residues forming the active site of purple acid phosphatases, a binuclear Fe(3+)-Me(2+)-containing center, as well as a beta(alpha)beta(alpha)beta motif as a typical secondary structure signature. Furthermore, the known biochemical properties of the bacterial phosphodiesterase encoded by the cpdA gene, such as the requirement of iron ions and a reductant for maintaining its catalytic activity, support this hypothesis developed by computational analysis. In addition, the availability of atomic coordinates for several purple acid phosphatases and related proteins allowed the generation of a three-dimensional model for class III cyclic nucleotide phosphodiesterases.     

Chemical synthesis and biological activities of partially inosine-substituted 2',5'-oligoadenylates: functional discrimination of three adenine amino groups on 2-5A for the binding to and activation of 2-5A-dependent endonuclease

Binding and activation efficacies to the 2-5A-dependent endonucease by chemically synthesized partially inosine-substituted 2-5A analogs, namely, pppI2'p5'A2'p5'A, pppA2'p5'I2'p5'A and pppA2'p5'A2'p5'I were compared with that of native 2-5A in mouse L cell and human lymphoblastoid cell extracts. The results obtained in this study indicated that the first adenine amino group from the 5' terminus of 2-5A molecule plays critical role in binding to the endonuclease, whereas the third adenine amino group has a function for the activation of this enzyme.