Hans Sternbach

The antiviral activity of thiophosphate-substituted polyribonucleotides in vitro and in vivo

Abstract As shown before for the alternating poly r(A-U) (riboadenylic-ribouridylic acid), substitution of thiophosphate for phosphate in the alternating poly r(I-C) (riboinosi-nic-ribocytidylic acid) resulted in a significant increase of its ability to induce in vitro resistance to virus infection and interferon production in vitro and in vivo. The antiviral activity of the partially substituted polynucleotides poly r(A s U ) and poly r ( s I-C ) was intermediate between the antiviral activities of the unsubstituted poly-nucleotides poly r(A-U) and poly r(I-C) and those of the completely substituted analogs poly r ( s A s U ) and poly r ( s I s C ) . The thiophosphate-substituted polyribonucleotides showed an increased resistance to degradation by nucleases present in fetal calf serum. The nucleolytic activity of the serum resembled pancreatic rather than T1 ribonuclease. The increased resistance to breakdown by ribonuclease and serum paralleled an increased capacity to confer cellular resistance to virus infection, suggesting that protection against premature enzymatic degradation might underly the increased antiviral activity of the thiophosphate analogs in vitro and in vivo.

[11] Enzymic modification of the C-C-A terminus of tRNA

Publisher Summary The chapter discusses the enzymic modification of the C-C-A terminus of tRNA. The tRNA species with an altered C-C-A terminus are used for investigation of the mechanism of aminoacylation and ribosomal protein biosynthesis, as well as for spectroscopic and X-ray crystallographic studies. ATP(CTP):tRNA nucleotidyltransferase (EC 2.7.7.25), which catalyzes the incorporation of CMP and AMP into tRNA lacking the C-C-A part of its 3 terminus, is used for preparation of modified tRNA species as it has been shown that some analogs of ATP and CTP are also substrates for this enzyme. An essential prerequisite for the preparation of uniformly modified tRNAs via incorporation of AMP and CMP analogs by ATP(CTP) : tRNA nucleotidyltransferase into the 3 end of tRNA is a tRNA species with a uniformly shortened 3 terminus. Furthermore, a highly purified enzyme free of nuclease is required because long incubation times and high enzyme concentrations usually have to be applied owing to the higher Km values and lower reaction velocities with the ATP and CTP analogs. Procedures for the isolation of ATP(CTP), tRNA nucleotidyltransferase from yeast, preparation of tRNAPhe-A, tRNAPhe-A-C, and tRNAPhe-A-C-C from yeast, enzymic synthesis of modified tRNAs, and their analysis are described.

The antiviral activity of certain thiophosphate and 2'-chloro substituted polynucleotide homopolymer duplexes.

Abstract Poly(rI)·poly(rsC) (polyriboinosinic acid) (polythiophosphate ribocytidylic acid) was found to be a slightly less potent interferon inducer than poly(rI)·poly(rC). The rate of degradation for poly(rI)·poly(rsC) and poly(rI)·poly(rC) by serum nucleases also proved to be very similar. In contrast, substitution of the 2′-hydroxyl by a chlorine atom in poly(rU) (polyribouridylic acid) and poly(rC) greatly increased their resistance to serum nucleases. Poly(rA)·poly(2′-ClU) (polyriboadenylic acid) (poly2′-chlorouridylic acid) and poly(rI)·poly(2′-ClC) were incapable of producing interferon although they possessed as great or greater thermal stability and much greater nucleolytic resistance than the unsubstituted interferon producing duplexes. This lack of interferon production by the 2′-Cl duplexes could not be explained by the failure of human foreskin fibroblasts (HFF) to take up these polymers. In addition, poly(rI)·poly(2′-ClC) did not compete with the interfering activity or the cellular association of poly(rI)·poly(rC). Substitution of the 2′ue5f8OH by a 2′ue5f8NH2 in poly(rU) also produced a polymer incapable of producing interferon.

[36] 5-Formyl-UTP for DNA-dependent RNA polymerase

Publisher Summary In order to determine which subunits of DNA-dependent RNApolymerase contain the catalytic site, affinity labeling has been employed by various groups. This chapter details the synthesis of α- and β-fo 5 UTP and enzyme purification and assay. The chapter observes that the analog of β-fo 5 UTP proved to be a potent inhibitor of the enzyme, and was hence analoged. Although this no longer has the usual fl configuration of those triphosphates that are substrates for RNA-polymerase, Rhodes and Chamberlin have shown that the elongation site in the ternary complex has a general affinity for the triphosphate moiety. The inhibition of RNA polymerase by the binding of α-fo 5 UTP to the β subunit has been shown to satisfy several criteria for affinity labeling: (1) it forms a noncovalent complex prior to covalent attachment; (2) the inhibition and covalent attachment of α-fo 5 UTP can be suppressed by the presence of nucleoside triphosphates; (3) the triphosphate α-fo 5 UTP is a more potent inhibitor than the nucleoside α-fo 5 UTP; and (4) the α-fo 5 UTP stoichiometrically labels RNA polymerase (after a 20-sec preincubation).