Enzymic Synthesis, Characterisation and Nuclear‐Magnetic‐Resonance Spectra of pppA2′p5′A2′p5′A and Related Oligonucleotides: Comparison with Chemically Synthesised Material

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Abstract
We have previously shown that a low‐molecular‐weight inhibitor (pppA2′p5′A2′p5′A) of cell‐free protein synthesis, effective at subnanomolar concentrations, is formed on incubation of extracts from interferon‐treated cells or rabbit reticulocytes with double‐stranded RNA and ATP. The inhibitors synthesised with the reticulocyte or interferon‐mediated enzymes, i.e. (2′‐5′)An synthetases, behave identically on thin‐layer chromatography and electrophoresis and have the same biological activity in the inhibition of cell‐free protein synthesis. Here we show that they also behave identically on chromatography on DEAE‐Sephadex in the presence of urea and yield identical products on enzymic and sequential degradation by periodate oxidation and β‐elimination. As previously shown with the interferon inhibitor, preparations of the reticulocyte pppA2′p5′A2′p5′A also include the corresponding biologically inactive dimer, pppA2′p5′A, and biologically active tetramer, ppp(A2′p)35′A, and occasionally higher oligomers in decreasing amounts. Further purification of the pppA2′p5′A2′p5′A yielded small amounts of the corresponding 5′‐diphosphates and 5′‐monophosphates. The 5′‐diphosphate has the same specific biological activity in the inhibition of cell‐free protein synthesis as the triphosphate. The 5′‐monophosphate is essentially inactive.Milligram quantities of the reticulocyte inhibitor can be conveniently synthesised by incubating the (2′‐5′)An synthetase from these cells bound either to poly(I) · poly(C)‐Sepharose or poly(I) · poly(C)‐paper with ATP. Simple methods for the fractionation of the resultant (2′‐5′)An, into its individual components are described. Proton and phosphorus nuclear magnetic resonance spectra were obtained for the putative pppA2′p5′A2′p5′A and the corresponding 5′‐monophosphate and 5′‐diphosphate from this material and for the A2′p5′A2′p5′A ′core′ derived from these by treatment with bacterial alkaline phosphatase. The spectra were compared with those for chemically synthesised A2′p5′A2′p5′A and commercially available A3′p5′A3′p5′A, AMP, ADP and ATP. The results are in accord with the proposed structure.Phosphorylation of chemically synthesised A2′p5′A2′p5′A yielded material with chromatographic properties and a specific biological activity in the inhibition of cell‐free protein synthesis apparently identical to those of biologically synthesised pppA2′p5′A2′p5′A.