Jiro Imai

Assay of 2′,5′-oligoadenylate phosphodiesterase activity in mouse L-cell extracts

A rapid and convenient assay for adenylyl(2 leads to 5)adenosine(A2p5A) or adenylyl(3 leads to 5)adenosine(A3p5A) phosphodiesterase activities is described. The dinucleotides A3p5A and A2p5A were labeled to a high specific activity by means of a catalytic-exchange procedure. Degradation studies of each of these labeled dinucleotides showed an asymmetrical distribution of label between the two adenine bases. Enzymatic degradation of [3H]A3p5A or [3H]A2p5A could be quantitated by first digesting the reaction products with bacterial alkaline phosphatase and then adding a slurry of DEAE-Sephadex. Under conditions described, adenosine did not adsorb to the resin, whereas dinucleotides as well as AMP did adsorb. As a consequence, when liquid scintillation fluid was added to the DEAE-Sephadex reaction mixture slurry, the radioactivity of the dinucleotides and AMP was severely quenched. This permitted a direct estimation of the amount of adenosine liberated during the phosphodiesterase degradation and subsequent alkaline phosphatase digestion. This method was applied to the measurement of A2p5A degrading activities in extracts of mouse L cells. Extracts from control mouse L cells were as active in degrading A2p5A as extracts from interferon pretreated cells.

[30] An efficient chemical synthesis of adenylyl(2′ → 5′)adenylyl(2′ → 5′)adenosine [(2′-5′)-oligo(A)]

Publisher Summary Adenylyl(2→5)adenylyl(2→5) adenosine or its 5-triphosphate is prepared by several different synthetic approaches, some of which are ambiguous. The chapter describes an eight-step synthesis that provides a 37% overall yield based on starting protected nucleosides. This triester approach uses tert-butyldimethylsilyl protecting groups. A new chiral center is generated upon formation of the internucleotide bond because it is a phosphotriester synthesis. The diastereomers thus formed are referred to as “a” and “b” for each numbered compound in the synthetic sequence. In this synthesis, the diastereomers are separated and synthetic sequences are carried out on each separately. This procedure uses zinc-copper coupled with acetylacetone in dimethylformamide to deblock the trichloroethyl groups of the intermediate trimer. This procedure results in a higher yield and cleaner reaction than with previously reported methods for removal of the trichloroethyl group. Use of Chelex in the procedures considerably improves the purification.

A sensitive immunoenzymometric assay for 2′,5′-oligoadenylate. Detection of elevated 2′,5′-oligoadenylate synthetase in human peripheral mononuclear cells

A competition immunoenzymometric assay for 2,5-oligoadenylate was developed and employed to measure the interferon-inducible enzyme 2,5-oligoadenylate synthetase in cell extracts. Microtiter plates coated with a novel conjugate of p5A2p5A2p5A and N-(2-aminoethyl)-carbamylmethylated-Ficoll (AECM-Ficoll) bound rabbit polyclonal or mouse monoclonal antibody directed against 2,5-oligoadenylate. Binding was inhibited by soluble 2,5-oligoadenylate. Estimates of 2,5-oligoadenylate concentrations based on inhibition of antibody binding compared favorably with those obtained using a protein synthesis inhibition assay. Low concentrations of 2,5-oligoadenylate synthesized in vitro by extracts of human peripheral mononuclear cells were conveniently estimated using less than or equal to 10(6) cells. Virtually identical results were obtained when either total extract or synthetase bound to poly(I) . poly(C)-agarose was used for the in vitro incubation. When peripheral mononuclear cells were incubated in vitro with interferon, the normally low levels of 2,5-oligo(A) synthetase rose dramatically. The assay was employed to measure synthetase levels in peripheral mononuclear cells isolated from patients with systemic lupus erythematosus. Some of these patients were found to have elevated levels of 2,5-oligoadenylate synthetase.

Strategies in the Design of Oligonucleotides as Potential Antiviral Agents

Polynucleotides have enjoyed considerable success as antiviral or antitumor agents due to their interferon-inducing ability1,2, their immunoadjuvant activity3, their reverse-transcriptase inhibitor properties4 or their capacity to inhibit virion-associated transcriptases5. On the other hand, the small sequence-defined oligonucleotides, until recently, have attracted little interest in this regard. Nonetheless, the potential of oligonucleotides as chemotherapeutic agents has been long appreciated. Levene and Stollar6 obtained partial inhibition of systemic lupus erythematosus sera by tetra- and pentanucleotides, and Shen7 suggested the design of high affinity oligonucleotide inhibitors of antigen-antibody complex formation. Oligonucleotides also have been employed as prodrug forms8, but we will not deal with this particular application.

Antagonism of 2–5 A-mediated inhibition of protein synthesis in intact cells by 2',5'-(pA)3

In vitro studies have shown that the translational inhibitory activity of 2–5 A can be blocked by the oligoribonucleotide 2,5‐(pA)3. We have examined the effect of simultaneous introduction of inhibitor and antagonist into intact mouse cells using calcium phosphate coprecipitation. Upon introduction of 10−4 M 2,5‐(pA)3 and 10−6 M 2–5 A, inhibition of protein synthesis was prevented. Efficiency of calcium phosphate precipitation of 2–5 A in the presence or absence of 2,5‐(pA)3 was comparable. Introduction of 2,5‐(pA)3 analogs showed that nucleotides which do not bind well to the 2–5 A dependent endonuclease do not prevent 2–5 A inhibitory activity. Thus, 2,5‐(pA)3 functions as an antagonist of 2–5 A in vivo.

Modes of action of interferon and analogues of 2-5A, a mediator of interferon action

In the last few years, several different interferons have been described, cloned, sequenced, expressed in heterologous systems and purified to homogeneity (Weissmann et al., 1982; Kingsman & Kingsman, 1983). In particular for human interferons there exists at least thirteen subtypes of IFN-α, one IFN-β and one IFN-γ. All these molecular species and some of their recombinant hybrids show a number of common biological properties but also distinctive antiviral (Weck et al., 1981; Kingsman & Kingsman, 1983) and other activities (Rehberg et al., 1982; Pestka et al., 1983b). Recombinant DNA technology has substantially contributed to the study of the mechanism of action of interferon and will probably continue to do so in the future by allowing a detailed analysis of the regulation and site-directed mutagenesis of IFN-induced proteins.